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Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania
ABSTRACT I. INTRODUCTION II. MOLECULAR PROPERTIES OF THE INOSITOL TRISPHOSPHATE RECEPTOR A. Identification of the InsP3R B. InsP3R Diversity 1. Gene expression 2. Alternative splicing 3. Heteroligomerization 4. Functional implication of InsP3R diversity A) GENERAL CONSIDERATIONS. B) ISOFORM DIFFERENCES. C) SPLICE VARIANTS. D) PROTEIN INTERACTIONS. E) BIOPHYSICAL PROPERTIES. III. STRUCTURE OF THE INOSITOL TRISPHOSPHATE RECEPTOR A. Overview B. Structural Properties of the InsP3R Molecule 1. InsP3 binding region 2. The transmembrane region A) THE PORE. B) THE GATE. 3. The coupling region 4. The COOH-terminal tail 5. Regulatory Ca2+ binding sites C. Structural Properties of the Tetrameric InsP3R Channel 1. The tetrameric structure 2. ER localization determinants 3. Structural insights from limited proteolysis 4. Three-dimensional structures IV. ELECTROPHYSIOLOGICAL STUDIES OF INOSITOL TRISPHOSPHATE RECEPTOR CHANNELS V. PERMEATION PROPERTIES OF THE INOSITOL TRISPHOSPHATE RECEPTOR A. Overview B. Monovalent Cation Conductance Properties C. Ion Permeability of the InsP3R D. Divalent Cation Conductance E. Physiological Ca2+ Current Through the InsP3R F. Molecular Models of the InsP3R Pore VI. REGULATION OF INOSITOL TRISPHOSPHATE RECEPTOR CHANNEL GATING A. Overview B. Cytoplasmic Ca2+ Regulation of InsP3R Channels 1. Biphasic regulation of InsP3R channel gating by Ca2+ 2. Po exhibited by maximally activated InsP3R channels 3. Use of biphasic Hill equation to describe Ca2+ regulation of InsP3R channel activity 4. Ca2+ activation of InsP3R channels 5. Ca2+ inhibition of InsP3R channels C. InsP3 Activation of InsP3R Channels 1. InsP3 regulates InsP3R channel activity through modulation of its sensitivity to Ca2+ inhibition 2. Physiological significance of InsP3 modulation of Ca2+ inhibition of InsP3R channel 3. Inadequate characterization of the InsP3 dependence of InsP3R channel 4. A different kind of InsP3 dependence D. InsP3 and Ca2+ Regulate InsP3R Channel Activities Through Multiple Ca2+ Sensors 1. The InsP3-independent inhibitory Ca2+ sensor 2. The InsP3-dependent Ca2+ sensor 3. The InsP3-independent activating Ca2+ sensor E. Regulation of InsP3R Gating by Luminal Divalent Cations F. Regulation of InsP3R Channel Gating by ATP 1. ATP potentiation of Ca2+ activation of InsP3R channel activity 2. Significance of ATP potentiation of InsP3R channel activity 3. Inadequate characterization of ATP potentiation of InsP3R channel 4. Identification of functional ATP sensors regulating InsP3R channel activity 5. Potentiation of InsP3R channel activity by other nucleotides 6. Effects of ATP on Ca2+ inhibition of InsP3R channel 7. Inhibition of InsP3R channel by millimolar ATP? 8. Other effects of ATP on InsP3R channel activity G. Ligand Regulation of InsP3R Channel Mean Open and Closed Durations H. Activation of InsP3R Channel by Adenophostin and Its Analogs I. Ligand-Dependent, InsP3-Induced InsP3R Channel Inactivation J. Ligand-Dependent InsP3R Channel Recruitment K. Ca2+ Sensors Regulating InsP3R Channel Activity L. Channel Regulation by Phosphorylation 1. Phosphorylation by PKA 2. Phosphorylation by PKG 3. Phosphorylation by PKC and CaMKII 4. Phosphorylation by Akt kinase 5. Phosphorylation by tyrosine kinases 6. Phosphorylation by cdks/CyB M. Regulation by Redox Status N. Regulation by Interacting Proteins 1. Calmodulin 2. CaBP and CIB1 3. RACK1 and Gbetagamma 4. IRBIT 5. Chromogranins 6. ERp44 7. FKBP12 8. Glyceraldehyde 3-phosphate dehydrogenase 9. Bcl-2 proteins 10. Cytochrome c 11. Huntingtin 12. Proteases (caspase-3 and calpain) 13. Adapter proteins (protein 4.1N, ankyrin, and Homer) 14. TRPC channels and Na+-K+-ATPase VII. CONCLUDING REMARKS GRANTS ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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and PLC-
by a wide variety of stimuli including ligand interaction with G protein- or tyrosine kinase-linked receptors. PLC hydrolyzes the membrane lipid phosphatidylinositol 4,5-bisphosphate, generating inositol 1,4,5-trisphosphate (InsP3) (27, 377). InsP3 diffuses in the cytoplasm and binds to its receptor (InsP3R), which is an intracellular ligand-gated Ca2+ release channel (136, 270) localized primarily in the endoplasmic reticulum (ER) membrane (132, 354, 400). The ER is the major Ca2+ storage organelle in most cells. ER membrane Ca2+-ATPases accumulate Ca2+ in the ER lumen to quite high levels. Because the lumen contains high concentrations of Ca2+ binding proteins, the total amount of Ca2+ in the lumen may be >1 mM; the concentration of free Ca2+ has been estimated to be between 100 and 700 µM (8, 21, 69, 330, 355, 373, 491). In contrast, the concentration of Ca2+ in the cytoplasm of unstimulated cells is between 50 and 100 nM, 3–4 orders of magnitude lower than in the ER lumen. This low concentration is maintained by Ca2+ pumps and other Ca2+ transporters located in the ER, as well as plasma, membranes. Upon binding InsP3, the InsP3R is gated open, providing a pathway for Ca2+ to diffuse down this electrochemical gradient from the ER lumen to cytoplasm. Ca2+ in the cytoplasm moves by passive diffusion, at a rate that is reduced by mobile and immobile Ca2+ binding proteins acting as buffers. As a consequence, microdomains with steep Ca2+ concentration gradients can rapidly form and dissipate near the mouth of an InsP3R Ca2+ channel. The Ca2+ concentration adjacent to the open channel may be 100 µM or more, whereas concentrations as close as 1–2 µm from the channel pore may be below 1 µM (342, 343, 390). Therefore, Ca2+ has only a restricted "range of action," on the order of 5 µm (7). The distribution and concentrations of Ca2+ binding proteins and the release