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Physiological Reviews, Vol. 80, No. 1, January 2000, pp. 173-210
Copyright ©2000 by the American Physiological Society
Institut für Pharmakologie, Universität Köln, Köln; and Institut für Pharmakologie und Toxikologie, Universität Münster, Münster, Germany
PREFACE
I. INTRODUCTION
II. STRUCTURE AND NOMENCLATURE OF SERINE/THREONINE PROTEIN PHOSPHATASES
A. Biochemical Classification of Protein Phosphatases
B. Primary Structure of the Catalytic Subunits of Protein Phosphatase and Subcellular Localization
III. MODULATORS OF PROTEIN PHOSPHATASE ACTIVITY
A. Protein Inhibitors
B. Nonprotein Inhibitors
C. Activators
IV. METHODOLOGICAL CONSIDERATIONS
V. EFFECTS OF PHOSPHATASES ON ION CHANNEL ELECTROPHYSIOLOGY
A. Voltage-Dependent Ca2+ Channels
B. Voltage-Dependent Na+ Channels
C. K+ Channels
D. Ligand-Gated Cation Channels
E. Anion Channels
VI. SUMMARY AND PERSPECTIVES
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ABSTRACT |
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Herzig, Stefan and
Joachim Neumann.
Effects of Serine/Threonine Protein Phosphatases on Ion
Channels in Excitable Membranes. Physiol. Rev. 80: 173-210, 2000.
This review
deals with the influence of serine/threonine-specific protein
phosphatases on the function of ion channels in the plasma membrane of
excitable tissues. Particular focus is given to developments of the
past decade. Most of the electrophysiological experiments have been
performed with protein phosphatase inhibitors. Therefore, a synopsis is
required incorporating issues from biochemistry, pharmacology, and
electrophysiology. First, we summarize the structural and biochemical
properties of protein phosphatase (types 1, 2A, 2B, 2C, and 3-7)
catalytic subunits and their regulatory subunits. Then the available
pharmacological tools (protein inhibitors, nonprotein inhibitors, and
activators) are introduced. The use of these inhibitors is discussed
based on their biochemical selectivity and a number of methodological
caveats. The next section reviews the effects of these tools on various
classes of ion channels (i.e., voltage-gated Ca2+ and
Na+ channels, various K+ channels,
ligand-gated channels, and anion channels). We delineate in which
cases a direct interaction between a protein phosphatase and a given
channel has been proven and where a more complex regulation is likely
involved. Finally, we present ideas for future research and possible
pathophysiological implications.
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PREFACE |
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Tu ne quaesieris, scire nefas, quem mihi, quem tibi finem di dederint, Leuconoe, nec Babylonios temptaris numeros. Ut melius, quidquid erit, pati! Seu pluris hiemes seu tribuit Iuppiter ultimam, quae nunc oppositis debilitat pumicibus mare Tyrrhenum. Sapias, vina liques, et spatio brevi spem longam reseces. Dum loquimur, fugerit invida aetas: carpe diem quam minimum credula postero.
Q. Horatius Flaccus: Carmen 1.11.
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I. INTRODUCTION |
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Nearly all aspects of cell function involve phosphorylation of the amino acids serine/threonine. It has been estimated that one-third of cellular proteins are reversibly phosphorylated (95). Phosphorylation can regulate proteins that induce very short-term or very long-term effects like ion channels and transcription factors. Specifically, cell division, cell differentiation, neuronal activity, muscle contraction, and metabolic functions are regulated by phosphorylation. Here, we are mostly concerned with phosphatases in mammalian cells that possess excitable membranes. At present, it seems that many important dephosphorylation reactions in these cells are catalyzed by a limited number of catalytic subunit isoforms. However, the important concept emerges that the substrate specifity and function of phosphatases are mainly regulated by ancillary proteins. Therefore, we address their putative function where required.
The past decade witnessed rapid progress on the physiological role of phosphatases. The advent and widespread experimental use of new inhibitors as pharmacological tools hastened this process. Ion channels are ideal candidates for studying the dynamics of phosphorylation and dephosphorylation of proteins, because their molecular properties can be measured on-line in single-channel experiments. Based on these considerations, we try to address the structural and biochemical properties of phosphatases and their regulatory subunits first. The tools available for physiological experiments are then described, and a paragraph is devoted to methodological problems regarding the use of such compounds. The present electrophysiological knowledge about regulation of ion channels is also summarized. We are aware of the vast amount of data in this field but have chosen to restrict our presentation to those types or families of ion channels studied in more detail. Emphasis is placed on experiments where the molecular target of the phosphatase is probably the channel itself, or where a more complex but interesting signaling cascade is involved. It should be stated here that electrophysiological experiments will usually fail to prove the exact molecular nature of the dephosphorylated protein or even the exact amino acid. Up to now, most studies have employed native cells. Space limitations necessitate us to focus the present review along several dimensions. Preference is given to cite more recent work, mainly covering the 1990s (until 4/99). For in depth discussion of earlier literature, excellent reviews are available (375). The role of phosphatases in cell division is covered by another review in this journal (319). This review is concerned with serine/threonine-specific phosphatases. However, additional phosphatases (mitogen-activated protein kinase phosphatases) exist that dephosphorylate both threonine and tyrosine residues within the same substrate protein (432). Tyrosine phosphatases, which may also be relevant for ion channel regulation, are also not covered here. Even within the remaining area of ion channel regulation by serine/threonine phosphatases, we have to skip a considerable number of valuable papers. We apologize for any omission that will have to be considered a regrettable flaw.
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II. STRUCTURE AND NOMENCLATURE OF SERINE/THREONINE PROTEIN PHOSPHATASES |
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A. Biochemical Classification of Protein Phosphatases
Philipp Cohen (77, 79) suggested to divide
phosphatases solely on an enzymatic basis into protein phosphatase (PP)
1 and PP2. This classification holds true for mammalian phosphatases that are the subject matter here. In contrast, bacterial phosphatases do not neatly follow this scheme (81). Mammalian type 2 PP
were subdivided into PP2A, PP2B, and PP2C (77). Type 1 PP
are characterized by their inhibition by protein inhibitors 1 and 2. Type 1 PP dephosphorylates preferentially the
-subunit of
phosphorylase kinase. Type 2 PP are not inhibited by inhibitors 1 and 2 (of PP1) and dephosphorylate mainly the
-subunit of phosphorylase
kinase. PP2B is characterized by its requirement for Ca2+
and calmodulin, whereas PP2C requires Mg2+ for activity
(375). Both PP1 and PP2A do not require divalent cations
for their enzymatic activity. More recently, new serine/threonine phosphatases have been cloned and sequenced (78). New
mammalian phosphatases are PP4 (or PPX), PP5, PP6 (or Sit4), and PP7
(see sects. IIB, 5-9). In contrast to
the other PP, PP7, when expressed in vitro, is inactive against the
widely used substrate phosphorylase a (it is unknown whether
this is also true in vivo); however, phosphorylated histone can be used
as substrate. PP7 is dependent on Mg2+ but not calmodulin
and is activated by Ca2+ (208).
B. Primary Structure of the Catalytic Subunits of Protein Phosphatase and Subcellular Localization
The catalytic subunits of phosphatases that dephosphorylate serine and threonine are encoded by the PPP and PPM gene families (78). These families are defined by distinct amino acid sequences and crystal structures. The PPP family includes the prototypical types 1, 2A, and 2B phosphatases. It also comprises novel phosphatases like PP4, PP5, and PP6 (78). The PPM family includes Mg2+-dependent phosphatases like PP2C. Hence, PP2C stands quite apart (375). This view is also supported by the fact that most inhibitors of PP1 and PP2A affect PP2B at high concentrations, but not PP2C (see sects. IIB4 and IIIB). An overview of the various catalytic subunits for the PP in this review is given in Table 1. In contrast to the monomeric PP2C, the PP1, PP2A, and PP2B are multimeric forms. The multimeric forms consist of the catalytic subunit and one or two accessory proteins. These accessory proteins can confer substrate specificity, can regulate enzyme activity, and can control the subcellular localization of the holoenzyme (127).
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1. PP1
A) CATALYTIC SUBUNITS.
Cloning revealed that the catalytic subunit of type 1 phosphatases is
differentiated into types 1
, 1
, and 1
derived from three
different genes (364). Protein phosphatase 1
codes for a protein of 330 amino acids (e.g., in rat and rabbit, Refs. 19, 31,
364). The catalytic subunit of PP1
is comprised of 327 amino acids
(rat, Ref. 364). The catalytic subunit of PP1
has two splice
variants called PP1
1 and PP1
2 that code
for 323 and 337 amino acids, respectively (364). Sequences
are highly conserved between species. For instance, human
PP1
1 is 93% nucleotide sequence similar and identical
in protein sequence to the rat homolog (334).
1 (380). In the
heart, where PP1
, PP1
, and PP1
1 are
immunologically detectable in whole tissue homogenates, the
myofibrillar fractions contain mainly PP1
(70). This
should be considered when comparing biochemical or
(electro)physiological data from whole tissues and cells.
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2, an isoform of PP1 enriched in testis. The
functional role of this association is unknown (71). PP1
can bind to the retinoblastoma gene product. This may contribute to the
function of PP1 in cell division (112). However, binding
has not yet been shown in vivo. With the use of the yeast
two-hybrid system it is was revealed that PP1
binds to p53BP2.
The latter is a protein that binds to the p53 protein that acts as a
tumor repressor. p53BP2 also inhibits potently PP1 activity against
phosphorylase a (176). PP1 also binds to a
splicing factor. The factor was identified as polypyrimidine
tract-binding protein-associated splicing factor (PSF). PP1 was
inhibited by recombinant PSF (see Table 4). It is conceivable that the
inhibitory action of PSF is potentiated by phosphorylation of PSF. The
interaction may play a role in the physiological regulation of splicing
in the spliceosome (192). In porcine aorta, PP1 could be
inhibited by a protein of ~20 kDa. The inhibition was proportional to
its phosphorylation by an unknown endogenous kinase or exogenous
protein kinase C (PKC). The IC50 was within the
physiological concentration of the inhibitory protein of 20 kDa and
might thus be functionally relevant (125). This protein
has been sequenced and was called CPI17 (C-kinase activated PP
inhibitor; apparent molecular mass 17 kDa; Ref. 126). It is expressed
in smooth muscle like aorta or bladder but not in skeletal muscle or
nonmuscle tissue (Table 3; Ref. 126). A new 36-kDa protein was
identified that binds and inhibits PP1 activity. It was termed PPP1R5
(105). This protein is related to GL, but it is, in contrast to GL, ubiquitously expressed (Table 3;
Ref. 105).
The inhibitory or stimulatory actions of the accessory subunits of PP1
are compared in Table 4.
C) DIRECT ALTERATION OF PP1 ACTIVITY. A
more direct way to modulate phosphatase 1 activity is by
posttranslational modification of their catalytic subunits.
Phosphorylation and methylation have been reported. All mammalian PP1
share a -TPPR- sequence (364), which is a
recognition site for phosphorylation by cyclin-dependent protein
kinases. Indeed, PP1
and PP1
1 are phosphorylated on threonine-320 and threonine-311, respectively, by cell
cycle-dependent protein kinases (cyclin-dependent protein kinases),
and this phosphorylation inhibits their phosphorylase phosphatase
activity (103). The phosphorylation led to the
incorporation of 0.5 mol phosphate/mol protein within 30 min. At this
time point, phosphatase activity decreased to 50% of the initial value
(103). This phosphorylation was first observed in vitro
but later also in a cell cycle-dependent manner in intact cells and
was accompanied by changes in cell cycle-dependent phosphatase
activity in cytosol and nucleus (264).
2. PP2A
A) CATALYTIC SUBUNIT.
The catalytic subunits of PP2A (see also Tables 1 and 2 for synopsis)
exist in two isoforms called PP2A
and PP2A
. They have been cloned
from many species (15, 84, 85, 161, 182, 248, 249, 392,
393). Rat liver PP2A
cDNA codes for a 309-amino acid protein.
PP2A
was different from PP2A
in 8 amino acids, but the cDNA coded
also for 309 amino acids and is coded by a different gene. The
expression on protein and mRNA is higher for PP2
than PP2
. For
instance, in porcine heart, the ratio is 8:1 (392). PP2A
is apparently mainly cytosolic. However, some PP2A was also detectable
in the nucleus (418).
and A
forms encode
proteins of 589 and 602 amino acids, respectively. The effect of the A
subunit on PP2A activity is substrate dependent. Recombinant A
inhibited the activity (using phosphorylase a or myosin
light chains as substrate) of the C subunit from the bovine heart with
high potency (IC50 = 0.1 nM) (235). In
contrast, in rabbit reticulocytes, the A subunit could stimulate the
enzymatic activity of the C subunit when using eukaryotic elongation
initiation factor 2 as substrate (61).
