Structure of the M2 Transmembrane Segment of GLIC, a Prokaryotic Cys Loop Receptor Homologue from Gloeobacter violaceus, Probed by Substituted Cysteine Accessibility

The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.

Prokaryotic members of the Cys-loop receptor ligand-gated ion channel superfamily were recently identified. Previously, Cys-loop receptors were only known from multicellular organisms (metazoans). Contrary to the metazoan Cys-loop receptors, the prokaryotic ones consist of an extracellular (ECD) and a transmembrane domain (TMD), lacking the large intracellular domain (ICD) present in metazoa (between transmembrane segments M3 and M4). Using a chimera approach, we added the 115-amino acid ICD from mammalian serotonin type 3A receptors (5-HT(3A)) to the prokaryotic proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC). We created 12 GLIC-5-HT(3A)-ICD chimeras by replacing a variable number of amino acids in the short GLIC M3M4 linker with the entire 5-HT(3A)-ICD. Two-electrode voltage clamp recordings after expression in Xenopus laevis oocytes showed that only two chimeras were functional and produced currents upon acidification. The pH(50) was comparable with wild-type GLIC. 5-HT(3A) receptor expression can be inhibited by the chaperone protein RIC-3. We have shown previously that the 5-HT(3A)-ICD is required for the attenuation of 5-HT-induced currents when RIC-3 is co-expressed with 5-HT(3A) receptors in X. laevis oocytes. Expression of both functional 5-HT(3A) chimeras was inhibited by RIC-3 co-expression, indicating appropriate folding of the 5-HT(3A)-ICD in the chimeras. Our results indicate that the ICD can be considered a separate domain that can be removed from or added to the ECD and TMD while maintaining the overall structure and function of the ECD and TMD.

Protein function depends on conformational flexibility and folding stability. Loose packing of hydrophobic cores is not infrequent in proteins, as the enhanced flexibility likely contributes to their biological function. Here, using experimental and computational approaches, we show that eukaryotic pentameric ligand-gated ion channels are characterized by loose packing of their extracellular domain β-sandwich cores, and that loose packing contributes to their ability to rapidly switch from closed to open channel states in the presence of ligand. Functional analyses of GABA(A) receptors show that increasing the β-core packing disrupted GABA-mediated currents, with impaired GABA efficacy and slowed GABA current activation and desensitization. We propose that loose packing of the hydrophobic β-core developed as an evolutionary strategy aimed to facilitate the allosteric mechanisms of eukaryotic pentameric ligand-gated ion channels.

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.

Although many of the details of GlyR dynamics still remain elusive, the extensive studies conducted on GlyR and other members of the nicotinicoid superfamily in recent years provide us with an emerging picture of the structure and function of these receptors. As novel studies continue to examine these receptors, we are confident that these ion channels will reveal their molecular mechanisms. These details are significant in that they will allow us to develop novel therapeutics and pharmacological tools to modulate channel activity in the central nervous system.

We have used a homology model of the extracellular domain of the 5-HT(3) receptor to dock granisetron, a 5-HT(3) receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 A from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [(3)H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.

A large cytoplasmic domain accounts for approximately one-third of the entire protein of one superfamily of ligand-gated membrane ion channels, which includes nicotinic acetylcholine (nACh), gamma-aminobutyric acid type A (GABA(A)), serotonin type 3 (5-HT3), and glycine receptors. Desensitization is one functional feature shared by these receptors. Because most molecular studies of receptor desensitization have focused on the agonist binding and channel pore domains, relatively little is known about the role of the large cytoplasmic domain (LCD) in this process. To address this issue, we sequentially deleted segments of the LCD of the 5-HT3A receptor and examined the function of the mutant receptors. Deletion of a small segment that contains three amino acid residues (425-427) significantly slowed the desensitization kinetics of the 5-HT3A receptor. Both deletion and point mutation of arginine 427 altered desensitization kinetics in a manner similar to that of the (425-427) deletion without significantly changing the apparent agonist affinity. The extent of receptor desensitization was positively correlated with the polarity of the amino acid residue at 427: the desensitization accelerates with increasing polarity. Whereas the R427L mutation produced the slowest desensitization, it did not significantly alter single channel conductance of 5-HT3A receptor. Thus, the arginine 427 residue in the LCD contributes to 5-HT3A receptor desensitization, possibly through forming an electrostatic interaction with its neighboring residues. Because the polarity of the amino acid residue at 427 is highly conserved, such a desensitization mechanism may occur in other members of the Cys-loop family of ligand-gated ion channels.