channels, as well as the complex properties of the release channels, enable InsP3R-mediated [Ca2+]i signals to have diverse spatial and temporal properties that can be exploited by cells, making this signaling system remarkably robust. Consequently, despite its expression in probably all cells in the body, this signaling system can provide specific signals that regulate diverse cell physiological processes. Analyses of InsP3-mediated [Ca2+]i signals in single cells has revealed them to be complex. In the temporal domain, this complexity is manifested as repetitive spikes or oscillations, with frequencies often tuned to levels of stimulation, suggesting that [Ca2+]i signals may be transduced by frequency encoding as well as amplitude. In the spatial domain, [Ca2+]i signals may initiate at specific locations and remain highly localized or propagate as waves (27, 28, 89, 466). Thus InsP3-mediated [Ca2+]i signals are often organized to provide different signals to discrete parts of the cell. High-resolution optical imaging of fluorescent Ca2+ indicator dyes in intact cells have suggested that InsP3-mediated [Ca2+]i signals are organized at three broad levels. Each level can provide different signaling functions and serve as a building block for [Ca2+] signals at the next level (Fig. 1) (26, 49, 358). At the first level, "fundamental" signals result from openings of individual InsP3R Ca2+ channels. Weak activation by low [InsP3] evokes localized elevations of cytoplasmic [Ca2+] that arise stochastically and autonomously at discrete release sites. They have variable size, with the smallest, called "blips" (358), possibly involving Ca2+ flux through one or, more likely, a few InsP3Rs (Fig. 1A). At the next level, "elementary" signals arise from the concerted opening of several channels. Larger events ("puffs") involve the concerted opening of multiple InsP3R channels organized within a cluster (446). The coordinated opening of several channels is triggered by Ca2+ release from one channel acting as an activating ligand to stimulate gating of nearby channels through a process of Ca2+-induced Ca2+ release (CICR) (see discussion below about activating ligands of the InsP3R channel) (Fig. 1B). Appropriate colocalization with effector proteins enables spatially restricted fundamental and elementary signals to provide specificity of cellular responses (49, 291). At the third level, with higher [InsP3] associated with stronger extracellular agonist stimulation, Ca2+ released at one cluster site can trigger Ca2+ release at adjacent sites by CICR, leading to the generation of Ca2+ waves (Fig. 1C) that propagate in a saltatory manner (48, 71, 86) at velocities of a few tens of microns per second by successive cycles of Ca2+ release, diffusion, and CICR (26, 101).
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InsP3-mediated Ca2+ signals are an example in which finite fluctuations at the microscopic (single channel) level give rise to signals that are observable at the macroscopic (cytoplasmic) level (230). The ability to trigger global signals depends strongly on InsP3R single-channel properties. Detailed knowledge of the microscopic properties of single InsP3R Ca2+ release channels is therefore necessary for an understanding of the diverse Ca2+ signals elicited by the InsP3 pathway. The focus of this review is on the permeation and gating properties of single InsP3R Ca2+ release channels. As such, we review recent studies that have provided new information regarding structural features of the InsP3R, the mechanisms of ion permeation, and channel gating and its regulation by InsP3, Ca2+, and other cellular factors including interacting proteins.
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II. MOLECULAR PROPERTIES OF THE INOSITOL TRISPHOSPHATE RECEPTOR |
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The glycoprotein receptor for InsP3 was first purified from rat cerebellum (443). Binding of InsP3 to the purified protein had high affinity (Kd 
100 nM) compared with other inositol phosphates and was inhibited by heparin, properties that were similar to those of the InsP3 receptor in crude cerebellar microsomes (520). Electrophoretic analysis revealed that the receptor had an apparent molecular mass of 260 kDa, whereas gel fractionation indicated a molecular mass of the native protein of 1 MDa, demonstrating that the receptor was a tetramer (443), a result that was later confirmed by cross-linking (270). Immunostaining of cerebellar Purkinje cells revealed that the receptor was expressed in the ER, nuclear envelope, and portions of the Golgi complex, but not mitochondria or plasma membranes (400). Subsequent studies have indicated that the plasma membrane in some cell types may also contain InsP3R (108, 457). Two groups simultaneously cloned full-length (150) and partial (313) type 1 InsP3R (InsP3R-1) cDNAs from mouse cerebellum. The full-length rat cerebellar InsP3R-1 cDNA was cloned shortly thereafter (311). The full-length mouse cDNA sequence encoded for a protein of 2,749 amino acids with a predicted molecular mass of 313 kDa (150), whereas an additional 2,734-amino acid protein was discovered as a splice variant in the rat (311). Expression of the recombinant proteins enhanced the magnitudes of InsP3 binding and InsP3-induced Ca2+ release from isolated membrane fractions (321). Reconstitution of the purified receptor into lipid vesicles showed that it mediated Ca2+ release in response to InsP3, with half-maximal flux activated by 40–80 nM InsP3 (136, 271). Furthermore, reconstitution of purified InsP3R into planar bilayer membranes resulted in the appearance of Ca2+-permeable ion channels (270, 302). Taken together, the data suggested that the InsP3R was itself an intracellular ligand-gated Ca2+ release channel.