B) B subunit. Three major variants of the B subunit have
been termed B (52 kDa, also called B/PR55), B' (53 kDa, also called B'/PR54), and B'' (72 kDa and a splice variant of 130 kDa). For overview and tissue distribution, see Tables 1 and
5. The binding of the B subunits is
mutually exclusive. Three isoforms called
(447 amino acids),
(443 amino acids), and
of the B subunits have been cloned
(174, 308, 469). The B' subunit (PR 53) is further divided
into
,
,
, and
. B'
can be alternatively spliced to
generate B'
1 and B'
2, coding for proteins
of 514 and 475 amino acids, respectively. Four variants (probably
splice variants) of the B'
isoform can be further distinguished. The B'' subunit is comprised of two isoforms of 72 kDa (PR 72) and 130 kDa
(PR 130) (319). PR 72 and PR 130 isoforms contain 529 and
1,150 amino acids, respectively. Like for the A subunit, the effect of
the B subunit on the enzymatic acitivity of PP2A is substrate
dependent. Reconstituted dimers of A and C subunits were
inhibited by the B subunit purified from bovine heart with an
IC50 of 0.59 nM (235). In contrast, the B
subunit (PR55) stimulated the dephosphorylation of cdk1-phosphorylated
histone H (3).
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3. PP2B
PP2B, alternatively called calcineurin, is a Ca2+/calmodulin-dependent protein phosphatase. The enzyme consists of two subunits, the catalytic A subunit (see Table 1) of ~60 kDa (CNA) and the regulatory B subunit (CNB) of ~19 kDa. Calcineurin is present in nearly all mammalian cells studied. However, it is most highly expressed in the brain (see Table 2 for tissue distribution).
A) CATALYTIC SUBUNIT (A SUBUNIT). Cloning from rat brain
indicated a length of 521 amino acids for the A subunit. There are three mammalian genes for the A subunit, giving rise to the CNA
, CNA
, and CNA
isoforms. CNA
and CNA
are highly expressed in brain, whereas CNA
is testis specific. Differential splicing of
CNA
generates two transcripts (
1 and
2). The CNA
gene is alternatively spliced to three
transcripts CNA
1, CNA
2, and
CNA
3. CNA
1 and CNA
2 are
highly expressed in neuronal tissue, whereas CNA
is specific for the
testis (419). The catalytic subunit (A subunit) of PP2B
shows autoinhibition that is relieved by interaction with the B
subunit. PP2B is quite different from PP1 and PP2A. It is the only PP
clearly regulated by a second messenger, namely, Ca2+. The
inhibition of B on A is relieved if B binds Ca2+. This
explains why the enzyme is dependent on Ca2+ for activity.
Using proteolysis of the autoinhibitory COOH terminus of the A subunits
generates a Ca2+-independent isoform. This can be used
experimentally to understand the function of PP2B.
B) REGULATORY SUBUNIT (B SUBUNIT). The B subunit was
sequenced at the protein level and found to comprise 168 amino acids. It shows sequence similarity to calmodulin. Like calmodulin, it binds 4 mol Ca2+/mol (5). Two different B subunit
genes are known that are called CNB
and CNB
. CNB
gives rise to
one isoform expressed in many tissues named CNB
1 (170 amino acids) and, by means of a different promotor, leads to another
testis-specific isoform called CNB
2 (216 amino
acids). Similarly, CNB
(179 amino acids) is only expressed in the
testis (52, 419).
The substrate specificity of PP2B is quite high. Well-investigated substrates include a subunit of phosphorylase kinase, inhibitor 1 (of PP1), DARPP-32, the type II regulatory subunit of the cAMP-dependent protein kinase, and the site 2 of the glycogen binding subunit of PP1 (RGL; Ref. 213). Data with inhibitors for PP2B (see also sects. IIIB, 7 and 8) indicate that the activity of the PP2B is substrate dependent. Although these inhibitors decreased the activity toward a 19-amino acid peptide, they caused a stimulation of activity toward p-nitrophenylphosphate (287). PP2B might play a role in signal transduction especially in the brain, where its expression is very high.
C) ACCESSORY PROTEINS. A) AKAP 79. A protein kinase A anchoring protein (AKAP 79) was able to bind PP2B. AKAP 79 inhibited in a noncompetitive manner PP2B activity with an IC50 of ~4 µM. It did not inhibit PP1 or PP2A. The interaction was studied in bovine brain (75).
B) Cain. Another protein called calcineurin inhibitor (cain) was more recently studied (266). Cloning predicted a sequence of 2,182 amino acids. The IC50 was ~0.4-0.5 µM. Hence, cain is more potent than AKAP 79. Cain was highly expressed on RNA and protein level in brain, kidney, and testis. It was least detectable in heart, spleen, lung, liver, and skeletal muscle. It is mainly cytosolic. It was speculated that cain may target inactivated PP2B to specific intracellular regions where its release would provide Ca2+-regulated phosphatase activity to specific signaling pathways (266).
4. PP2C
PP2C is monomeric. In mammalian cells, PP2C
and PP2C
are
known (404, 437). PP2C
is comprised of 382 amino acids.
Several isoforms of PP2C
, namely, PP2C
1,
PP2C
2, and PP2C
3, have been reported
(302). PP2C
1 is the most abundant isoform.
Alternative splicing seems to generate the isoforms
PP2C
1 and PP2C
2. They were cloned from a
mouse library and show differences in COOH termini and the
3'-untranslated region. PP2C
1 is expressed in all mouse
tissues studied, whereas PP2C
2 is confined to heart and
brain where they might subserve special functions (408). PP2C
1 and PP2C
2 code for 390 and 389 amino acids, respectively. Tissue distribution is included in Table 2.
PP2C was originally assumed to be exclusively cytosolic
(375). More recent work identified PP2C also in the
nucleus of mammalian cells (87).
5. PP3
A PP3 has been suggested to exist (203). It was described as a particulate protein in the bovine brain that was inhibited by okadaic acid but stimulated by inhibitor 2 and inositol phosphates (471). Because there has not been any recent report on PP3, it seems likely that this enzyme may have been artifactual.
6. PP4
PP4, also called PPX (44), is expressed highly in testis; however, it was also detectable in all other tissues investigated (see Table 2). Its structure, like that of PP6, is reminiscent of the paradigmatic PP2A. PP4 is comprised of 307 amino acids (rabbit); PP4 is mainly localized in the nucleus, although smaller amounts are also present in the cytosol (44). Regulatory subunits of PP4 are thought to exist but have not been clearly identified (78).
7. PP5
PP5 is ubiquitously expressed in human tissues (see Table 2). The calculated molecular mass of the protein is ~58 kDa (the 5'-end of the sequence was incomplete in the inital report, Ref. 58). PP5 contains an autoinhibitory domain. Polyunsaturated fatty acids can relieve this inhibition (57). PP5 was detectable mainly in the nucleus, although some immunoreactivity was also present in the cytosol (56, 58, 67). PP5 interacts with the atrial natriuretic peptide receptor and was isolated in complex with a glucocorticoid receptor (56). These associations might indicate some kind of regulatory interaction.
8. PP6
PP6 is structurally related to PP2A. PP6 (303) has so far been identified in all mammalian tissues examined (see Table 2). Sit 4, the Saccharomyces homolog of PP6, has regulatory subunits (130). Therefore, regulatory subunits of PP6 are thought to exist but have not been clearly identified (78).
9. PP7
PP7 is comprised of 653 amino acids. With a comparison of RNA from various human tissues, PP7 was only detectable in the retina and not, for instance, in the heart (see Table 2).
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III. MODULATORS OF PROTEIN PHOSPHATASE ACTIVITY |
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In contrast to some tyrosine phosphatases, serine/threonine phosphatases are probably not located transmembranic but subplasmalemmal. This is no problem when using cell-free extracts. In more intact systems, inhibitors or activators have to pass the membrane. This is easiest if the compounds are freely permeable. Otherwise, the membrane has to be broken. This can be done mechanically (injection through pipette), electrically (gene pulser), chemically (transfection reagents), or by viral gene transfer. Hence, if information is available on cell permeability, it will be provided for the compounds listed in this section.
A. Protein Inhibitors
All these inhibitors are comprised of amino acids and are thus not expected to pass cell membranes easily.
1. Inhibitor 1 PP1
Inhibitors 1 and 2 of PP1 were identified by Huang and Glinsmann (206, 207). They share unusual physical properties. Both are heat stable and are not precipitated by 1% trichloroacetic acid, in contrast to most other proteins. The sequence of I1 has been reported first by direct protein sequencing from rabbit skeletal muscle and then from a rat skeletal muscle library and a human brain library by cloning and results in a predicted protein of 171 amino acids (4, 117, 119). The NH2-terminal region is highly conserved. Only in the COOH termini were differences between rabbit, rat, and human noted (119).
I1 from rabbit skeletal muscle and human brain has a
calculated molecular mass of 18.7 and 19.2 kDa, respectively (4,
119). The apparent molecular mass is ~26 kDa on SDS-PAGE.
This discrepancy has been explained by a low degree of order in the
protein. I1 binds to and inhibits PP1 only after being
phosphorylated on threonine-35 by cAMP-dependent protein kinase or
cGMP-dependent protein kinase (181). It is very
selective for PP1. For instance, phosphorylated recombinant human
I1 inhibited PP1 and PP2A with IC50 values of 1.1 and 21,000 nM, respectively (119). Phosphorylation of
I1 occurs in vivo in skeletal muscle, heart, and
cardiomyocytes after
-adrenergic stimulation (76, 168,
329). In vitro-phosphorylated I1 has been used
as a tool to study in permeabilized preparations whether a process
involves PP1. For instance, I1 phosphorylated by
cGMP-dependent protein kinase induced force generation in
permeabilized smooth muscle cells (413). As expected,
mutagenesis of threonine-35 to alanine yielded a mutant I1
that could not be phosphorylated by I1 and that did not
inhibit PP1. Mutation of threonine-35 to aspartic acid, which is
intended to mimic phosphorylation, led to a mutant form of
I1 that inhibited both PP1 and PP2A with IC50 values of 24 and 25 µM, respectively (119). Immobilized
I1 binds ~10 times better to PP1 than to PP2A,
independent of its phosphorylation state. Amino acids 9-12 KIQF are
conserved in rat, rabbit, and human, and they seem to be crucial for
binding and inhibition of PP1 (114). If this sequence is
deleted, phosphorylation of I1 is unable to inhibit PP1
activity (119). Interestingly, I1 is present
in the liver of rabbits, guinea pigs, and sheep but absent from mouse
and rat liver (210, 294). I1 is present, for example, in skeletal muscle, heart, kidney, uterus, and adipose tissue
(117, 294). I1 is a cytosolic protein.
2. DARPP-32
DARPP-32 is similar to I1 in function but derived from a different gene, and it is mainly expressed in the brain (178). It has been sequenced on protein and cDNA level from bovine brain (262, 444) and rat brain (115). DARPP-32 from bovine brain has a predicted molecular mass of 22.6 kDa and, like I1, a higher apparent molecular mass of 32 kDa on SDS-PAGE. The same threonine residue on DARPP-32 is phosphorylated by cAMP-dependent protein kinase but also by cGMP-dependent protein kinase. Phosphorylation of DARPP-32 changes its IC50 for PP1 from 1 µM to 2 nM (96, 97), underscoring its high selectivity. Under unphysiological conditions (mM Mn2+ in the assay), I1 and DARPP-32 are dephosphorylated and inactivated by PP1. Both I1 and DARPP-32 are dephosphorylated by PP2A and even better by PP2B (98, 178, 180). The dephosphorylation by PP2B is dependent on the presence of Ca2+. Hence, it was suggested that this might be a way for Ca2+ levels to control protein phosphorylation (214). DARPP-32 is a cytosolic protein.
Thiophosphorylated I1 or DARPP-32 is not (easily) dephosphorylated and has been successfully used to study the physiological role of PP1-mediated phosphorylation in muscle contraction.
3. Inhibitor 2 PP1
I2 from rabbit skeletal muscle is comprised of 204 amino acid and has a calculated molecular mass of 22.9 kDa (198). Similarly to I1, its apparent molecular mass on SDS-PAGE is larger and amounts to ~31 kDa. It is unrelated in sequence to I1. Its tissue distribution has been studied (294, 354). It binds to and inhibits PP1 regardless of phosphorylation. Therefore, it has been used to study the effect of PP1 in intact cells. It forms complexes with PP1 and is therefore discussed in depth above. A caveat is noteworthy. I2 inhibits the free catalytic subunit of PP1 in the nanomolar range. However, the glycogen-associated PP1 is poorly inhibited, and the smooth muscle myosin-associated PP is not inhibited at all (7, 213, 214). However, it is known that glycogen-associated PP and the myosin-associated PP contain the catalytic subunit of PP1 (95). Hence, inability to block a process by addition of I2 does not prove that a PP1 is not involved but requires additional experimentation. The binding site for I2 is probably close to that of okadaic acid (460). Mutational analysis suggests that I2 inhibits via interaction with the amino acid tyrosine-272 on PP1 because its IC50 is changed from 13 to 180 ng/ml in the mutant Y272K (458).