The gamma-aminobutyric acid, type A (GABAA), receptor ion channel is lined by the second membrane-spanning (M2) segments from each of five homologous subunits that assemble to form the receptor. Gating presumably involves movement of the M2 segments. We assayed protein mobility near the M2 segment extracellular ends by measuring the ability of engineered cysteines to form disulfide bonds and high affinity Zn(2+)-binding sites. Disulfide bonds formed in alpha1beta1E270Cgamma2 but not in alpha1N275Cbeta1gamma2 or alpha1beta1gamma2K285C. Diazepam potentiation and Zn2+ inhibition demonstrated that expressed receptors contained a gamma subunit. Therefore, the disulfide bond in alpha1beta1E270Cgamma2 formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-A resolution structure, the distance between alpha carbon atoms of 20' aligned positions in non-adjacent subunits is approximately 19 A. Because disulfide trapping involves covalent bond formation, it indicates the extent of movement but does not provide an indication of the energetics of protein deformation. Pairs of cysteines can form high affinity Zn(2+)-binding sites whose affinity depends on the energetics of forming a bidentate-binding site. The Zn2+ inhibition IC50 for alpha1beta1E270Cgamma2 was 34 nm. In contrast, it was greater than 100 microM in alpha1N275Cbeta1gamma2 and alpha1beta1gamma2K285C receptors. The high Zn2+ affinity in alpha1beta1E270Cgamma2 implies that this region in the beta subunit has a high protein mobility with a low energy barrier to translational motions that bring the positions into close proximity. The differential mobility of the extracellular ends of the beta and alpha M2 segments may have important implications for GABA-induced conformational changes during channel gating.

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a Cl channel that is an essential component of epithelial Cl transport systems in many organs, including the intestines, pancreas, lungs, sweat glands, and kidneys. In the Cl secretory intestinal epithelium, Cl enters the cells through a Na-K-2Cl cotransporter in the basolateral membrane and exits through CFTR in the apical membrane; water follows osmotically (1). Absorptive epithelia use similar transporters and channels, but their polarized distribution between the apical and basolateral membranes is usually reversed. A major determinant of the transepithelial Cl transport rate is the level of activation of CFTR (2, 3), which depends on the extent to which it is phosphorylated. This is determined by the relative activities of kinases and phosphatases, the activities of which are often hormonally regulated (1). Defects in the gene encoding CFTR that reduce either its Cl transport capacity or its level of cell surface expression cause cystic fibrosis (CF) (4–6) as well as a form of male sterility due to congenital bilateral absence of the vas deferens (7). CF is the most common lethal genetic disease in Caucasians, with about 30,000 CF patients in the United States. In contrast, in intestinal epithelial cells overstimulation of CFTR because of the activation of protein kinases by bacterial enterotoxins causes secretory diarrhea (1, 8). Secretory diarrhea is the second largest cause of infant mortality in the developing world, causing 3 million deaths per year of children under the age of 5. Thus, although CFTR was named because of its association with CF, as a cause of disease, its relationship to secretory diarrhea is a more widespread public health problem. The cloning of CFTR in 1989 (9) has facilitated studies of its structure, function, regulation, biogenesis, and degradation, which will be reviewed in this article. Issues reviewed elsewhere and not discussed here include the mechanisms by which mutations in CFTR cause CF (5, 6) and the possible role of CFTR in regulating the pH within intracellular organelles (10).

Epithelial Na+ channels facilitate the transport of Na+ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na+ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.

Members of the Cys-loop superfamily of ligand-gated ion channels, which mediate fast synaptic transmission in the nervous system, are assembled as heteropentamers from a large repertoire of neuronal subunits. Although several motifs in subunit N-terminal domains are known to be important for subunit assembly, increasing evidence points toward a role for C-terminal domains. Using a combination of flow cytometry, patch clamp recording, endoglycosidase H digestion, brefeldin A treatment, and analytic centrifugation, we identified a highly conserved aspartate residue at the boundary of the M3-M4 loop and the M4 domain that was required for binary and ternary gamma-aminobutyric acid type A receptor surface expression. Mutation of this residue caused mutant and partnering subunits to be retained in the endoplasmic reticulum, reflecting impaired forward trafficking. Interestingly although mutant and partnering wild type subunits could be coimmunoprecipitated, analytic centrifugation studies demonstrated decreased formation of pentameric receptors, suggesting that this residue played an important role in later steps of subunit oligomerization. We thus conclude that C-terminal motifs are also important determinants of Cys-loop receptor assembly.