Subsequently, it was established that three genes (39, 103, 150, 289, 313, 399, 439) encode for a family of InsP3Rs in mammalian cells, including humans, and other vertebrates. The three full-length amino acid sequences are 60–80% homologous overall, with regions, including the ligand-binding and pore domains (discussed below), having much higher homology (363, 460). In contrast, invertebrates appear to express only a single InsP3R, most closely related to the type 1 isoform (196, 200). In mammals, the InsP3R is ubiquitously expressed, perhaps in all cell types (104, 146, 149, 415, 460). The three channel isoforms have distinct and overlapping patterns of expression, with most cells outside the central nervous system expressing more than one type (68, 104, 105, 340, 345, 418, 451, 460, 493). InsP3R isoform expression levels can be modified during development and differentiation (129, 242, 340, 394, 419, 450, 460) and in response to various normal and pathological stimuli (20, 61, 70, 218, 226, 250, 305, 403, 418, 460, 502, 526). Furthermore, InsP3R protein expression levels can be downregulated by a use-dependent mechanism that involves InsP3- and Ca2+-dependent channel ubiquitination, and subsequent degradation involving the proteasome (9, 10, 35, 515).
Further diversity of InsP3R expression is created by alternative splicing (99, 138, 311, 340, 348). The type 1 channel has three main splice regions, denoted SI, SII, and SIII. The SI site is located within the core InsP3 binding region, comprising residues 318–332 (Fig. 2B) within a loop connecting -strands 6 and 7 of the second -trefoil domain (the structure is discussed in sect. IIIB1). The SII site is located near the middle of the protein sequence in the coupling domain, comprising residues 1693–1731 (Fig. 2B). It was proposed that the SII+ variant (the "long" form) is the neuronal form, while peripheral tissues express primarily the SII– short form (99). The SII sequence is absent in the types 2 and 3 isoforms. SIII corresponds to a 9-amino acid insert after residue 917 (Fig. 2B). Until recently, none of the splice forms had been cloned from tissues. A detailed analysis of the InsP3R-1 transcriptome has revealed a previously unrecognized and remarkable diversity of expression in the brain (386). SII splicing can come in four varieties (339, 386), with the result that the type 1 transcript can vary at six segments within the open reading frame, which can give rise to 48 possible channel subunits. Seventeen variants were detected in cerebellum, with each brain tissue and developmental stage generating 11–13 forms. A biased stochastic model for splicing regulation could quantitatively account for the multiple forms expressed at each developmental stage.
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-trefoil of the InsP3-binding region in the so-called suppressor domain (discussed in sect. IIIB1) (199). The deletion of this sequence eliminates two of the -strands of the domain, which would be expected to severely disrupt its structure. The SIm2– mRNA comprised 7–20% of the total type 2 transcripts in various mouse tissues, with the submandibular salivary gland expressing it at 41% (199). Another mouse type 2 splice variant termed TIPR was detected in skeletal and heart muscle that codes for a truncated protein of only the NH2-terminal 181 residues (151). It shares the splice acceptor site with the SIm2 variant. Although invertebrates appear to express only a single InsP3R isoform, the Caenorhabditis elegans channel exists as six alternatively spliced forms (22, 159) and the Drosophila channel exists as two (427).
A final level of channel diversity is generated by heteroligomeric interactions among different isoforms. The InsP3Rs are 2,700–2,800 amino acid intracellular membrane proteins that exist as homo- or heterotetramers (209, 210, 212, 270, 311, 328, 363, 443, 536). By analogy with other cation channels and some structural information, the ion-conducting pore is believed to be created at the central axis of the tetrameric structure (Fig. 3). Evidence for the existence of heterotetramers of two isoforms has come primarily from the ability of isoform-specific antibodies to coprecipitate other isoforms (363). Cross-linking studies (352) and the ability of mutant channels to exert dominant negative effects (40, 433) also support the existence of heterotetrameric channels. The results to date indicate that two different InsP3R forms can exist in the same tetramer. Whether all three isoforms and/or multiple splice variants ever exist in a single tetrameric complex is however unknown. If they do, then the diversity of channels could be quite impressive. For example, adult cerebellum, a source of InsP3R for many biochemical and functional studies, expressed 13 splice forms of the InsP3R-1. Twenty percent of the transcripts were SI+, whereas 98% contained three of four possible SII varieties, and SIII was absent in 73% of transcripts. For tetrameric channels with no bias against heteromultimerization among different forms, the presence of 12 transcript variants is predicted to give rise to nearly 5,900 channel isoforms (386)!