4. Inhibitor 1 PP2
Inhibitor 1 PP2 has been isolated from bovine kidney. It is thermostable and not inactivated by 1% trichloroacetic acid. Its apparent molecular mass was 30 kDa. (277). Later, it was identified as putative class II human histocompatibility leukocyte-associated protein (PHAP) I (279). PHAP I had been cloned and sequenced before by another group (421). It seems to inhibit the catalytic subunit directly (279). It is present in the nucleus but more abundant in the cytosol (421). The tissue distribution has not been rigorously tested. However, its transcript was also detectable in Northern blots from rat heart and skeletal muscle (J. Neumann and H. Lüss, unpublished observations). It is not known whether its activity is regulated by a posttranslational modification like I1 of PP1 (277).
5. Inhibitor 2 PP2
This inhibitor has been isolated from bovine kidney. It is also
thermostable and not inactivated by 1% trichloroacetic acid. Its
apparent molecular mass was initially reported as 20 kDa
(277). Protein sequencing revealed that the protein had
been described before as SET (278, 426, 427), PHAP II
(421), and template activating factor-1
(322). SET has a predicted molecular mass of 32,100 Da and
an observed molecular mass of ~39 kDa and is largely located in the
nucleus (138). Thus proteolysis should account for the
lower molecular mass reported initally. Ubiquitous expression of SET
was reported (2). Interestingly, SET was phosphorylated on
serine in intact cells. Whether this alters phosphatase inhibitory
function remains to be established (2). These inhibitors
might be useful tools to study the physiological function of PP2A. It
has been speculated that I1 and I2 of PP2A might be involved in signal transduction. Specifically, they were suggested to mediate the effects of insulin on PP2A (277).
6. Simian virus 40 small tumor antigen
Simian virus 40 (SV40) is a member of the papova family of small
DNA tumor viruses. Its lytic cycle takes place in permissive monkey
cells. SV40 infection leads to the production of proteins that are
immunogenic and that were called tumor antigens. One such antigen is
the SV40 small tumor antigen. It can inhibit PP2A activity with an
IC50 of 10-15 nM. This has been reported for substrates
such as myosin light chains, phosphorylated by myosin light-chain
kinase (453). As mentioned above, PP2A can occur as a
monomeric, dimeric, or trimeric species: C (catalytic subunit), AC, or
ABC. SV40 inhibits PP2A activation after forming a complex with the AC
species (453). This occurs with brain PP2A or in CV-1
cells that all contain the B
form of the the B subunit of PP2A.
Here, SV40 small tumor antigen displaces the B subunit from the PP2A
holoenzyme (386, 453). In cells the interaction of small
tumor antigen with PP2A leads to deinhibition and thus activation of
MAP kinase and MEK which induces cell proliferation (386). SV40 small tumor antigen can conceivably be used in cell extracts to
quantify the amount of PP2A activity. The protein is not expected to
pass through intact cell membranes. However, it can be injected into
cells or alternatively might be delivered to cells as their coding DNA
by various means of transfection including virus vectors (386,
387).
B. Nonprotein Inhibitors
1. Okadaic acid and derivatives
The history of okadaic acid (OA) is paradigmatic
(80). Okadaic acid is a polyether compound with a C-38
structure, isolated from the black sponge Halichondria
okadai named in honor of Yaichiro Okada (146).
Shibata et al. (376) noted that OA increased tone in
smooth muscle preparations. Erroneously, this was interpreted as
opening of Ca2+ channels and activation of a
Ca2+-dependent kinase and phosphorylation of regulatory
proteins. Moreover, OA was studied as a skin tumor promoter in mice
(396). However, Takai et al. (400) were the
first to report that OA is a potent phosphatase inhibitor in smooth
muscle preparations (34). Okadaic acid inhibited PP1,
PP2A, and PP2B with IC50 values of 272, 1.6, and 3,600 nM,
respectively (35). PP2C, phosphotyrosyl phosphatase, acid
phosphatase, and alkaline phosphatase were not inhibited by up to 10 µM OA. Inhibition was noncompetitive, mixed competitive, and
reversible (35). Hescheler et al. (190) noted that OA inhibited PP activity in skeletal muscle preparations and
increased currents through cardiac L-type Ca2+
channels. It is currently thought that tumor promotion by OA and
related compounds like calyculin A and microcystin LR is due to
inhibition of PP1 and PP2A (for review, see Ref. 146). The OA binding
site of PP is not the substrate binding site because OA inhibits PP1
and PP2A noncompetitively (35). However, a caveat is
warranted. Okadaic acid in concentrations <2 nM inhibits also PP4,
PP5, and PP6 that are present in mammalian cells (78). At least 16 derivatives of OA are known, and potent inhibitors include,
in addition to OA, dinophysistoxin-1 and acanthifolicin (146). Dinophysistoxin-1 was first isolated from the
hepatopancreas of the mussel Mytilus edulis. It caused
shellfish poisoning in Japan. Its name is derived from its source in
the dinoflagellate Dinophysis fortii (146).
Chemically, it is 35-methylokadaic acid (199).
Acanthifolicin is an episulfide derivative of okadaic acid. It occurs
naturally in the sponge Pandaros acanthifolium. Treatment of
acantifolicin with diazomethane led to acanthifolicin-methyl ester
(146). Of importance, some derivatives are inactive and can be used as negative controls. These include OA methyl ester, nor-okadaon, acanthifolicin methyl ester. Interestingly, chemical degradation products of OA, namely, OA spiroketal I and II, were still
able to inhibit type PP2A, indicating that it may be possible to design
simpler but still active OA derivatives (146). Okadaic acid increases the phosphorylation state of a number of proteins. Some
have been identified; these include vimentin and the 27-kDa heat shock
protein (human fibroblasts, Refs. 170, 454). Okadaic acid increased the
phosphorylation state of the epidermal growth factor receptor
(185), histone H3 (300), a progesterone
receptor (92), the 2. Cantharidin and analogs
Cantharidin, cantharidic acid, palasonin, and endothall are
structural analogs (268). Cantharidin (CA) is the vesicant
in blister beetles (beetle in Greek is 3. Calyculin A
Calyculin A (CyA) was isolated from another marine sponge,
Discodermia calyx. It is an octamethylpolyhydroxylated C-28
fatty acid linked to two 4. Microcystins
Whereas OA and CyA are fatty acid derivatives, microcystin and
nodularin are peptide toxins. Microcystins are of toxicological relevance. They cause death in cattle and humans exposed to water contaminated by certain algae. These include colonical and filamentous algae and cyanobacteriae like Microcystis aeruginosa
(48, 49, 146). Algae and prokaryotes seem not to contain
PP1 or PP2A; hence, they can survive these toxins in contrast to other
phyla. Microcystins are cyclic heptapeptides containing five constant (some of which are unique) and two variable amino acids. The variable amino acids in microcystins are given in the one-letter code. Hence, their short-hand notation is microcystin-LR, -YR, and
-RR. More than 40 additional microcystins have been identified
(146). Microcystin-LR inhibits PP1 and PP2A with
IC50 values of 0.1 nM each (298) or 1.7 and
0.04 nM, respectively (202). It inhibits PP2B with an
IC50 of 0.2 µM and does not inhibit PP2C up to 4 µM
(298). Okadaic acid prevents the interaction of
microcystin with PP2A, implying a similar site of action. Moreover,
binding of inhibitor 2 to PP1 prevented the binding of
microcystin-LR to PP1. It is important to keep in mind that the
newer mammalian PP4 and PP5 are also inhibited by <2 nM microcystin.
Hence, it is possible that some effects thought to result from PP1 or
PP2A inhibition actually result from PP4 or PP5 inhibition (44,
58, 303). Moreover, inhibition of PP6 by microcystin has not
been tested, and other phosphatases will likely be cloned in the
future. Microcystin is the most potent (and toxic) PP inhibitor. As
expected for a peptide, cell permeation is a problem. In fibroblasts,
microcystin-LR did not increase phosphorylation. However, it led to
hyperphosphorylation in hepatocytes (123). This is
consistent with the clinical observation that intoxications with
microcystin-contaminated water led to hepatic necrosis and
subsequent death, as shown recently by an epidemic caused by
microcystin-contaminated dialysis fluid (230). Peroral
microcystin is taken up with a bile acid transport system across the
ileum into hepatocytes (123). When radioactive microcystin was given intravenously to mice, label was detected mainly in liver but
also in kidney, gut, and lung. No label was found in spleen and heart.
Hence, no transport system for microcystin seems to exist in
cardiomyocytes or splenocytes (357). Interestingly, these
investigators clearly demonstrated that hepatic radioactive label
persisted after single administration and that the label was covalently
bound to a protein that they did not identify further but that is
expected to be a PP. Permeability problems can be overcome by
permeabilizing preparations with, e.g., The three-dimensional structure of microcystin-LR bound
to PP1 has been determined (18, 155). The modified amino
acid N-methyl-dehydroalanine (Mdha) in microcystin was bound
to cysteine-273 in PP1 (155). In a two-step mechanism,
microcystin-LR first binds to and inactivates PP1 or PP2 within
minutes. Thereafter, a covalent modification of Mdha in microcystin was
formed (within hours) with PP1 or PP2A (82). Mutation of
cysteine-273 in PP1 to alanine impeded covalent binding of microcystin
to PP1. However, this mutation does not reduce the potency of
microcystin, OA, nodularin, tautomycin, and inhibitor 2 to inhibit PP1
activity. This strongly implies a two-step mechanism and indicates
that binding to PP1 and inhibition of activity are distinct processes
(360). Likewise, another laboratory also identified
cysteine-273 in PP1 5. Nodularin
Nodularin was isolated from the toxic water cyanobacterium
Nodularia spumigenia (49). It is a cyclic
pentapeptide. Like microcystin, it is toxic to the liver. It does not
penetrate into fibroblasts, but it is active in hepatocytes, like
microcystins (146). Nodularin R and its derivative called
motuporin (nodularin V) potently inhibit PP1 as well as PP2A with
IC50 values of 1.6 and 0.03 nM, respectively
(201). Hence, it inhibits PP1 and PP2A ~10 times more
potently than OA. It is ~70-fold selective for PP2A. It inhibits PP2B
with an IC50 of 8.7 µM but does not affect PP2C
(201). Hence, the IC50 values are comparable
to those of microcystin-LR. In contrast to microcystins, nodularin
R or V does not covalently bind to PP1 or PP2A (82).
Mutational analysis suggests that nodularin inhibits PP activity via
interaction with amino acid tyrosine-272 on PP1 because its
IC50 is changed from 0.5 to 150 nM in the mutant Y272S
(458). The three-dimensional solution structure of
nodularin closely resembles that of microcycstin-LR (13). 6. Tautomycin
Tautomycin was isolated from Streptomyces
spiroverticillatus as an antibiotic because it is toxic to yeast
and fungi. Its structure as a polyketide resembles somewhat that of OA
(205, 296). It inhibits PP1 and PP2A with IC50
values of 0.7 and 0.65 nM, respectively (146), or 0.16 and
0.4 nM, respectively (296). Others reported
IC50 values of 0.4 and 34 nM for PP1 and PP2A, respectively
(401). The latter results might be interpreted as selectivity for PP1. However, p-nitrophenylphosphate was
used as substrate, whereas other laboratories that reported more potent inhibition of PP2A used the more conventional and perhaps more relevant
substrate phosphorylase a. Tautomycin does not inhibit PP2C,
and its IC50 for PP2B is 100 µM. Okadaic acid prevents
the interaction of tautomycin with the catalytic subunit of PP2A. Unlike microcystin, tautomycin led to hyperphosphorylation in all cell
types tested, like keratinocytes and K562 cells (146). Hence, it is apparently cell membrane permeable. Mutational analysis suggests that tautomycin inhibits via interaction with amino acid tyrosine-272 on PP1 because its IC50 is changed from 1.1 to
2,600 nM in the mutant Y272K (458). Like CyA, it can be
used in comparison with OA. This might indicate whether a physiological
effect is mediated by PP1 or PP2. 7. Ciclosporin
Ciclosporin, or cyclosporin A (a cyclic undecapeptide), and FK-506
(a macrocyclic lactone) inhibit PP2B (342). This
inhibition is not direct. First ciclosporin and FK-506 bind to
cyclophilin and a FK-506-binding protein (FKBP), respectively.
Thereafter, they interact with the latch region of the CNB subunit of
PP2B (74, 314). Then inhibition of CNA, the catalytic
subunit, occurs. The three-dimensional structure of the
calcineurin-FK-506 and FKBP complex supports this notion (162,
247). Rapamycin, an immunosuppressant fungal metabolite, also
binds to FKBP but does not inhibit PP2B because it cannot interact with
CNB for steric reasons (162). In contrast, it targets to a
rapamycin-associated protein (FRAP) that leads to inactivation of
some specialized protein kinases like p70s6k (6,
346). Work with ciclosporin is complicated by the fact that it
requires special solvents like Tween and ethanol mixtures that tend to
be cell toxic. However, it is cell membrane permeant. 8. Cypermethrin
Type 2 pyrethroids like cypermethrin are used as insecticides
because they modulate ion channel activity, but they have also been
reported to inhibit PP2B in nanomolar concentrations (118) independent of mediator proteins like cyclophilin (see sect.