The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by alpha-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors.

Hsp90 was originally identified as one of several conserved heat shock proteins. Like the other major classes of heat shock proteins, Hsp90 exhibits general protective chaperone properties, such as preventing the unspecific aggregation of non-native proteins (1Wiech H. Buchner J. Zimmermann R. Jakob U. Nature. 1992; 358: 169-170Crossref PubMed Scopus (417) Google Scholar). However, Hsp90 seems to be more selective than the other promiscuous general chaperones, as it preferentially interacts with a specific subset of the proteome (2Picard D. CMLS Cell. Mol. Life Sci. 2002; 59: 1640-1648Crossref PubMed Scopus (645) Google Scholar). Another specific feature of Hsp90 is its regulatory role of inducing conformational changes in folded, native-like substrate proteins that lead to their activation or stabilization (3Jakob U. Lilie H. Meyer I. Buchner J. J. Biol. Chem. 1995; 270: 7288-7294Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Recently, the three-dimensional structures of full-length Hsp90 from Escherichia coli, yeast, and the endoplasmic reticulum were solved (4Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 5Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (683) Google Scholar, 6Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 7Pearl L.H. Prodromou C. Annu. Rev. Biochem. 2006; 75: 271-294Crossref PubMed Scopus (875) Google Scholar). Together with sequence data, these showed that, although Hsp90 maintained its general domain structure from bacteria to man, distinct changes seem to have adapted Hsp90 to the more complex protein environment of the eukaryotic cell. Concomitant with the occurrence of a long charged linker connecting the N- 3The abbreviations used are: N-domain, N-terminal domain; M-domain, middle domain; SHR, steroid hormone receptor; TPR, tetratricopeptide repeat; PPIase, peptidylprolyl cis/trans-isomerase; eNOS, epithelial nitric-oxide synthase. 3The abbreviations used are: N-domain, N-terminal domain; M-domain, middle domain; SHR, steroid hormone receptor; TPR, tetratricopeptide repeat; PPIase, peptidylprolyl cis/trans-isomerase; eNOS, epithelial nitric-oxide synthase. and M-domains, the eukaryotic protein exhibits an extension of the C-terminal domain, which includes the conserved amino acid motif MEEVD at the C terminus (8Chen S. Sullivan W.P. Toft D.O. Smith D.F. Cell Stress Chaperones. 1998; 3: 118-129Crossref PubMed Scopus (166) Google Scholar). This region serves as the major interaction site for a cohort of co-chaperones (Table 1) (9Richter K. Meinlschmidt B. Buchner J. Buchner J. Kiefhaber T Protein Folding Handbook. Wiley-VCH Verlag GmbH & Co., Weinheim, Germany2005: 768-829Crossref Scopus (1) Google Scholar), which apparently support Hsp90 in the folding and activation of its substrate proteins in eukaryotes. In this review, we summarize the current knowledge on the functional principles of this molecular machine, including the ATP-driven chaperone cycle of Hsp90 and its regulation by co-chaperones and post-translational modifications.TABLE 1Selected Hsp90 cofactors Open table in a new tab Hsp90 is a flexible dimer. Each monomer consists of three domains: the N-domain, connected by a long linker sequence (in eukaryotes) to an M-domain, which is followed by a C-terminal dimerization domain (Fig. 1). The N-domain possesses a deep ATP-binding pocket (10Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar), where ATP is bound in an unusual kinked manner. ATP hydrolysis by Hsp90 is rather slow: Hsp90 from yeast hydrolyzes one molecule of ATP every 1 or 2 min (11Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (610) Google Scholar, 12Scheibel T. Weikl T. Buchner J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1495-1499Crossref PubMed Scopus (229) Google Scholar), and human Hsp90 hydrolyzes one molecule of ATP every 20 min (0.04 min–1) (13McLaughlin S.H. Smith H.W. Jackson S.E. J. Mol. Biol. 2002; 315: 787-798Crossref PubMed Scopus (210) Google Scholar). The ATPase activity is essential for the function of Hsp90 in yeast (11Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (610) Google Scholar, 14Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (479) Google Scholar). The slow hydrolysis suggests that complex conformational rearrangements of Hsp90 are coupled to the ATPase reaction and that these