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A) GENERAL CONSIDERATIONS.
This diversity of channel expression is impressive, but the functional implications of this diversity, both at the single-channel as well as cellular levels, are still only poorly explored. This diversity suggests that cells require distinct InsP3Rs to regulate specific functions. Cerebellar Purkinje neurons express the type 1 isoform predominately (although many different splice variants of it), whereas insulin-secreting -cells express primarily the type 3 channel (460), and cardiac myocytes express predominately the type 2 isoform (256, 368). Genetic knockout of the mouse type 1 receptor causes neurological defects and early death (297), consistent with the dominant expression of the type 1 isoform in the cerebellum. Similarly, genetic deletion of the mouse type 2 receptor abolishes the positive ionotropic and arrhythmogenic effect of endothelin in cardiac atrial myocytes (253) and endothelin-induced HDAC5 nuclear translocation in ventricular myocytes (521). It is therefore perhaps surprising that genetic diseases as a direct consequence of mutations of the human InsP3R have not yet been discovered, whereas several (malignant hyperthermia, central core disease, arrhythmogenic right ventricular cardiomyopathy, catecholaminergic polymorphic ventricular tachycardia) have been identified as consequences of mutations in ryanodine receptors (RyR), the other major family of intracellular Ca2+ release channels (reviewed in Ref. 376). It is possible that disease-causing InsP3R mutations will be discovered and that some InsP3R mutations are invariably embryonically lethal. On the other hand, the lack of identified InsP3R mutations in humans may suggest that expression of multiple InsP3R isoforms in most cell types provides functional redundancy that is necessary because of the critical importance of InsP3-mediated Ca2+ signaling for many cell physiological processes. Indeed, evidence suggests that there is functional redundancy among the InsP3R isoforms in cells that express more than one. For example, knock-out of both types 2 and 3 isoforms together were required to create a pancreatic acinar cell secretion phenotype (152), genetic deletion of the type 1 isoform was without effect on T-cell Ca2+ signaling or development (188), and genetic deletion of all three InsP3R isoforms was necessary to generate an apoptosis phenotype in chicken DT40 B cells (440).
B) ISOFORM DIFFERENCES. Despite these considerations, the molecular diversity of InsP3R expression nevertheless suggests that it is likely that InsP3R isoform-specific permeation, gating, or localization and their regulation by ligands or interacting proteins confers specificity required for specific cell physiological processes. Many different biochemical, Ca2+ release, and channel properties have been proposed to exist among the different isoforms. For example, the three channel isoforms may be differentially sensitive to activation-induced, ubiquitin-mediated downregulation (516). It has been proposed that different channel isoforms have distinct InsP3 binding properties. However, the order of InsP3 affinity is variable among studies [type 2 > type 1 > type 3 (122, 318, 345, 439); type 1 > type 2 > type 3 (517); type 3 > type 2 > type 1 (344)], and the differences in affinity among the isoforms in some of the studies are modest. Thus the physiological relevance of different InsP3 affinities among isoforms has not been clearly established. It has been proposed that cADP-ribose and the oxidizing agent thimerosal regulate InsP3 binding or Ca2+ release differentially among the three channel isoforms (67, 488, 489) and that InsP3 binding to different isoforms is differentially regulated by [Ca2+]i or calmodulin (77, 487, 530). Nevertheless, the literature regarding all these studies is conflicting (reviewed in Ref. 461). In part, the divergent results likely reflect the different preparations used, for example, fragments of the recombinant InsP3R as bacterially expressed fusion proteins versus full-length channels in microsomes, as well as the different assays used, for example, InsP3 binding to microsomes versus Ca2+ flux from permeabilized cells. Differences among isoforms in Ca2+ release rates may not necessarily reflect intrinsic differences in the properties of the channels, since the state of phosphorylation or association with interacting proteins are usually unknown and are likely different in different cell preparations. It has also been suggested that divergent results could arise from "dominance" of a single subunit within a heterotetramer (461). In addition, the variability of published results likely stems from the presence of different conformational states of the channel present in the different studies. As a highly allosteric protein that is regulated by several heterotropic ligands (including InsP3, Ca2+, ATP, H+, and interacting proteins) as well as by redox and phosphorylation status, apparent binding affinities of each ligand will be strongly influenced by the conformational state of the channel, which is in turn dependent on the state of binding of all the other ligands. Even under identical experimental conditions, apparent differences in otherwise identical ligand binding properties between isoforms may be caused by such allosteric effects. Consequently, various reported ligand-binding properties may have been strongly influenced by the channel conformational state, which could be different among studies. It is therefore quite difficult to interpret much of the literature that has attempted to compare channel isoforms when only limited sets of experimental conditions are employed, as in most published studies.
C) SPLICE VARIANTS. A consistent observation relates to the effects of the SII splice site on the ability of the type 1 channel to be phosphorylated by protein kinase A (PKA) (99, 497, 498). Deletion of the SII region creates a novel ATP binding site (137) (ATPC; Fig. 2B). ATP binding to that site modulates the ability of the channel to be phosphorylated by PKA (496). Channel phosphorylation in turn allosterically modifies the channel sensitivity to InsP3 (497, 498, 541) (discussed in sect. VIL). The SII+ channel as well as the types 2 and 3 channels lack this ATP binding site and are therefore expected to respond differently in response to elevated cAMP, although both the types 2 and 3 channels can be phosphorylated by PKA at different sites (432, 436, 518).