IIIB7). However, subsequently others
reported that up to 1 mM cypermethrin did not affect PP2B, PP1, or PP2A
(297). 9. Apomorphine
Somewhat surprisingly, apomorphine and SKF-38393
(2,3,4,5-tetrahydro-7,8-dihydroxy-1H-3-benzazepine) inhibited
PP2A1 (trimeric ABC) from rat brain with IC50
values of 1 and 50 µM, respectively. In contrast, apocodeine was
inactive. It has apparently not been reported whether PP2A from other
tissues or other PP are inhibited by apomorphine (238).
Apomorphine is cell membrane permeant. 10. Fostriecin
The antitumor antibiotic fostriecin (CI-920) is a type II DNA
topoisomerase-directed anticancer drug, like doxorubicin or etoposide (41). Other phosphatase inhibitors are tumor
promotors (see above). Phase I clinical trials of fostriecin are being
conducted in the United States. It is a naturally occurring compound
from Streptomyces pulveraceous subspecies fostreus, an
actinomycete found in a Brazilian soil sample. It is a
water-soluble polyene lacton with a phosphate ester. Fostriecin
inhibited both PP1 and PP2A with IC50 values of 4 µM and
40 nM, respectively, but it did not inhibit tyrosine phosphatases
(356). Fostriecin led to histone phosphorylation and
vimentin phosphorylation in baby hamster kidney cells (88,
167). Fostriecin is very polar, and hence, it must be actively
transported into cells. The mechanism is poorly understood but may
involve the reduced folate carrier (88). It is has been
suggested that the PP inhibition at least contributes to its efficacy
against solid tumors in humans. 11. Heparin
Heparin inhibits and binds to PP1 but not PP2A (120, 121,
153), The compound can actually stimulate PP2A (see below). This property has led to a procedure to separate these phosphatases using a
heparin-based affinity column chromatography. Spermine inhibits
both PP1 and PP2A with similar potency (375). It was speculated that polycationic compounds might mimic the action of some
unknown intracellular factor. Their use as tools to study PP function
is hampered by their lack of membrane permeability. 12. Thyrsiferyl 23-acetate
This compound is, like OA, a polyether fatty acid and contains a
squalene carbon skeleton. It was isolated from the red alga L. obtusa. It is unique because it is a selective inhibitor of PP2A.
Up to 1 mM it does not inhibit PP1, PP2B, PP2C, or tyrosine phosphatase
activity. Its IC50 for PP2A is ~4 µM. Hence, it is several orders of magnitude less potent than OA or CyA. However, it can
be used in cell extracts to distinguish between type 2A and other
phosphatases. It is expected to be cell membrane permeant (307). The potency of inhibitors is usually 10-100 times less in intact
tissue or cells than in enzymatic inhibition assays (54). This has been explained by reduced uptake into cells, their
preferential localization within the lipid phase, or by the high
intracellular concentrations of the targeted phosphatases (76, 122; see
also sect. IV). C. Activators
1. 2,3-Butanedione monoxime
2,3-Butanedione monoxime (BDM) was initially described as a
chemical phosphatase. However, it was demonstrated that BDM does not
directly dephosphorylate substrates like phosphorylase a. Instead, BDM activates the phosphatase holoenzyme of PP1 and/or PP2A
(468). A caveat is in order. BDM is probably a poor tool because it is no specific phosphatase activator but exhibits various effects on additional proteins. Indeed, there are numerous examples where BDM directly blocks ion channels but does not cause
dephosphorylation (9, 116, 367, 384, 457). 2. Sphingosine derivatives
Ceramide can stimulate the activity of trimeric PP2A. However, the
activity of the dimer (CA) or the free catalytic subunit (C) cannot be
stimulated by ceramide (101). Ceramide might be an
important second messenger for cell membrane-located receptors (20, 252). 3. Other activators
PP2A is activated by polylysine, protamine, polybrene, and histone
H1 (120, 343). Histone H1 is only present in the nucleus and might thus be a physiological stimulator of PP2A that is detectable in the nucleus in small amounts (see above). Protamine did not stimulate the activity of the purified catalytic subunit of PP2A, but
the PP2A1 (trimeric ABC) was stimulated. Protamine could also stimulate
the dimeric form of PP2A reconstituted with recombinant SV40 tumor
small antigen (234). Protamine can stimulate or inhibit PP2A, depending on the substrate studied. Protamine stimulated and
inhibited trimeric PP2A when myosin light chain or histone-1 were used
as substrates, respectively (234). In constrast, heparin can stimulate trimeric PP2A independently of the substrate under study
(234). The activation probably does not result from
dissociation of trimeric PP (64). However, the subtype of
B subunit is important. Heparin did not displace B
-subunit of the inhibitory
nucleotide binding protein Gi-2 (46), and
cardiac regulatory proteins (326). In the nuclei, OA
caused sustained activation of gene expression as well as
hyperphosphorylation of suppressor gene products (145,
170). Okadaic acid induced apoptotic death (39). In
other systems, OA inhibited heat-induced apoptosis
(25). Nuclear effects of OA include transcription of
c-fos and c-jun, the classical early response
genes (243). NF
B was induced and dissociated from I
B
(410). Okadaic acid reduced the expression of the myogenic
determination gene MyoD1 (242). Okadaic acid
activated the 70-kDa heat shock protein promoter (53). The
permeation of OA through cells seems to be rather poor. It has been
estimated that OA penetrates the cell membrane 100-fold less readily
compared with calyculin A (128). Peroral application of
radioactive OA led only to 1% absorption. Intraperitoneal application
of radioactive OA indicated that OA is excreted through hepatobiliary
circulation (146). However, OA freely permeated through
the lipid membrane of multilayer vesicles in a liquid-crystalline state, indicating that OA gains access to receptors in the cytosol (325). Okadaic acid (458, 460) binds probably
to YRCG (amino acids 267-270) or the vicinity. Mutational
analysis suggests that OA inhibits via interaction with amino acid
tyrosine-272 on PP1 because its IC50 is changed from 200 to
50,000 nM in the mutant Y272S (458).




o
) and
present in Spanish flies, palasonin is an anthelmintic in seeds of a
medicinal tree, and endothall is a synthetic herbicide. The toxic
action of cantharidin is thought to be due to phosphatase inhibition.
Initially CA was reported to bind to PP2A in mouse liver
(281). However, CA inhibits PP1 and PP2A with
IC50 values of ~500 and 40 nM (200, 282,
330). Palasonin and cantharidic acid exhibited similar
inhibitory activity (282). Endothall inhibited both PP1
and PP2A with IC50 values of ~5,000 and 1,000 nM
(282). Neither compound inhibited PP2B (>30,000 nM) or
PP2C (>1 mM). All compounds are herbicides. However, endothall is more
potent, possibly due to the expression of other PP in plant or to
permeability differences (282). Cantharidin is a
terpenoid. It is cell membrane permeable. It caused phosphorylation of
regulatory proteins in rhabdomyocytes and leiomyocytes (251, 330). This phosphorylation was accompanied by contraction of papillary muscles and coronary arterial preparations (250, 286, 330). It is less potent and selective than okadaic acid but is inexpensive. Mutational analysis indicates that OA and CA might act on
different amino acids on PP1 (460). In mutational analysis of amino acids 274-277 of PP1, no change in the IC50 for
cantharidic acid was noted, in contrast to OA, which was much more
active when GEFT was changed to the mutant YRCG (460). It
has been claimed that CA and endothall are not readily permeable
through cell membranes but are taken up by hepatocytes (122,
124). However, others reported that endothall is membrane
permeable. Okadaic acid displaced cantharidin from PP2A
(281).
-amino acids and esterified with phosphate. How it is phosphorylated and dephosphorylated and whether this changes
its activity has apparently not been reported. It is cell membrane
permeable. Calyculin A induced muscle fiber contraction (226) and contraction of cardiac preparations
(327). In addition to CyA, seven other related compounds
were isolated termed alphabetically calyculin B to H
(146). They have different substitutions with cyano groups
and methyl groups. There is an inactive acetonide derivative that might
be a useful negative control. Like OA, CyA increased phosphorylation of
vimentin, phospholamban, the inhibitory subunit of troponin, and C
protein (54, 327, 327a). Calyculin A is equally potent against PP1 and
PP2A (146, 225), with IC50 values from 1 to 14 nM. Okadaic acid and CyA seem to compete for the same inhibitory site
on PP2A (401). Mutational analysis suggests that CyA
inhibits the enzyme via interaction with amino acid tyrosine-272 on
PP1, because its IC50 is changed from 0.5 to 3000 nM in the mutant Y272K (458).
-toxin or
-escin. With
the use of this approach, microcystin increased the tone in isolated
preparations from guinea pig femoral artery, guinea pig ileum, rabbit
femoral artery, and rabbit portal veins (158).
1 as the amino acid that is covalently bound to microcystin. However, in their hands, mutation of
cysteine-273 to alanine increased the IC50 of microcystin
on PP1 from 0.2 to 4.0 nM. They argued that this opposite finding could
be due to different dilutions of the PP1 between laboratories (299). It was extrapolated that microcystin should
covalently bind to cysteine-266 in PP2A (360). Mutational
analysis suggested that microcystin and other toxins like OA,
nodularin, and CyA competed for the same inhibitory site on PP1 near
amino acids 273-276 (460). Mutational analysis suggests
that microcystin inhibits via interaction with amino acid tyrosine-272
on PP1 because its IC50 is changed from 0.3 to 14 nM in the
mutant Y272K (458).
/PR55
from the
trimeric form (the main bovine brain isoform) but did displace
B
/PR55
. In both cases, the PP2A activity was enhanced. This
implies that dissociation is not necessary for stimulation of
phosphatase activity by heparin (234). Heparin, protamine,
and polylysine are not present within the cytosol of mammalian cells.
However, they may mimic the effect of an endogenous activator.
Arachidonic acid inhibits PP1 but activates PP2A (159) and
PP5 (57).
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IV. METHODOLOGICAL CONSIDERATIONS |
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The vast majority of studies cited below, which address the role of protein phosphatases in the regulation of ion channels, make use of phosphatase inhibitors in more or less intact biological systems, i.e., cells, tissue slices, isolated organs, or channels in isolated membrane patches or bilayers. It is important to point out the limitations of those results for a proper assessment of the present state of knowledge. A number of caveats apply to the above-mentioned approach, and these will be briefly outlined in this chapter. Table 6 compiles a selection of frequently used phosphatase inhibitors, together with their subtype selectivity as known from the cited biochemical studies. It may serve as a guideline for choosing the appropriate tools to investigate selected phosphatases, but "cookbook" advice is strongly discouraged. The so-called selectivity (i.e., difference between inhibition constants against various molecular targets) applies to the comparison between phosphatase isoforms. Effects on other proteins may have to be taken into account. For example, fluoride ions (forming aluminum fluoridate in the absence of chelators in any physiological solution) are known to affect G proteins, and orthovanadate is a well-known inhibitor of Na+-K+-ATPase. Such risk may be reduced when using high-affinity agents such as the microcystins or okadaic acid derivatives, but not all potential candidates (protein kinases, nucleotide-binding proteins, ATPases, or unknown) for unspecific action have been thoroughly studied.
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A way to minimize this problem would be to undertake the appropriate control experiments. Several examples can be given. It is reasonable to use a second or third compound from a different chemical class, but with a similar selectivity profile, as a positive control, e.g., results with OA suggesting a role for PP2A can be substantiated using cantharidin or protein inhibitors of PP2A if possible. Data with CyA can be complemented with nanomolar OA (PP2A), or with PP1 inhibitor 2. Protein preparations of inhibitor 2 of PP1 should still be active after heat inactivation of possible contaminants. PP2B inhibition can be achieved with ciclosporin, and PP2B activity should be negligible when intracellular Ca2+ is strongly buffered with EGTA or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Negative controls are possible if inactive derivatives are available, as with OA (i.e., norokadaone, okadaic acid methyl ester). Studies with peptide inhibitors should be accompanied by inactive peptides of the same physicochemical properties, e.g., by using a scrambled sequence.
A second point of concern relates to the comparison between biochemical
inhibition constants and functional effects and their concentration
dependency. First, little is known about the extent and time course of
intracellular accumulation of those compounds that penetrate cell
membranes. There may be considerable differences among drugs (see, for
example, Ref. 128). Even if the cellular content would be known, the
free concentration might be much lower than expected due to
accumulation in subcellular compartments or binding to membrane or to
specific targets. Accordingly, biochemical inhibition constants are
often far lower than concentrations necessary for functional effects
(see, for example, Refs. 326, 327, 330). Another point is of importance
here. When measuring the phosphorylation state of a protein or, even
more indirectly, the activity of an ion channel, a steady state is
observed, which may vastly differ from the (pseudo)equilibrium
conditions of an enzyme activity assay. The fraction of phosphoprotein
here is a result of the balance between underlying protein kinase and
phosphatase activities, as illustrated by a simple model calculation.
