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.

	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.

	II. STRUCTURE AND NOMENCLATURE OF SERINE/THREONINE PROTEIN PHOSPHATASES

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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).

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).

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 PP1₁ and PP1₂ that code for 323 and 337 amino acids, respectively (364). Sequences are highly conserved between species. For instance, human PP1₁ is 93% nucleotide sequence similar and identical in protein sequence to the rat homolog (334).

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).

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 CNA₁, CNA₂, and CNA₃. CNA₁ and CNA₂ 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 CNB₁ (170 amino acids) and, by means of a different promotor, leads to another testis-specific isoform called CNB₂ (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 (R_GL; 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).

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, PP2C₁, PP2C₂, and PP2C₃, have been reported (302). PP2C₁ is the most abundant isoform. Alternative splicing seems to generate the isoforms PP2C₁ and PP2C₂. They were cloned from a mouse library and show differences in COOH termini and the 3'-untranslated region. PP2C₁ is expressed in all mouse tissues studied, whereas PP2C₂ is confined to heart and brain where they might subserve special functions (408). PP2C₁ and PP2C₂ 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).

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.

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).

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.

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).

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).

	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.

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.

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.

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).

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).

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).

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 -subunit of the inhibitory nucleotide binding protein G_i-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). NFB was induced and dissociated from IB (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).

Cantharidin, cantharidic acid, palasonin, and endothall are structural analogs (268). Cantharidin (CA) is the vesicant in blister beetles (beetle in Greek is 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).

Calyculin A (CyA) was isolated from another marine sponge, Discodermia calyx. It is an octamethylpolyhydroxylated C-28 fatty acid linked to two -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).

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., -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).

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 PP1₁ 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).

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).

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.

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 p70^s6k (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.

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).

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.

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.

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.

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).

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).

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/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).

	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.

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

(1)

where PP1 and PP2 are the total activity of two phosphatases, PK is the total kinase activity (all used in arbitrary units), and K₁ and K₂ are the remaining fractions of phosphatase activity in the presence of a selective inhibitor I, also following simple mass action

(2)

where K_D1 is the true inhibition constant of PP1 against I (and analogous for K_D2 and PP2).

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). K_D1 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 (K_D1 = 1 nM, log = 0, and K_D2 = 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,