D) PROTEIN INTERACTIONS.
Many protein interactions with InsP3R have been described (discussed in sect. VIN and reviewed in Refs. 267, 365). Most interactions have been examined for only one isoform. Some proteins, including CaBP/caldendrin (527), Bcl-2-related proteins (83, 349, 508), and Na+-K+-ATPase 
-subunit (540) have been shown to interact with all three InsP3R isoforms, whereas others, including AKAP9 (480) and protein 4.1N (301, 544), appear to interact specifically with the type 1 isoform. Because the stoichiometry of these interactions is unknown, it is unknown whether isoform-specific interactions can also form with heterotetrameric channels containing fewer than four copies of the type 1 channel molecule. It seems likely that many more isoform-specific protein interactions will be discovered and that these interactions may in turn be regulated. Thus a diversity of regulated isoform-specific protein interactions may confer yet further InsP3R channel diversity through mechanisms involving InsP3R localization and gating.
E) BIOPHYSICAL PROPERTIES. Finally, the different channel isoforms and their splice variants may have different biophysical properties related to gating and permeation. As discussed in detail in section V, the permeation properties of different InsP3R channel isoforms are quite similar, likely reflecting the conserved amino acid sequences in the pore region of the different isoforms. The gating properties of different isoforms of homotetrameric InsP3R channels have either been inferred from Ca2+ release studies or examined directly by single-channel electrophysiology. By analysis of Ca2+ release and agonist-induced Ca2+ signals in DT40 chicken B cells with either one or two InsP3R isoforms genetically deleted, it was concluded that the type 2 isoform is required for long-lasting regular [Ca2+]i oscillations, that the type 1 receptor mediated less regular [Ca2+]i oscillations, and that the type 3 channel generated only monophasic [Ca2+]i responses (318). Knockdown of the type 1 channel by RNA interference in HeLa and COS-7 cells abolished agonist-induced [Ca2+]i oscillations, whereas knockdown of the type 3 resulted in long-lasting [Ca2+]i oscillations (173). It was concluded that the two receptors have opposite roles in generating Ca2+ signals (173). Three points should be noted regarding these studies. First, even if these types of measurements provide insights into roles of different isoforms in generating distinct Ca2+ signals, they provide little mechanistic insight into the molecular features that distinguish the different channels. Gating of the InsP3R channel involves channel activation, inhibition, inactivation, stochastic attrition, and sequestration, and all these processes are complicated functions of ligand (Ca2+, InsP3, ATP) sensitivities and concentrations, interactions with proteins, phosphorylation state, etc. (discussed in sect. VI). Which gating properties in channels formed by different isoforms can account for different Ca2+ release behaviors have not been elucidated in these studies. Second, it is also important to note that, because the curves that describe the transient kinetics of Ca2+ release observed in cells and rapid perfusion experiments are reminiscent of the biphasic curves that describe the cytoplasmic Ca2+ concentration dependencies of steady-state channel activity, there has been a tendency in such cell studies to equate the two and to account for kinetic features of Ca2+ signals in terms of observed effects of Ca2+ concentration on steady-state single-channel gating activity. For example, the purported lack of high-Ca2+ inhibition of type 2 or type 3 InsP3R steady-state channel gating (163, 382) has been invoked to account for either the presence or absence of [Ca2+]i oscillations in such studies (173, 318). However, as pointed out (430), the bell-shaped or otherwise biphasic shape of the steady-state open probability (Po) versus [Ca2+]i curve has "very little, if anything" to do with the fact that the InsP3R exhibits complex rapid kinetic behaviors. Third, complex Ca2+ signals in cells are determined not only by "intrinsic" permeation and gating features of each isoform, but by many other factors as well, including the absolute channel density and the spatial distribution of the channels, and the influence of these factors within the context of complex cellular machinery, including pumps and buffers, that participate in regulating cytoplasmic Ca2+ concentration. Thus interpretation regarding the roles of, and differences among, different channel isoforms in such Ca2+ release/Ca2+ signaling studies is complicated and requires considerable caution.
More detailed mechanistic insights into the intrinsic differences among different channel isoforms can be obtained from single-channel recordings. Unfortunately, the channel properties observed in planar bilayer reconstitution studies have been quite variable among studies, even for the same isoforms from the same laboratories, and different from those observed by nuclear patch-clamp electrophysiology. This experimental variability limits the degree to which generalizations can be made regarding distinctions among different channel isoforms or splice variants. In patch-clamp studies of isolated Xenopus oocyte nuclei, it was concluded that the permeation and gating properties of the expressed recombinant rat type 3 channel were similar to those of the endogenous type 1 channel (284), but that the Ca2+ activation properties of the two channels uniquely distinguished them (283). In reconstitution studies of Sf9 cell-produced recombinant rat types 1, 2, and 3 channels, numerous differences were noted (482). First, the maximum channel open probability (Pmax) in 1 mM ATP was considerably smaller for type 3 channels (<5%) compared with the other two isoforms (30%). However, the Po for the type 1 channel recorded in this study was considerably higher than that observed in other reconstitution studies (generally <5%). Furthermore, another study found that the Pmax of the type 2 channel exceeded that of the type 1 channel (382). Of note, the maximum Po values for all three channels in all reconstitution studies were considerably lower than that measured in the patch-clamp studies (80%) (42, 196, 278, 282, 283). Second, the InsP3 sensitivities differed over fourfold, with the order as follows: type 2 > 1 > 3. In addition, the isoforms had different apparent sensitivities to ATP free acid. It is important to note that the determinations of channel Po in these studies were made at a single Ca2+ concentration (200 nM). However, as discussed in detail in section VIF, the interplay between the effects on channel Po of the cytoplasmic concentrations of Ca2+ and InsP3 or ATP as heterotropic ligands of the InsP3R, an allosteric protein, can be manifested as apparent differences in ligand sensitivity depending on the concentration of the other heterotropic ligands (282). Therefore, to determine the effective ligand sensitivity of a channel isoform, it is necessary to examine the effect of InsP3 or ATP concentrations on the Ca2+ concentration dependence of channel Po over a wide range of cytoplasmic Ca2+ concentrations.