Assume a phosphoprotein phosphorylated at one site by one protein
kinase, and dephosphorylated by one or two different protein
phosphatases. The steady-state phosphoprotein level PrP (fraction
of 1) is then defined by the law of mass action as
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(1) |
|
(2) |
A quantitative problem arises even in the simple case where only one phosphatase is involved (PP2 = 0) (see Fig. 1, A and B). Data were calculated (thin, noisy lines) and fitted (solid lines, using 1- or 2-site models of a Langmuir isotherm, Eq. 2). KD1 was 1 nM (log = 0), and the kinase activity was always set to PK = 1. The difference between the calculations in Figure 1, A and B, resides in the counteracting phosphatase activity (A: PP1 = 0.5, B: PP1 = 10). In both cases, the apparent half-inhibitory concentration (arrow) is higher than predicted (log = 0). This shift amounts to more than 10-fold in case B. The apparent inhibitory concentration approximates the true value I when PK >> PP1. However, then phosphoprotein levels at baseline are close to 1, and a phosphatase inhibitor would have nearly no absolute effect. The problem is even more serious if a subtype-selective inhibitor is used to assess the relative proportion of two different phosphatases in dephosphorylating a common substrate. In Figure 1, C and D, a second phosphatase is included in the model, and a pronounced subtype selectivity was chosen (KD1 = 1 nM, log = 0, and KD2 = 1 µM, log = 3). The half-inhibitory constants are right-shifted as above, but the proportions of the two components also depend on the relationship between kinase activity and total phosphatase activity; PK was set 1, and PP1 and PP2 were equieffective (C: PP1 = PP2 = 0.25, D: PP1 = PP2 = 10). The fraction attributed to the more sensitive PP1 is seriously underestimated, especially in Figure 1D, where baseline PrP level is low. Thus, in cases where two phosphatases act in an intact cellular system, their relative contribution cannot be easily estimated from inhibitor experiments. These aspects can account in part for the fact that functional inhibition constants are much higher than expected based on in vitro data (Table 6).
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Different phosphatases can operate sequentially under physiological conditions, e.g., PP2B dephosphorylates inhibitor 1 of PP1 (see sects. IIA2 and IIB3). To check whether a phosphatase acts at the channel itself, and not somewhere upstream a signaling pathway, measurements of channels in bilayers or inside-out patches are often performed. However, it has to be taken into consideration that endogenous enzymes may coexist (and even copurify) with the channels, as known not only for protein kinases (see Ref. 89), but also for PP2B (371). Examples suggesting close association between phosphatases and channels are given below. Another point of interest is that biochemical data (as in Table 6) usually relate to skeletal muscle phosphatase preparations, with model phosphoprotein substrates such as phosphorylase a (see above). It is not clear whether these inhibition constants depend on the substrate, although this problem refers more likely to the high-molecular-weight protein inihibitors with (allo)steric mechanisms of inhibition, rather than active-site inhibitors such as the microcystins or OA (see above). Tissue or species variations of inhibition constants for a given catalytic subunit have only scarcely been addressed but can profoundly hamper interpretation of data (186). It is therefore recommended, at least for results that are hard to interpret, to check the biochemical activity of a phosphatase inhibitor by purifying and using the same source of enzyme as present in the functional experiments. Finally, the effects of phosphatase inhibitors and interpretation of their concentration-response curve critically depend on proper preparation and storage of stock solution or choice of solvent (16). Rather than relying on manufacturers information, solutions should be checked using a well-defined and robust assay, e.g., an isolated phosphatase enzyme assay.
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V. EFFECTS OF PHOSPHATASES ON ION CHANNEL ELECTROPHYSIOLOGY |
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A. Voltage-Dependent Ca2+ Channels
1. L-type Ca2+ channels
Similar to the paradigm of cAMP-dependent phosphorylation,
cardiac L-type channels served as a classical model system to study regulation of voltage-dependent ion channels by protein
phosphatases and their inhibitors. PP1 and PP2A reduced the
isoproterenol-stimulated whole cell current in guinea pig myocytes
(189). In rat heart cells (109), PP2A, but
not PP1, reduced the current also under basal conditions. Inhibitor 2 of PP1 and OA at micromolar concentrations elevated baseline current
density and potentiated the stimulatory response to isoproterenol in
the guinea pig (189, 190). At the single-channel
level, OA (5 µM) slowed channel rundown after patch excision
(339). Wang et al. (435) measured cardiac
channels in lipid bilayers. Here, OA (0.1 µM) dramatically increased
channel activity, suggesting that endogenous phosphatases are still
associated with the channels after reconstitution in the bilayer
system. Neumann and co-workers (326, 327, 330)
published a series of papers on the phosphatase inhibitors OA, CyA, and
CA, respectively. All compounds increased single-channel activity
and enhanced the phosphorylation of other, regulatory proteins like
phospholamban, troponin inhibitor, and myosin light chains in myocytes.
Contractile force of multicellular preparations was also increased.
Notably, the concentrations eliciting these functional effects were
consistently higher than those required for inhibition of cardiac
phosphatases in vitro. Incomplete cellular penetration and the more
complex situation of an intact phosphorylation-dephosphorylation
system (see sect. IV) may account for this discrepancy. In
expression systems using cardiac Although L-type channels from heart are consistently stimulated by
inhibitors of PP1 and PP2A (see above and Refs. 173, 253), the
situation can be more complex. In intestinal smooth muscle cells, CyA
was reported to stimulate (420) or depress
(436) Ca2+ currents, and the direction of the
effects of OA differed between intracellular dialysis and bath
application (270). Later, Obara and Yabu
(335) noted a biphasic response of whole cell currents to
increasing concentrations of both OA and CyA. They concluded that PP2A
inhibition reduces and PP1 inhibition stimulates the channels in these
cells. In vascular smooth muscle cells, OA antagonized the current
reduction exerted by PKC inhibitors (336). When studied at
the single-channel level, preferential inhibition of PP1 using tautomycin (1-100 nM) led to a reduction in the channel availability, i.e., the probability that a voltage step will elicit channel openings
and lead to an active sweep (163). On the other hand, preferential inhibition of PP2A (by 1 µM OA) increased open
probability within active sweeps by promoting long openings (mode 2),
and PP2A reduced open probability. Although these studies differ in the
assignment regarding phosphatase subtype to function, they show that
qualitatively different effects are mediated by PP2A and PP1 in smooth
muscle L-type channels. This could indicate the presence of
distinct regulatory phosphorylation sites with differential sensitivity
toward phosphatases, as shown biochemically for skeletal muscle
Ca2+ channels (267). In rabbit heart, Ono and
Fozzard (340) noted two types of effects of OA at the
single L-type channel level. Here, both availability and open
probability (open times) were raised as expected (see above), but in
different concentration ranges of OA (availability being more
sensitive, suggesting PP2A to be involved). Allen and Chapman
(8) used BDM to assess the effect of dephosphorylation on
single cardiac L-type channels. Open probability was reduced by the
proposed "chemical phosphatase," mainly due to lengthening of long
closures. In addition, availability was reduced, and kinetic analysis
of the slow gating process revealed that the oxime reduced the lifetime
of the available state (as expected for increased dephosphorylation),
but also increased the lifetime of the unavailable state. The use of
BDM as a tool is complicated, however, because of nonspecific
inhibitory effects unrelated to channel phosphorylation
(9). Wiechen et al. (443) compared the
effects of OA, its inactive derivative norokadaone, and CyA (1 µM
each) on single channels in guinea pig cardiomyocytes. Availability was
stimulated by OA and by CyA, and a more complex analysis of slow gating
was compatible with a selective drug effect on the dephosphorylation
rate. Okadaic acid, but not CyA, increased open probability, mode 2 gating, and prepulse-facilitated mode 2. They concluded that in
these cells, like in vascular smooth muscle, PP2A controls open
probability and modal gating, whereas PP1 governs availability (see
also Ref. 187). In summary, several lines of evidence indicate
differential modulation of L-type channels by PP1 and PP2A,
although the exact single-channel mechanism may differ between
species, tissues, and channel isoforms. Relatively little is known about the effects of PP2B on cardiac
L-type channels, although this phosphatase is abundant in heart and
capable of dephosphorylating various sarcolemmal substrates (301). Inhibition of PP2B by peptide inhibitors
(140) or by ciclosporin (186) had little or
no stimulatory effect, respectively. One has to consider, however, that
whole cell studies are inherently associated with some level of
intracellular Ca2+ buffering, which may affect basal PP2B
activity. Indeed, a liposomal preparation of PP2B was reported to
affect action potentials in embryonic chick ventricle
(417), but this effect has not yet been confirmed at the
Ca2+ current level. In GH3 cells, neuronal
L-type channel inactivation was unaffected by ciclosporin and
FK-506 (424), thus not supporting the "calcineurin
hypothesis" (50) originally raised for neuronal Ca2+ channels (see also Ref. 144). In smooth muscle cells,
however, both PP2B and ciclosporin strongly affect channel activity,
and PP2B seems to be one mediator of the inhibitory action of
Ca2+ on these channels by reducing open probability as well
as availability (370). L-type Ca2+ current in pinealocytes, carried by the
Modulation of protein phosphatase activity as a physiological mechanism
of Ca2+ channel regulation has been addressed in a number
of studies. Herzig et al. (187) have analyzed by a
discrete-time Markov analysis the slow gating process governing
availability. The prolongation by isoproterenol (337) of
the lifetime of the available state, thought to represent
phosphorylated channels, can be prevented by OA, supporting the concept
that PKA stimulation can indirectly inhibit PP1 by phosphorylation of
inhibitor 1 (329). A very similar model has been proposed
for the regulation of neuronal L-type, but also N- and P-type,
channels by D1 receptor-induced PKA activation with
subsequent phosphorylation of DARPP-32 and inhibition of PP1
(397). Muscarinic receptor-mediated reduction of
PKA-stimulated cardiac L-type currents, in addition to the
well-known cAMP-dependent mechanism (188), may partly
be due to phosphatase stimulation (186) in guinea pig
ventricular myocytes, but not in frog heart cells (233).
The proposed mechanism resembles the signal transduction cascade
(Gi/o-induced PP2A stimulation) described below for some K+ channels (258, 442), but the details of
possible intermediate steps are unknown. Inositol hexakisphosphate and
related mediators have been proposed to serve as second messengers to
increase Ca2+ currents and insulin secretion via protein
phosphatase inhibition (271). Finally, the peptide
chromastatin lowers Ca2+ entry, presumably through
L-type channels, into chromaffin cells via PP2A stimulation
(151), and an A2 adenosine receptor agonists exerts a similar effect via phosphatase stimulation (305).
There may also be developmental changes of protein phosphatases, e.g., in the heart, where a decreased sensitivity of the adult versus newborn
heart cells to various phosphatase inhibitors was found (157,
289). 2. Non-L-type Ca2+ channels
In central and peripheral neurons, whole cell Ca2+
current represents a mixture of low- and high-voltage-activated
currents. The latter can be dissected pharmacologically into L type
(dihydropyridine sensitive, encoded by There is strong evidence for a role of PP2B in the control of neuronal
Ca2+ channels. Immunoreactivity of this phosphatase is
colocalized with Ca2+ channel In summary, both PP1/2A and PP2B mechanisms control neuronal
high-voltage-activated Ca2+ currents. In most cases,
current is raised directly or made resistant against inhibitory
modulation by inhibition of these phosphatases. The work reviewed,
however, does not sufficiently address the exact molecular substrate
for phosphorylation. Given the prominent role for N-type and
P/Q-type channels in neurotransmitter release, it is important to
further investigate this question in reductionistic systems
(single-channel recording, reconstituted channels, and expression
systems allowing for site-directed mutagenesis and biochemical measurements). B. Voltage-Dependent Na+ Channels
Voltage-dependent Na+ channels are phosphorylated by
PKA and PKC. The functional consequences seem to be different among the respective channel subtypes from brain, heart, and skeletal mucle (26, 347, 385). The role of protein phosphatases has been studied in more detail for rat brain Na+ channels. Here,
PP2B and PP2A are active, but with different selectivities for the
various serine residues phosphorylated by PKA (321). These
results are in line with the presence of five different soluble
Na+ channel dephosphorylating enzymes in rat brain, which
have been pharmacologically and immunologically identified as either
2A-like or 2B-like (62). There may be an exception in
striatal neurons, where Na+ currents are reduced by the
PP1-specific inhibitor phosphorylated DARPP-32 (366).
Interestingly, a convergence of PKC- and PKA-mediated phosphorylation (280) of Na+ channels could be
due to an inhibition of dephosphorylation of PKA sites by PP2A or PP2B
in the presence of PKC (254). C. K+ Channels
1. Inward rectifier K+ channels
Inward rectifier channels may be activated or inhibited by
phosphorylation, depending on the particular type and system under study. In nucleus basalis neurons, substance P leads to a
PKC-mediated suppression of currents, an effect which can be
potentiated and rendered irreversible by 100 nM OA (402).
This suggests a dephosphorylation of the PKC site by PP2A or PP1. In
the pituitary GH3 cell line, thyrotropin inhibits inward
rectifiers by a Ca2+-dependent mechanism, and this effect
can be reverted by the catalytic subunit of PP2A, but not of PP1
(21). The cardiac ventricular inward rectifier current is
inhibited by stimulation of 2. Transient outward K+ channels
Only a few studies have addressed the role of dephosphorylation
with respect to transient outward currents. Recombinant neuronal channels have been postulated (196) to be dephosphorylated
by PP2B, which resulted in reduction of currents. As shown in a later study, the rate of inactivation was markedly accelerated by PP2B and
slowed by calmodulin kinase II (358). In rat heart, BDM
was used to infer a role of phosphatases in the control of cardiac transient outward currents. Indeed, the BDM-induced reduction of
current was reversed by cAMP-dependent stimulation
(448), but a nonspecific effect of BDM was not excluded,
and the subtype of the putative endogenous phosphatase is not known.
FK-506 reduced Ito in rat cardiomyocytes,
but this effect appears unrelated to PP2B inhibition
(108). In pituitary GH4C1 cells, a
subpopulation of slowly inactivating channels was characterized at the
single-channel level, which was depressed by a
membrane-permeant cAMP derivative. Recovery was observed after
somatostatin (72), which stimulates Ca2+-dependent K+ channels in these cells via
PP2A activation (442), but it remains to be elucidated
whether this mechanism is also involved in the control of transient
inward currents. 3. Delayed rectifier K+ channels
In the classical studies on cardiac Ca2+ currents,
Hescheler and co-workers (189, 190) also noted some
role of phosphatases on the cardiac delayed rectifier current,
iK. Although OA (5 µM, bath applied) amplified
the increase of this current after isoproterenol (190),
cell dialysis with PP1 by itself did not affect the current but
prevented isoproterenol-induced stimulation (189). In
frog cardiac myocytes, intracellular dialysis with micromolar OA or microcystin concentrations reduced a slowly activating delayed rectifier current (140), an effect opposite to that of
isoproterenol and similar to that of ATP depletion. The authors
concluded that delayed rectifier currents are regulated by at least two
phosphorylation sites, one which has to be phosphorylated for channel
activity, and another that mediates inhibition by phosphatase
inhibitors. The apparent discrepancy between the studies may be due to
species differences or to the heterogeneity of
iK (362).
Unfortunately, none of these experiments reveals which phosphatase
subtype mediates channel dephosphorylation under physiological
conditions. Duchatelle-Gourdon et al. (110) suggested
a role of a Mg2+-stimulated (i.e., PP2C) phosphatase to
revert catecholamine stimulation of cardiac delayed rectifiers, but
direct biochemical or pharmacological evidence was not given.
Regrettably, no specific tools are available to inhibit PP2C. In an attempt to characterize phosphatase regulation of an expressed
delayed rectifier potasssium channel, Lopatin and Nichols (288) studied effects of BDM on Kv2.1 channels expressed
in Xenopus oocytes. Unfortunately, they found reversible
inhibition by the drug, regardless of the phosphorylation conditions,
and had to conclude that direct block rather than chemical phosphatase
activity was the most likely mechanism. Kang et al. (237)
investigated in detail the mechanism of angiotensin II-induced
increase in delayed rectifier current of rat hypothalamic neurons. This
effect was pertussis toxin sensitive and inhibited by nanomolar OA.
Involvement of PP2A was further substantiated by the effect of
antibodies directed against PP2A, which blocked the angiotensin II
effect. This is yet another example of a signaling chain leading from receptors via PTX-sensitive G proteins to PP2A (212). 4. ATP-dependent K+ channels
In rabbit heart cells, ATP-dependent K+ currents
are modulated by PKC phosphorylation (284, 285). This
effect is stabilized by nanomolar OA and reverted or prevented by
addition of PP2A to inside-out patches. This finding suggests that
the PKC site involved is dephosphorylated by a PP2A located in close
vicinity of the channel. The direction of PKC effects apparently
depends on the ambient ATP concentration. At low ATP, where channels
are basally active, PKC inhibits the current, whereas at higher ATP, where channels usually are silent, PKC activates the current. This is
consistent with a less steep ATP concentration dependence of
phosphorylated channels (284). These data (at low ATP
concentrations) are somewhat in contrast to those of Kwak et al.
(263) obtained in rat cardiomyocytes. They demonstrated
that 100 nM OA slowed spontaneous rundown of channel activity, whereas
orthovanadate accelerated the decrease in current observed in
inside-out patches in the absence of ATP. The opposite effects were
exerted by PP2A and a tyrosine phosphatase, and exact nature of the
serine/threonine kinase involved was not investigated. In the absence
of PKC activity, a PKA site might thus be responsible for long-term
stability of channel activity. Along similar lines, in smooth muscle
cells of the gallbladder, glibenclamide-sensitive currents are
enhanced by PKA activation, and this effect is stabilized by 5 µM OA
(457). In this preparation, an increase of channel
activity (under physiological conditions) is caused by PKA
phosphorylation, and the dephosphorylation may be catalyzed by PP2A or
PP1. In renal tubule cells, the ATP-dependent K+
currents run down after excision of the patches in low-ATP
solution. Rundown is blocked by Mg2+ withdrawal,
orthovanadate, and 1 µM OA (260). Because PP2A, but not
Ca2+, PP1, or PP2B reduced channel activity, the authors
concluded that rundown is caused by a membrane-associated PP2A.
Interestingly, McNicholas et al. (310), using the ROMK1
(195) channel expressed in Xenopus oocytes,
reproduced the prevention of rundown by Mg2+ withdrawal and
orthovanadate, but 1 µM OA or CyA was not equally effective in their
hands. They concluded that rundown is mediated by a
Mg2+-dependent dephosphorylation (by PP2C?) of a PKA site.
However, it seems conceivable that the differences between their
results and those of Kubokawa et al. (260) are due to lack
of a native channel-associated 2A phosphatase in the expression
system. Alternatively, the rundown was so rapid (>50% in 30 s)
that the time of exposure to the organic phosphatase inhibitor may have
been insufficient for steady-state effects. The interpretation of
the orthovanadate effects is certainly even more difficult, due to the
indiscriminate character of its effects (for instance, inhibition of
protein kinases, Na+-K+-ATPases, or tyrosine
phosphatases; Ref. 263). 5. Ca2+-activated K+ channels
There are a considerable number of studies on dephosphorylation of
Ca2+-activated K+ channels, probably due to
their vast abundance and important physiological role for regulation of
the activity of neuronal, endocrine, and smooth muscle function. No
unifying picture can be drawn based on this information. This is not so
much related to the gross structural heterogeneity of these channels,
classically signified by their conductance (small-conductance
SKCa channels, intermediate-conductance IKCa
channels, and large-conductance, "maxi" or "big"
BKCa channels). Rather, the latter group of
BKCa channels has been examined in the majority of studies,
with results differing between tissues or cell types, protein kinases,
and phosphatase subtypes. This may of course be related to the
respective regulatory scenario of the cell and/or the particular
channel isoform (one of the many possible splice variants), but this
idea has yet to be confirmed at the level of an in vitro expression system. Up to now, most experiments have been done with native channels, and kinase regulation is not easily reconstituted in channel
expression systems (464). As a starting point, the study of Reinhart et al. (352) is
worthwhile of detailed consideration. They found that BKCa
channels prepared from rat brain and reconstituted in lipid bilayers
showed two different biophysical and regulatory phenotypes:
so-called type 1 channels (fast gating, high sensitivity to
charybdotoxin, activated by low Ca2+) are stimulated by PKA
in the majority of cases, and this effect was reversed by PP2A, but not
by PP1. Type 2 channels (slower open and closed times, less sensitve to
charybdotoxin and Ca2+) are downregulated by PKA, an effect
also specifically reverted by PP2A. The same group (73)
showed that these type 2 channels can also be stimulated by
phosphorylation, mediated by an endogenous kinase present in the
bilayer system, because Mg2+ plus ATP and adenosine
5'-O-(3-thiotriphosphate) were sufficient to cause
stimulation. This ATP effect could be reverted by PP1 (10-40 nM), but
not PP2A (60 nM), also suggesting a different site involved compared
with the site that mediates suppression of channel activity. In another
study, exogenous PKC could mimic and thiophosphorylated inhibitor 1 of
PP1 could stabilize this type of upregulation (353). The
conclusions from these systematic experiments are manyfold.
1) Within the same experimental system, different channel
subtypes reveal qualitatively distinct responses (stimulation vs.
inhibition of type 1 vs. type 2 channels by PKA). 2) The
same type (here, the so-called type 2 BKCa channels) is bidirectionally modulated by PKA and PKC, and 3) the
backward reactions are also catalyzed in a phosphatase subtype-specific manner (PP2A vs. PP1). Finally, both kinases and phosphatases must
exist in close association with the channel molecule, because their
influences are still present after channel reconstitution in the
bilayer. Based on kinetic studies of BKCa channels in
excised patches from the pituitary, Bielefeldt and Jackson
(36) argued that a kinase (involved here in maintaining
channel activity) is in very close asssociation with the channel
("intramolecular model"), more so than the corresponding
phosphatase (diffusion-limited "intermolecular model"). It is
therefore tempting to speculate that slow, spontaneous oscillations in
the open probability of BKCa channels, already described by
Reinhart et al. (352), and termed "Wanderlust"
kinetics by Silberberg et al. (382) may reflect phosphorylation-dephosphorylation reactions catalyzed by
endogenous, channel-associated enzymes. In the papers mentioned subsequently, dephosphorylation of
Ca2+-activated K+ channels (or associated
regulatory proteins) leads to an increase in activity. In the rat
pituitary tumor cell line GH4C1, Armstrong's group (441) showed that atrial natriuretic peptide
stimulates BKCa channels through an elevation of cGMP,
stimulation of cGMP-dependent protein kinase (PKG), and
activation of a phosphatase sensitive to 10 nM OA (PP2A). This final
mechanism resembles, but the initial cascade differs from the effect of
somatostatin in the same cells (442). Somatostatin does
not induce a rise in cGMP, and it requires a pertussis
toxin-sensitive G protein, but ultimately also leads to a channel
stimulation inhibited by OA. Later studies provided evidence that the
signaling cascade initiated by the activation of somatostatin receptor
leads to accumulation of lipoxygenase metabolites of arachidonic acid
(111). A biochemical link between eicosanoids and the
activation of PP2A (159) has already been mentioned, and
stimulation of this phosphatase may be of broader physiological
importance. Zhou et al. (463) demonstrated that BKCa channels from bovine tracheal smooth muscle are
stimulated by PP2A in excised patches and that an indirect stimulation
exerted by an active fragment of PKG is blocked by the appropriate
concentrations of phosphatase inhibitors. Interestingly, a similar type
of regulation was shown in the same paper for
intermediate-conductance KCa channels in Chinese
hamster ovary cells expressing recombinant PKG Ia. In both cases, PP1
was ineffective at equimolar concentrations (350-700 nM), and PP2A was
detected in the membranes by Western blot analysis. Activation of PKG,
PP2A, and finally BKCa channels was again proposed as the
common pathway of cGMP-dependent regulation in these systems.
Similar mechanisms may operate in some neurons. In hippocampal neurons,
secreted On the other hand, protein dephosphorylation may also reduce
BKCa channel activity, as already mentioned above. A number
of such examples come from studies in above smooth muscle cells. Archer
et al. (14), using rat pulmonary artery, showed that nitric oxide increased whole cell BKCa currents. Based on
pharmacological evidence (inhibition by methylene blue, mimicking by
Sp-guanosine 3',5'-cyclic monophosphothioate and by 2 µM
OA), they proposed that this effect is mediated by phosphorylation of
the channels. Along similar lines, Stockand and Sansom
(389) and Sansom et al. (363) described a
cGMP-dependent stimulation of BKCa channels from human
mesangial smooth muscle cells (see Ref. 390 for review). The effect was
exerted by PKG and by cell-permeant cGMP derivatives. The
activation was biphasic, and the second declining phase of activity was
abolished by low concentrations of OA and cantharidin, but not by CyA.
Conversely, PP2A but not PP1 inhibited channel activity in excised
patches. These results are most easily explained by a PKG site that
must be phosphorylated to support channel activity. This site also
seems to be specifically dephosphorylated by PP2A. Note that
arachidonic acid activates BKCa channels in this system, but in a manner independent of phosphorylation (391).
Phosphorylation and stimulation by PKA and cAMP derivatives have been
addressed by Schubert et al. (369) in myocytes from rat
tail artery. Astonishingly, the presence of 1 µM OA in the whole cell
pipette did not modify the response to iloprost, a prostacyclin analog
shown to act via PKA activation. It is feasible that longer incubation
times or larger pipette diameters would have been required to achieve
sufficient intracellular dialysis of the phosphatase inhibitor. In
gastrointestinal smooth muscle cells, phosphatase inhibitors led to an
increase in channel activity (47, 272). These effects were
observed in excised patches (47), where PKA also had
stimulatory effects. The phosphatase subtype could not be identified
unequivocally, since CyA appeared to have a lower potency and/or
efficacy than OA. In neuroendocrine and neuronal cells, there are some
examples (36, 228, 274) of BKCa channel
stimulation by phosphorylation and inhibition by phosphatases, but in
none of these studies have the respective enzyme isoforms been
precisely identified. FK-506 increases single-channel activity in
cultured hippocampal neurons, but again (see sect.