The Ca2+ concentration dependencies of the channel Po of the different reconstituted InsP3R isoforms were narrow bell-shaped, centered around 200 nM for all three channels (481). In agreement, patch-clamp electrophysiology of the endogenous Xenopus (282) and Sf9 (196) and recombinant rat type 1 (42) and type 3 (283) channels indicated that their steady-state activities are indeed both activated and inhibited by Ca2+, but the high [Ca2+]i inhibition was exerted at much higher concentrations in the patch-clamp studies (half-maximal inhibition 20–40 µM in patch-clamp experiments versus 0.5–1 µM in bilayers). However, very different cytoplasmic Ca2+ concentration dependencies of channel Po have been observed in other single-channel reconstitution studies. In contrast to the observations in Reference 481, it was reported that the activities of the type 3 (163) and type 2 (380, 382) channels are monophasic functions of cytoplasmic Ca2+ concentration, with little or no evidence of high-[Ca2+]-mediated inhibition. Furthermore, the "width" of the biphasic Po versus [Ca2+] curve was distinct for the same reconstituted type 1 SII+ isoform obtained from different sources by the same lab (229, 478), with the reasons for the variability not clear to the authors (478). The reasons for the widely divergent and inconsistent permeation and gating properties and their regulation observed in InsP3R channel reconstitution studies are unclear, but are important to resolve to bring clarity to the field.
In summary, a remarkable diversity of InsP3R isoforms exists, but insights into the functional implications of this diversity are still rudimentary. Although single-channel electrophysiology promises to provide the most detailed insights into the distinct properties of different isoforms, the divergent results obtained within different studies of reconstituted channels and from nuclear patch-clamp studies indicate a need to define more optimal systems for expression and recording of different single InsP3R variants. Furthermore, it is expected that appreciation of the molecular diversity of InsP3R will likely also be enhanced by use of other approaches that address channel localization and interaction with molecular partners in protein complexes.
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III. STRUCTURE OF THE INOSITOL TRISPHOSPHATE RECEPTOR |
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Each InsP3R molecule contains 2,700 amino acids with a molecular mass of 310 kDa. Structurally, each InsP3R molecule contains a cytoplasmic NH2 terminus comprising 85% of the protein mass, a hydrophobic region predicted to contain six membrane-spanning helices that contribute to the ion-conducting pore of the InsP3R channel, and a relatively short cytoplasmic COOH terminus (Fig. 2A). Functionally, the NH2-terminal region can be divided into a proximal InsP3 binding domain and a more distal "regulatory"/"coupling" domain (Fig. 2A). InsP3 binding to the InsP3R is stoichiometric and localized by mutagenesis and an X-ray crystal structure to a region within residues 226–578 (Fig. 2B). Because of the similarity among channel isoforms, to facilitate discussion of various structural aspects of the InsP3R in this review, we refer throughout to specific amino acids in the sequence involved in ligand binding, protein interactions, etc., using numbering based on the rat type 1 SI+, SII+, SIII– InsP3R sequence. The InsP3R channel is a tetramer of four InsP3R molecules (Fig. 3). Approximately 2,000 amino acids separate the InsP3-binding domain from the pore. This intervening region between the InsP3 binding domain and the pore contains consensus sequences for phosphorylation and binding by nucleotides and various proteins. It may function to integrate, through allosteric coupling, other signaling pathways or metabolic states with the gating of the InsP3R.
B. Structural Properties of the InsP3R Molecule
The localization of the InsP3 binding region to the NH2 terminus of the InsP3R was first proposed by Mignery and co-workers based on the discovery that deletion of the first 410 residues of the protein completely eliminated InsP3 binding (311) and that soluble monomeric proteins with COOH-terminal boundaries between residues 519 and 788, that lacked the transmembrane regions, efficiently bound InsP3 (312). Similar experiments subsequently established the NH2 terminus as the site of InsP3 binding in all three isoforms (289, 322, 439). Binding of InsP3 to the receptor is stoichiometric (271, 443) with an apparent Kd usually in the range of 10–80 nM. Binding of InsP3 to recombinant InsP3R proteins containing only the NH2-terminal 586 residues had similar affinity, pH sensitivity, and inositol phosphate selectivity as the native channel (345). Of note, deletions from either the NH2 or COOH terminus of this construct eliminated binding, indicating that this region contained the complete InsP3 binding domain (345). Further deletion mutagenesis confirmed that even small NH2-terminal deletions abolished InsP3 binding to the ligand-binding region. However, binding was restored when more substantial deletions were made, with a mutant construct with the first 225 residues deleted having 10- to 100-fold higher affinity than the full-length construct (538). Thus the region encompassing residues 226–576 was sufficient for InsP3 binding, forming an InsP3 binding "core," whereas the region containing residues 1–225 was referred to as the "suppressor" domain (538) (Fig. 2B). Within the core domain, site-directed mutagenesis identified 10 conserved arginine and lysine residues distributed throughout the domain as playing important roles in InsP3 binding, with residues Arg-265, Lys-508, and Arg-511 critically important (538).