VC2), this effect is unrelated to PP2B
inhibition (409). In collecting duct cells of the rat
nephron (194), both small- and
intermediate-conductance KCa channels are activated by
phosphorylation, evidently via PKG. The phosphatase inhibitors CyA (10 nM) and OA (1 µM) were tested and effective at concentrations that do
not firmly discriminate between PP2A and PP1. In summary, native Ca2+-activated K+ channels,
notably BKCa channels, display a variety of regulatory
pathways, including phosphorylation by PKG and PKA and
dephosphorylation by PP2A and PP1. These enzymes are, both functionally
and anatomically, in tight association with the channels, and a number
of physiologically important regulatory mechanisms converge at this
target. However, it is desirable to address the diversity of the
responses in various systems at the molecular level. In particular,
information about the particular sites of phosphorylation would be
required from future biochemical studies or from electrophysiological
studies after site-directed mutagenesis of the cloned channels. D. Ligand-Gated Cation Channels
1. N-methyl-D-aspartate receptor
channels
Glutamate receptors of the NMDA type are nonselective cation
channels critical for neuronal excitability and particularly for
Ca2+-dependent modulation of synaptic plasticity. They are
regulated by phosphorylation and dephosphorylation. Evidence for a more direct interaction between protein phosphatases and these channels can
be derived from single-channel studies. In cultured hippocampal neurons, both exogenous PP1 and PP2A depressed open probability, and
CyA and OA exerted opposing effects (434). Calyculin A (20 nM) also increased the whole cell current, with effects on kinetics depending on the glycine concentrations. In the mouse nucleus accumbens, DARPP-32 is essential for the dopamine-induced
phosphorylation of a NMDA receptor subunit (131). These
findings hint that endogenous PP1 or PP2A regulate channel
phosphorylation, activity, and kinetics. There is ample literature, on
the other hand, on NMDA receptor regulation by PP2B. Lieberman and Mody
(283) tested the effect of 10 µM OA on
single-channel currents of neurons from dentate gyrus. The
phosphatase inhibitor increased open times and open probability. These
effects were mimicked by FK-506 but not by lower concentrations of OA.
Stimulation of channels by phosphatase inhibition depended on
Ca2+ instead of Ba2+ as the charge carrier.
Calcineurin by itself had an inhibitory effect. At the whole cell
level, inhibitors of both PP1/2A and PP2B prevented either
long-term rundown (311) or short-term
desensitization/inactivation (414) or had no effects at
all on these phenomena (276, 359, 429). This leaves some
uncertainty on the important question whether or under which conditions
Ca2+ entering through the channel are regulating the
channel itself by activating Ca2+-dependent phosphatases.
This question may crucially depend on the type of neuron studied, since
PP2B expression and PP2B inhibitor effects are both absent in
hippocampal interneurons (381). Convincing data favoring
the idea that PP2B mediates NMDA receptor desensitization were obtained
by measurements of excitatory postsynaptic currents (EPSC) in
hippocampal cell cultures. Here, desensitization of NMDA receptors was
gauged by the change in EPSC amplitude after a train of repetitive
agonist applications in the absence or presence of an NMDA receptor
blocker. Strong Ca2+ chelation as well as various PP2B
inhibitors, but not CyA, completely abolished desensitization
(415). The kinase involved in this system is a tonically
active PKA, which can be modulated by 2. Other nonselective cation channels
As described for NMDA and AMPA (446) receptors,
various other ligand-gated cation currents are stimulated by
phosphatase inhibitors. Examples include 5-HT3
receptors (38), capsaicin receptors (102),
and P2x3 purine receptors (246), all of which apparently desensitize (i.e., current decreased in the continuous presence of agonist) via PP2B activity. In contrast, P4
purine receptors are less active in the presence of PP1/2A or PP2B
inhibitors (345). Okadaic acid induced phosphorylation of
the recombinant nicotinic acetylcholine receptor Cyclic nucleotide-gated nonselective cation channels may be
regulated by protein phosphatases, as demonstrated for the
cGMP-gated channel in the retina (160). Here, an
endogenous phosphatase seems to sensitize the channel toward cGMP, an
effect mimicked by exogenous PP1 but counteracted by PP2A. The hyperpolarization-activated nonselective cation current
if in heart tissues is regulated by
(de)phosphorylation, as shown by an increase in current amplitude after
application of CyA. In sinus node cells, this effect can be clearly
discriminated from the well-known direct regulation by cAMP: here,
the CyA response was not accompanied by a shift in the activation curve
along the voltage axis (1), although such a shift was
reported earlier (455) for ventricular myocytes. A
hyperpolarization-induced cation current in peripheral neurons,
which is also regulated by cAMP, does not respond to conditions
altering protein kinase or phosphatase activity (221). Other nonselective cation currents have been reportedly stimulated by
OA (43, 403), by microcystin-LR (445), or
by low Mg2+ and vanadate (222, 223),
suggesting regulation by PP1/2A or, perhaps, PP2C, respectively. In summary, phosphorylation sites coupled to increased channel activity
and agonist sensitivity apparently exist in a vast number of
nonselective cation channels. Physiological regulation pathways
involving the phosphatases encountered here remain to be resolved,
except for the obvious role of Ca2+-dependent PP2B activity
in some cases. E. Anion Channels
1. Cystic fibrosis transmembrane conductance regulator
channels
The cystic fibrosis transmembrane conductance regulator (CFTR)
gene encodes a cAMP-dependent Cl A role of another phosphatase has early been postulated by Hwang et al.
(216); in the presence of high OA concentrations, only
part of the cAMP-dependent current was preserved. A proposed candidate would be alkaline phosphatase, since blockers like
levamisole, bromotetramisole (but see Ref. 269), or methylxanthines can
activate CFTR currents (27-29, 219). Notably, an antibody
against alkaline phosphatase increased channel activity
(27), and the enzyme as well as its inhibitors affected
32P incorporation into CFTR protein (28).
However, it has been argued that the effect of alkaline phosphatase was
nonspecific, because channel activity was restored upon application of
fresh ATP solution (30), and this enzyme should not have
access to the phosphorylation sites in intact cells (150). Genistein, a known inhibitor of tyrosine kinases, stimulated CFTR
currents (65, 141, 351, 452), and it increases the sensitivity of the channel toward PKA-dependent activation
(204). The mechanism involved in these effects is
controversial. Reduced tyrosine phosphorylation would be expected to
increase PP2A activity (55). Indeed, genistein has been
proposed to inhibit a phosphatase distinct from PP2A (65,
351), possibly PP2C (452). However, the results of
French et al. (141) and Wang et al. (433)
argue against a dephosphorylation mechanism (150).
Importantly, the pattern of activation by CyA and genistein differs at
the single-channel level (452), and the two compounds
act in an additive manner (351). The report of Zhou et al. (462) demonstrates stimulation
of cardiac CFTR currents by the Cl 2. GABAA receptor Cl GABAA channel currents are downregulated by PKA, and
an endogenous PKA phosphorylates these channels (406). It
is likely, however, that another protein kinase (e.g., tyrosine kinase,
Ref. 211) is responsible for phosphorylation of a site associated with
increased channel activity. Several groups have shown that Ca2+ entering through activation of NMDA channels
diminishes GABAA currents. A likely mechanism is
Ca2+-dependent activation of PP2B, since the pathway was
blocked by nanomolar fenvalerate (211), cypermethrin
(388), or delthametrin (10, 355), by
calcineurin inhibitory peptide (60), and by Ca2+ buffering with BAPTA (60, 317). The
latter authors also found an effect of micromolar microcystin-LR or
OA concentrations but could not exclude indirect mechanisms
(317). An additional pathway of such inhibitory
cross-talk between NMDA receptors and GABAA channels
may be the nitric oxide system (355). 3. Other Cl Okadaic acid has been shown to activate a variety of
Cl On the contrary, Cl In a renal cell line, the open probability and, remarkably, the
single-channel conductance of an outwardly rectifying
Cl An outwardly rectifying Cl A voltage-dependent Cl
1C-subunits, several
investigators failed to detect significant PKA-mediated increase of
the Ca2+ current (344, 383, 470). On the other
had, PP1 reduced the current (383) in the
Xenopus model. Forskolin as well as OA elevated the currents
when previously inhibited by the PKA inhibitor H-89 (344).
Furthermore, prepulse-induced facilitation could be enhanced by PKA
and by OA in some (51, 372, 373) but not all studies (236). Gao et al. (152) were the first to
report robust cAMP-dependent modulation in an expression system.
Their data suggest that for appropriate PKA-dependent stimulation
and phosphorylation of
1C-subunits, the protein kinase
has to be anchored in the membrane by proteins like AKAP 79, both in
the expression system and in native cardiac cells. The results,
including site-directed mutagenesis of Ser-1928 to abolish
cAMP-dependent regulation, await confirmation by other groups.
There is still no firm evidence that the cardiac
1C-subunit itself serves as the substrate for regulatory
phosphorylation and dephosphorylation, although this has been shown
directly for neuronal and skeletal muscle L-type channels
(175, 461).
1D-pore subunit, is decreased by various protein
phosphatase inhibitors (66). Conversely, secretory
function of pancreatic
-cells can be enhanced by OA
(11), but the moderate increase found for L-type
currents can only partially explain this effect, possibly more at
threshold potentials for current activation (171). Okadaic acid may even decrease Ca2+ current (365) and
insulin secretion in isolated islets (365) or RINm5f cells
(12). Interestingly, activation of PP2B can mediate the
inhibitory effects of somatostatin, galanin, and an
2-agonist on insulin secretion. This effect is
independent of changes of the Ca2+ current and can be
blocked by deltamethrin and a PP2B inhibitory peptide, but not by OA.
1C- or
1D-subunits), N type (
-conotoxin GVIA sensitive,
encoded by
1B-subunits), P or Q type (sensitive to
-agatoxin IVA or
-conotoxin MVIIC, respectively, encoded by
1A-subunits), and R type (toxin resistant, probably
encoded by
1E-subunits). With the notable exception of
two studies in sympathetic neurons (42, 440), addition of
phosphatase inhibitors led to an increase in neuronal Ca2+
channel amplitude and/or a reduced extent of inactivation. Dolphin (106) demonstrated that cAMP-dependent stimulation of
currents in rat dorsal root ganglia is mimicked by an active fragment
of inhibitor 1 of PP1, suggesting that endogenous PP1 dephosphorylates a regulatory PKA site. Unfortunately, many studies employed OA in
concentrations too high to discriminate between PP1 and PP2A inhibition. This holds true for snail neurons, where a PKC-mediated stimulation of currents by serotonin was amplified by 500 nM OA (191), and, in another study, the rate of inactivation was
reduced by dialysis with either 50 µM OA or a peptide inhibitor of
CaM kinase II (451). Both kinases were apparently not
involved in the action of the muscarinic agonist carbachol
(156), as intracellular injection of OA and of
microcystin-LR, but not bath application of 50 µM ciclosporin,
mimicked and occluded the stimulation exerted by carbachol. In a
mammalian neuronal cell line, OA (100 nM) prevented part of the
inhibitory action of a dopamine D2 agonist on N-type currents (45). This part (the late or sustained component)
of inhibiton was not voltage dependent and likely reflects regulation of a PKA site, whereas the okadaic acid-insensitive part (on the rate of activation) represents voltage-dependent regulation by direct G protein interaction of the channels. Synaptosomes from rat
hippocampus were studied with fura 2 Ca2+ imaging
(23). Here, preincubation with 100 nM OA markedly
potentiated the stimulation by a PKC activator, suggesting a strong
basal PP1 and/or PP2A activity in this system. In cultured rat cortical neurons, Thomas et al. (411) demonstrated that
postsynaptic excitatory currents are increased in amplitude by 1 µM
CyA (in the bath) or microcystin (10 µM in the pipette). This effect
was not mimicked by PP2B inhibitors (10 µM ciclosporin or 2 µM
FK-506). These compounds had been shown earlier by the same group
(425) to increase the frequency but not amplitude of
postsynaptic currents, which seems to indicate a presynaptic mechanism
involving PP2B.
-subunits in dorsal root
ganglia (291). PP2B interferes with G protein inhibition
of N-type channels in sympathetic neurons (467), where
the response to
2-adrenergic or somatostatin receptor stimulation is inhibited by an autoinhibitory PP2B fragment or by
ciclosporin, but not by OA. High-voltage-activated currents in
lactotrophs from rat pituitary were stimulated by PKC activation, and
ciclosporin mimicked this effect in the absence of a PKC inhibitor (137). In rat cortical synaptosomes, ciclosporin and
FK-506 increased Ca2+ influx and glutamate release
(374), in line with the electrophysiological results of
Victor et al. (425). In the NG 108-15 cell line,
overexpression of PP2B reduced N-type currents, and this reduction
could be overcome by FK-506 (292). Calcium-dependent
dephosphorylation of neuronal channels may also be involved in several
other studies using OA (59, 256, 315, 466), where channel
stimulation was mimicked by calmodulin antagonists or impeded by
Ca2+, respectively. Because the OA concentrations used here
were in the micromolar range, it is impossible to tell whether a PP2B has been nonselectively inhibited here by the toxin or whether PP1 was
involved indirectly (e.g., through PP2B-mediated dephosphorylation of
endogenous PP1 inhibitors).
-adrenergic stimulation, an effect
mimicked by forskolin, cAMP derivatives, or the catalytic subunit of
PKA (257). This inhibition can be reversed by muscarinic
stimulation using acetylcholine. The muscarinic effect can be abolished
by 1 µM OA, both at the whole cell level and in the
single-channel configuration (258). Importantly, the effect of the phosphatase inhibitor was still obtained when channels were studied in the inside-out configuration [with guanosine
5'-O-(3-thiotriphosphate) or Gi
used to
antagonize PKA-induced inhibition]. This suggests a pathway
leading from muscarinic receptors via pertussis toxin-sensitive G
proteins to a protein phosphatase that is closely associated with the
channel and dephosphorylates a PKA site. Unfortunately, the
concentration of OA (1 µM) does not allow one to conclude whether
PP2A or PP1 is involved, but the authors checked the phosphatase specificity of their approach using the inactive derivative norokadaone.
-Adrenergic regulation is very different for the cardiac
acetylcholine-gated K+ channel, which also shows some
extent of inward rectification. Here, the directly G
protein-mediated activation due to muscarinic receptors is
inhibited by
-adrenergic stimulation or PKA (239), suggesting that the phosphorylated channel has a lower open-state probability. Alkaline phosphatase reverts this effect.
Dephosphorylation also seems to account for spontaneous desensitization
of the activated native (241) and recombinant (GIRK1/GIRK4
heteromultimeric) channels (244). The endogenously present
phosphatase seems to be located in the cytosol (241),
requires Ca2+, and is inhibited by 3 mM orthovanadate, but
not by OA (241). This is somewhat puzzling because PP2B
would be a good candidate for Ca2+-dependent
dephosphorylation, but this enzyme is relatively insensitive to
orthovanadate (375). In guard cells from plants, a PP2B is likely responsible for dephosphorylation and inhibition of inward rectifiers (290). In contrast, dephosphorylation (of a
site phosphorylated by unknown kinases) and inhibition of inward
rectifier currents in guinea pig chromaffin cells is probably catalyzed
by PP2A or PP1, because the effect is blocked by micromolar CyA or OA
concentrations (223, 224). Rundown of recombinant Kir 2.1 channels is also prevented by inhibition of PP2A and PP1 by
microcystin, but this effect is linked to a PKA site in the
Xenopus expression system used (361). In view
of the functional and structural diversity of inward rectifiers, it is
not astonishing that a common pattern of phosphorylation- or
dephosphorylation-linked events does not emerge from the literature.
-amyloid precursor protein causes hyperpolarization via
BKCa channels (148). The authors proposed that
cGMP, PKG, and a protein phosphatase (sensitive to 10 nM OA) are
involved. BKCa channels in a clonal pituitary cell line have similar sensitivity toward OA. Interestingly, PP2A regulation here
seems to get targeted to the channel by glucocorticoid treatment (412). Holm et al. (197) investigated
BKCa channels at the whole cell level in mouse cortical
neurons. Neurotrophin-3 and nerve growth factor activated the current,
and this effect was blocked by 30 nM OA, which leaves some uncertainty
about the phosphatase subtype involved. The nature of the upstream
mechanisms also remained somewhat unclear but likely involved a
tyrosine kinase and a phospholipase C. In summary, stimulation of
BKCa channels by dephosphorylation through PP2A seems to be
a common mechanism to reduce cellular excitability, present in various
tissues and initiated by a number of signaling cascades, most notably
by cGMP and PKG.
-adrenoceptor activation
(349), providing a physiological pathway between
noradrenergic input and synaptic strength at a glutamatergic synapse.
Phosphatases may also participate in physiological long-term
changes in NMDA receptor function such as long-term depression
(447) or long-term potentiation (37), but
the signaling pathway as well as the phosphatase subtype involved are
not known. In summary, for a better understanding of the multitude of
functional data, it would be important to define the susceptibility of
the phosphorylation sites of the cloned NMDA receptor subunits toward
protein phosphatases.
-subunit, but the
functional consequences have not yet been studied (255).
channel present in
heart, epithelia, and other tissues. Its regulation has been recently
reviewed in detail (150). The channel is activated by PKA
and phosphorylated on at least nine different consensus sites
(142, 149). However, other kinases like type II PKG
(142) and certain PKC isoforms (30) have also
been shown to phosphorylate and activate the channel. PP2A, but not PP1
or PP2B, inactivated and dephosphorylated the channel
(30). Exogenous PP2A and PP2C exerted different kinetic
effects on single CFTR channels expressed in baby hamster kidney cells
(293). Even at nanomolar concentrations (350), OA accelerated activation (323) and
slowed down or prevented inactivation when conditions were chosen to
minimize de novo phosphorylation (216, 348). PP2B
inhibitors increased CFTR currents when stably expressed in
fibroblasts, but not the currents endogenous to two epithelial cell
lines (135), suggesting that this mechanism has no
physiological significance.
channel blocker
anthracene-9-carboxylate via phosphatase inhibition. Their data suggest
that yet another unknown phosphatase, pharmacologically distinct from
PP2A, PP2C, or alkaline phosphatase, might be of some physiological
importance for CFTR regulation. Of note, in addition to appropriate
substrate specificity, intimate colocalization of phosphatases with
their substrate may be an important determinant of physiological
function (e.g., Refs. 27, 134).
channels
channels
channels, including a 50-pS channel from shark rectal
gland epithelia (265), a Ca2+-activated
Cl
channel from osteoclasts (147), and a
PKA-activated channel from Necturus gallbladder
epithelia (132, 133). In the latter case, low
concentrations of OA were sufficient, and PP2A, but not PP1, had an
opposite effect on channel activity after reconstitution in bilayers
(133).
conductance in skeletal muscle was
slightly diminished by 0.5 µM OA, and a stimulatory response to
insulin-like growth factor I was blocked by the phosphatase
inhibitor (90) and mimicked by a ceramide
(91), suggesting a role of PP2A (see sect.
IIIC2).
channel were increased by insulin. These effects were
mimicked by PP2B and by a tyrosine phosphatase, and they were blocked
by ciclosporin, but not by OA. The authors (304) propose
that insulin action in this case should be mediated by
calmodulin-dependent activation of PP2B, which in turn would be
able to dephosphorylate a regulatory tyrosine residue.
channel from a human
intestinal cell line is stimulated by calmodulin-dependent protein
kinase II. This activation is spontaneously reverted by an OA- and
microcystin-sensitive phosphatase. Interestingly, inositol
3,4,5,6-tetrakisphosphate exerted inhibitory effects, which depended on
uninhibited phosphatase activity, but were not mediated directly by
phosphatases, suggesting a complex regulatory scheme
(450).
channel present in the same
cells was increased in activity by low osmolarity. This regulation was abolished by OA (1 µM) or CyA (50 nM) (143).
Hypotonicity induced a Cl
current in chick cardiac
myocytes under whole cell but not perforated-patch recording
conditions (172). It became insensitive to osmotic challenge even under whole cell dialysis when intracellular cAMP was
raised or endogenous phosphatases were blocked by 100 nM OA. Okadaic
acid and 20 nM CyA also rendered expressed CLC-3 channels insensitive
to hypotonicity (107). When a critical serine residue (position 51) was mutated to alanine, the channel was tonically active,
not further stimulated by hypotonicity, and no longer inhibited by PKC
stimulation (107). These results indicate a direct
relationship between volume regulation by this Cl
channel
and its (de)phosphorylation.
| |
VI. SUMMARY AND PERSPECTIVES |
|---|
|
|
|---|
As exemplified in the preceding sections, ion channel regulation by phosphatases takes place through numerous more or less complex pathways, as scetched in Figure 2.
|
Some limitations of studies leading to this picture are obvious. If, for instance, 10 µM OA is applied in intact cells and an altered current through an ion channel is noted, this does not really prove direct modulation of the channel protein by phosphatase type 1 or 2. First, other phosphatases are also blocked by OA (see Table 6), and these might thus be involved (see Fig. 1). Moreover, it is conceivable that a protein kinase is stimulated, a kinase that is activated by phosphorylation, thus the effect of a change of kinase activity on channel function is actually measured. Thus a naive direct correlation of phosphatase inhibition and channel function by channel phosphorylation might lead to wrong conclusions. This indicates that an integrated approach based on biochemical, biophysical, and physiological methods will be important for progress in the field.
Clearly, first all channels mentioned above as putatively regulated by phosphatases have to be identified unambiguously. As mentioned in section I, the efforts should continue to express cloned channel proteins in vitro to use highly controlled systems to study their biophysical function. This approach has its pitfalls. Crucial posttranslational modifications might be missed in the expression system. Expression systems possess their own signal transduction machinery, which may fail to work at the expressed target protein, or lead to regulatory effects that have no physiological significance. Because expression systems have become very popular, some of the very recent studies illustrate these problems.
Ideally, the phosphorylation site of the channel protein should be identified by direct sequencing on the protein level. Here, the problem might arise that the physiological kinase is unknown. Hence, an unphysiological phosphorylation site might be identifed. Alternatively, putative phosphorylation sites can be identified from the predicted protein sequence. Then it would be possible to use site-directed mutagenesis to remove these phosphorylation sites and look whether the channel activity is now altered. One could also consider a biophysical approach. It would be possible to measure the protein-protein interaction of channels and phosphatases in bilayers. Ultimately, and possibly in the near future, a three-dimensional picture from cocrystallization of a channel and a phosphatase should yield clear-cut answers where they actually interact. However, the same interactions are likely but not necessarily realized in the intact cell.
Information can be gained by dialysis or injection of phosphatases or antibodies into intact cells, studying alteration in channel activity. This approach has already been used in the past. This method can be complicated if the site of injection, or appropriate targeting, is crucial for the function of the protein. Moreover, sufficient amounts of the protein of interest might not be readily available. Alternatively, expression vectors, namely, adenovirus constructs, could be useful to transfer the channel or phosphatase of interest into cells. These versatile methods would be helpful to establish by mutational analysis which parts of phosphatases are actually crucial for their function. However, most phosphatases do not function alone but in concert with their regulatory or targeting protein. It might be important to cotransfect these auxiliary proteins to get meaningful results. Regrettably, in expression systems is it is difficult to control the amount of overexpressed protein.
On the basis of transfection of isolated cells, there will be the
development of trangenic mice that overexpress phosphatases, channels,
regulators, and the combination thereof. Indeed, recently, PP1
(332) and PP2B (316) have been overexpressed.
Both transgenic models show altered physiological function. Soon, data
will become available whether channels are altered in these transgenic
lines. In addition, several groups are currently working on
knock-out mice for phosphatases or their regulators. Even here a
word of caution is warranted. Some knockouts are likely to be lethal, for instance, of PP2A
. Moreover, it is expected from precedence that
overexpression and knockout leads to compensatory changes of other
regulatory proteins (69). Hence, the results may be difficult to interpret. One possible route that will be used is the
conditional, or tissue-specific, overexpression and knockout of
phosphatases in animals.
In addition to their importance in fostering our understanding of nature, research on phosphatases and their action on channels is likely to have additional benefits in medicine. There is a growing body of evidence that phosphatase are altered in, for example, cardiovascular disease. Pathological states like hypertrophy (40), cardiac failure (328), and postischemic contractile dysfunction (17, 209) have been accompanied by alterations in phosphorylation of proteins and/or phosphatase function (22, 169), including channel regulation by phosphatases (368). As mentioned above, overexpression of phosphatases can lead to cardiac hypertrophy, strengthening the connection (316, 332). In addition to these pathophysiological changes, phosphatases, in part by altering channel activity, can change the contractility of muscle (heart or vasculature, Refs. 250, 251, 286). Hence, phosphatases might be candidate targets for drug therapy (398; but see also Refs. 99, 312, 459). It is conceivable that some of the many functions regulated by phosphorylation in humans might be favorably altered by application of phosphatase activators and inhibitors or by directly altering cellular protein phosphatase expression, using gene therapy.
| |
ACKNOWLEDGMENTS |
|---|
Research of the authors on protein phosphatases and ion channels is supported by the Deutsche Forschungsgemeinschaft.
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: S. Herzig,
Institut für Pharmakologie, Universität Köln,
Gleueler Stra
e 24, D-50931 Köln, Germany (E-mail:
stefan.herzig{at}uni-koeln.de).
| |
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