Crystal structures of both the core (52) and suppressor (54) domains of the mouse type 1 InsP3R have been solved (Fig. 4A). In the 2.2-Å resolution structure of the core domain in complex with InsP3, two distinct domains are present at right angles to each other in an elongated L-shaped structure. The region from 225–436 constitutes a -sheet-rich -trefoil domain, whereas the region from 436–600 is -helical, comprised of two partial and one complete armadillo repeats. InsP3 is present in the structure at the interface of the domains, in a deep cleft with important binding determinants contributed by both. The cleft is lined with basic residues that anchor InsP3 to the protein. The phosphates in the 1 (P1) and 5 (P5) positions of InsP3 interact primarily with residues from the -helical domain, whereas the phosphate at the 4 position (P4) interacts with the -trefoil domain (Fig. 4A). The most extensive interactions involve P4 and P5 through hydrogen bonding primarily with several basic residues, although nonbasic residues as well as water are also involved. P1 interacts with only two basic residues.
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NMR studies of the core domain revealed well-resolved peaks when the core domain protein was complexed with InsP3, whereas many broadened peaks in the spectrum appeared in the absence of InsP3 (53). It was suggested that a dynamic equilibrium might exist in the ligand-free domain as a result of motions around the hinge region that connects the two subdomains. InsP3 binding to this region stabilizes the conformational relationship of the two domains with each other, consistent with earlier studies that indicated that InsP3 binding to an NH2-terminal 1,800-residue fragment of the receptor caused a conformational change (312). However, these studies of the isolated core domain may not reflect the behavior of this region within the context of the whole channel, where interactions with other parts of the protein, for example, the suppressor domain, or other structural features may constrain the mobility of this region. Nevertheless, InsP3 binding undoubtedly stabilizes the observed structure of the two domains. By analogy with the mechanism of glutamate binding to its bidentate binding pocket in the glutamate receptor (269), it has been speculated that InsP3 might bind primarily to either the -trefoil or armadillo-repeat domain first, and then recruit and stabilize the other domain in the structure observed in the crystal (459).
The suppressor domain, encompassing residues 2–223 of the mouse type 1 InsP3R, was resolved at 1.8 Å (54) (Fig. 4A). The structure is comprised of a typical -trefoil domain, referred to as the "head" subdomain, that contains an unusual helix-loop-helix insert that protrudes away from the structure, referred to as the "arm" subdomain, with the overall appearance reminiscent of a hammerlike structure. Thus the complete ligand-binding region (1–586) contains a proximal pair of -trefoil domains and a distal armadillo repeat region. Whereas the sequence similarity between the two -trefoil domains is low, their structures superimpose well, excluding the helix-loop-helix insert in the first domain, and a long loop in the second domain that contains the SI splice site (54). Before the structure of the 2–223 suppressor domain fragment was solved, it had been noted (375) that this region has repeats that are recapitulated in what is now recognized as the (second) 225–436 -trefoil domain, so the discovery of the suppressor domain as a -trefoil domain was somewhat anticipated. It had been similarly noted that the NH2 terminus of the RyR also contains the same repeats (375). Molecular modeling is consistent with the presence of tandem -trefoil domains similarly present in the RyR (54). The ug3 mutation (109, 216) in the single Drosophila InsP3R gene (3, 490), a missense mutation that changes a serine to phenylalanine at position 217 (Fig. 2B) near the COOH terminus of the suppressor domain, enhances the sensitivity of activation, but not the binding affinity, of the reconstituted Drosophila InsP3R to InsP3 (434), suggesting that the suppressor region may allosterically couple InsP3 binding to gating activation. Interestingly, mutations within these domains in the RyR cause central core disease and malignant hyperthermia (54). Some of the residues are predicted to contribute to the -trefoil fold, so their mutation might be expected to have structural implications for the entire domain. Others, however, were predicted to be located in surface-exposed loops, suggesting that they are importantly involved in channel function. It is quite interesting that such striking structural homology between the two families of Ca2+ release channels, if confirmed, should be present in this region of the channels, since the InsP3 binding function in the InsP3R is a main feature that distinguishes InsP3R from RyR.
Although deletion of the suppressor domain enhances InsP3 affinity of the core domain, Ca2+ release activity of the channel could not be elicited by InsP3, suggesting that the suppressor domain is required for normal channel activation (486). It has been proposed that the suppressor domain may therefore couple InsP3 binding in the core domain to other regions of the channel that impinge on the gating mechanisms (486). A critical next step is to resolve details regarding the structures of the three domains together, both in the presence and absence of InsP3 and Ca2+. The mechanisms by which the suppressor domain modulates the affinity of the channel for InsP3 are not elucidated by these structures. A logical hypothesis is that the suppressor domain interacts directly with the core binding domain. Deletion of the unusual helix-loop-helix arm subdomain was without major effect on the ability of a recombinant NH2-terminal 604 residue ligand-binding domain to bind to InsP3, suggesting that it was not critical for the suppressor function of the suppressor domain (54). It was noted that one surface of the suppressor domain contains several conserved residues (54). Mutagenesis of particular residues located within the surface enhanced the InsP3 affinity of the recombinant binding domain (54), consistent with the notion that this region of the head might participate in a protein interaction with another region that modulates InsP3 binding affinity of the core domain. Genetic studies have shown that the single C. elegans InsP3R gene, itr-1 (22, 88), is important to the ultradian rhythm underlying defecation (97). One of two InsP3R alleles identified that disrupt the defecation cycle, n2559, characterized as a loss-of-function mutation because the defecation cycle was eliminated, was mapped to residue 103 as a missense alteration of Gly to Glu (G103E), corresponding to Gly-25 (Fig. 2B). This residue is located immediately adjacent to the residues identified (54) whose mutations enhance InsP3 binding. This result suggests that whereas this region might participate in regulating the InsP3 binding properties of the core domain, the suppressor domain is also required for the channel to function, consistent with the loss of channel activation by InsP3 binding when the entire domain is deleted (486).
Inspection of the two crystal structures could not identify a potential binding interface within the core domain that might constitute the interaction region with the suppressor domain (54). However, other regions of the InsP3R molecule have also been proposed to interact with the ligand-binding region. First, a direct association between the NH2-terminal 340 residues and the COOH terminus has been observed (40, 214). The 340-residue NH2-terminal construct contains the suppressor domain and part of the second -trefoil domain. Because truncation of the construct in the middle of the -trefoil domain likely severely disrupts its structure, the suppressor domain probably mediates the interaction with the COOH terminus. Thus the conserved patch observed in the crystal structure of the suppressor domain (54) could possibly be involved in interactions with the COOH terminus. Recently, the NH2-terminal interacting region in the COOH terminus of the channel was localized to the cytoplasmic linker that connects transmembrane helices 4 and 5 (S4-S5 linker) (411). The possible implications of this interaction for activation gating of the channel are discussed in section IIIB2B.
A) THE PORE. A six transmembrane topology of the InsP3R was established by immunocytochemical techniques and N-linked glycosylation analyses of full-length and truncated proteins (153, 309). These studies, together with analogy modeling of InsP3R and RyR with well-characterized cation channels, suggested that COOH-terminal transmembrane helices are involved in ion permeation, with helices 5 and 6 and intervening sequences in InsP3R critical for creating the basic pore structure (309, 459, 514) (Fig. 3). Deletion of the first four transmembrane helices from InsP3R, leaving transmembrane helices 5 and 6, resulted in a channel with normal conductance and selectivity properties (383), consistent with this model. Site-directed mutagenesis of two residues between TM5 and 6, and believed to be located in the putative selectivity filter (43), also suggested that such a model provides a rational basis for considering the roles of particular residues that contribute to conductance and selectivity properties of the InsP3R permeation pathway. Furthermore, homology of RyR and InsP3R sequences in the putative pore region suggests that insights from studies of the RyR can provide insights into important molecular determinants in InsP3R.
The bacterial K+ channel KcsA has been used as a template to successfully model the pore region of the InsP3R and RyR (414, 507). Based on homology, predicted secondary structure, surface area, hydrophobicity, and electrostatic potential, the assembled tetrameric TM5/6 region of RyR2 adopted an equivalent structure to that of KcsA (507). The validity of the model was demonstrated by its ability to quantitatively predict in molecular dynamics simulations empirical permeation results for RyR2. Recent electron microscopic structures of the RyR resolved at 13.6 Å (405) and 10 Å (261) provide details regarding the membrane domain, including the pore. In these studies, some helices were resolved that could be well fitted with the pore helices from crystal structures of bacterial K+ channels. These studies reinforce the hypotheses based on homology modeling that the pore of RyR and, by extrapolation because of their sequence, secondary structural and functional similarities, the InsP3R as well, are constructed in a manner believed to be similar in many types of cation channels (266). Additional details regarding the functional and structural properties of the InsP3R pore are discussed in section V that focuses on ion permeation properties of the channel.
B) THE GATE. InsP3 binding to the NH2 terminus of the channel induces conformational changes that are transduced to the activation gate that then enables ion flow through the channel. The molecular identity of the gate is unknown, and the mechanisms that couple ligand binding to opening and closing of the gate are unknown as well. Structural and functional studies in other cation channels indicate that activation gating can reside at two locations. First, the inner helices associated with the pore cross each other near the cytoplasmic surface of the membrane, at a so-called bundle crossing (115). The bundle crossing appears to either provide too narrow a passage for ion translocation or it is lined with hydrophobic residues at the narrow point that act effectively as a barrier to ion flow (115, 243, 416). The structures of bacterial MthK and KvAP K+ channels (205, 206), and functional accessibility and structural studies in other channels (106, 369, 370), indicate that activation gating is associated with bending and rotation of the inner helix, with consequent widening of the pore access region, creating the inner vestibule. Helix bending is conferred by highly conserved glycine residues located above the helix bundle crossing (205, 206, 266), or by inner-helix proline residues in some cases (106, 244). Alternatively, the activation gate appears to be located at the selectivity filter in some ion channels, including inward-rectifier and small-conductance K+ channels and CNG channels (62, 142, 143, 257, 260, 550). Furthermore, the selectivity filter may undergo conformational changes during gating (36, 143). It has been speculated that distinct channel kinetic states in Kir channels reflect gating at the two different gates (36).
Analyses and modeling of single-channel gating kinetics of patch-clamped InsP3R indicate that besides the ligand (InsP3 and Ca2+)-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel P