represent the rate-limiting step of the enzyme. The first steps of these conformational changes were elucidated recently in detail (15Richter K. Moser S. Hagn F. Friedrich R. Hainzl O. Heller M. Schlee S. Kessler H. Reinstein J. Buchner J. J. Biol. Chem. 2006; 281: 11301-11311Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar): upon ATP binding, a short segment of the N-domain called the “ATP lid” changes its position and flaps over the binding pocket (Fig. 1, steps 2 and 3). This releases a short N-terminal segment from its original position (16Richter K. Reinstein J. Buchner J. J. Biol. Chem. 2002; 277: 44905-44910Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In a subsequent reaction, this segment binds to the respective N-domain of the other subunit in the dimer, producing a strand-swapped, transiently dimerized N-terminal conformation (step 3) (5Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (683) Google Scholar, 15Richter K. Moser S. Hagn F. Friedrich R. Hainzl O. Heller M. Schlee S. Kessler H. Reinstein J. Buchner J. J. Biol. Chem. 2006; 281: 11301-11311Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). These N-terminal rearrangements result in further conformational changes throughout the entire Hsp90 dimer leading to a twisted and compacted dimer, in which N- and M-domains associate and the distance between M-domains is shortened by 40 Å (5Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (683) Google Scholar). The association of N- and M-domains completes the active site of this “split ATPase” (step 4). Recently, a similar progression of steps was shown to occur also for the endoplasmic homolog Grp94 (17Frey S. Leskovar A. Reinstein J. Buchner J. J. Biol. Chem. 2007; 282: 35612-35620Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), mitochondrial TRAP1 (6Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 18Leskovar, A., Wegele, H., Werbeck, N. D., Buchner, J., and Reinstein, J. (2008) 283, 11677–11688Google Scholar), and human Hsp90 (19Richter K. Soroka J. Skalniak L. Leskovar A. Hessling M. Reinstein J. Buchner J. J. Biol. Chem. 2008; 283: 17757-17765Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Therefore, the scenario outlined above seems to be the ubiquitously conserved ATPase mechanism for Hsp90. Interestingly, the unusual way in which ATP is bound by Hsp90 is perfectly mimicked by some natural compounds, such as geldanamycin and radicicol. These are highly specific and potent inhibitors of the Hsp90 ATPase (20Roe S.M. Prodromou C. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. J. Med. Chem. 1999; 42: 260-266Crossref PubMed Scopus (871) Google Scholar), blocking the maturation of substrate proteins and eventually resulting in their degradation (21Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1310) Google Scholar). As several Hsp90 substrate proteins are kinases, which can be deregulated in the development of cancer, derivatives of Hsp90 inhibitors are currently being investigated as anticancer therapeutics at the stage of clinical trials (22Sharp S. Workman P. Adv. Cancer Res. 2006; 95: 323-348Crossref PubMed Scopus (269) Google Scholar). Current models assume that the conformational changes associated with ATP hydrolysis are required for reaching or maintaining an activated state of a substrate protein. In well studied examples such as the SHRs, several cofactors interact with Hsp90 in a sequential manner to assemble a functional chaperone machinery (23Pratt W.B. Toft D.O. Exp. Biol. Med. (Maywood). 2003; 228: 111-133Crossref PubMed Scopus (1239) Google Scholar, 24Smith D.F. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar). The basis for this ordered succession of different assemblies can now be rationalized, as it turned out that several Hsp90 cofactors display a strong binding preference for specific Hsp90 conformations. The loading of an SHR onto Hsp90 requires the cooperation of Hsp90 with the chaperone Hsp70 and its cofactor Hsp40 (25Smith D.F. Sullivan W.P. Marion T.N. Zaitsu K. Madden B. McCormick D.J. Toft D.O. Mol. Cell. Biol. 1993; 13: 869-876Crossref PubMed Scopus (246) Google Scholar). Moreover, both chaperones become physically linked by an adaptor protein called Hop/Sti1 (Table 1). This co-chaperone binds via small helical TPR domains to the C-terminal ends of Hsp70 and Hsp90 (26Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (991) Google Scholar). It seems that Hsp70 stabilizes the SHR in a conformation that can be recognized and bound by Hsp90. However, experimental evidence for this notion is largely lacking. How the substrate in this complex is transferred from Hsp70 to Hsp90 is also still unclear. It might be that the bridging by Hop/Sti1 selects for Hsp90 molecules in a conformation competent for substrate binding in addition to increasing the local concentration of Hsp70 and Hsp90. For the progression of the chaperone cycle, empty Hsp70 and Hop/Sti1 have to dissociate, and other co-chaperones such as specific PPIases and p23/Sba1 enter the complex (Table 1) (24Smith D.F. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar). These PPIases also possess a TPR domain, which binds to the C-terminal end of Hsp90. The second cofactor, p23/Sba1, associates with the N-terminally dimerized conformation of Hsp90 (27Prodromou C. Panaretou B. Chohan S. Siligardi G. O'Brien R. Ladbury J.E. Roe S.M. Piper P.W. Pearl L.H. EMBO J. 2000; 19: 4383-4392Crossref PubMed Google Scholar, 28Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar), making it likely that the dramatic conformational rearrangement from the open to the closed state of Hsp90 occurs at this stage of the Hsp90 chaperone cycle (Fig. 1, steps 3 and 4). This closed conformation is metastable and upon ATP hydrolysis returns to the open state (Fig. 1, step 5) (28Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar). The bound substrate protein dissociates in turn from Hsp90, permitting a new round of the cycle. The first steps of the cycle for the maturation of signaling kinases is a variation of the scheme described above. Here, the kinase-specific Hsp90 cofactor Cdc37 seems to associate with substrate kinases in their inactive forms first. This complex may then be loaded onto Hsp90 (29Kimura Y. Rutherford S.L. Miyata Y. Yahara I. Freeman B.C. Yue L. Morimoto R.I. Lindquist S. Genes Dev. 1997; 11: 1775-1785Crossref PubMed Scopus (175) Google Scholar). The following steps are less clear yet. It may be that also for kinases, Hsp70, Hsp40, and Hop/Sti1 are additionally required (30Caplan A.J. Mandal A.K. Theodoraki M.A. Trends Cell Biol. 2007; 17: 87-92Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Because Cdc37 partially inhibits the ATPase of Hsp90 (31Siligardi G. Panaretou B. Meyer P. Singh S. Woolfson D.N. Piper P.W. Pearl L.H. Prodromou C. J. Biol. Chem. 2002; 277: 20151-20159Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), it is reasonable to speculate that a transient stalling of the ATPase facilitates substrate transfer onto Hsp90 in general. Up to now, more than a dozen distinct Hsp90 cofactors have been identified (9Richter K. Meinlschmidt B. Buchner J. Buchner J. Kiefhaber T Protein Folding Handbook. Wiley-VCH Verlag GmbH & Co., Weinheim, Germany2005: 768-829Crossref Scopus (1) Google Scholar). Their large number is not paralleled by other chaperone systems. Most bind Hsp90 with submicromolar affinities (Table 1). The major class of these is the TPR domain-containing cofactors, which include the proteins Hop/Sti1, PP5/Ppt1, and the large PPIases, among others (Table 1). Some of these cofactors could specifically facilitate the activation of a certain set of substrate proteins. In this context, the cofactor Unc45 has been shown to participate in early muscle development during the assembly of myosin filaments (32Barral J.M. Hutagalung A.H. Brinker A. Hartl F.U. Epstein H.F. Science. 2002; 295: 669-671Crossref PubMed Scopus (197) Google Scholar), and Xap2/AIP has been found in complex with the protein HbX from the hepatitis B virus (33Kuzhandaivelu N. Cong Y.S. Inouye C. Yang W.M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (94) Google Scholar) or the endogenous aryl hydrocarbon receptor (34Meyer B.K. Perdew G.H. Biochemistry. 1999; 38: 8907-8917Crossref PubMed Scopus (175) Google Scholar). It remains to be seen whether these cofactors are really highly specialized or whether they are just the first substrates identified. Interestingly, in yeast, only two cofactors of the Hsp90 system are essential for viability (in addition to Hsp90). These are Cdc37 and Cns1 (29Kimura Y. Rutherford S.L. Miyata Y. Yahara I. Freeman B.C. Yue L. Morimoto R.I. Lindquist S. Genes Dev. 1997; 11: 1775-1785Crossref PubMed Scopus (175) Google Scholar, 35Dolinski K.J. Cardenas M.E. Heitman J. Mol. Cell. Biol. 1998; 18: 7344-7352Crossref PubMed Google Scholar, 36Marsh J.A. Kalton H.M. Gaber R.F. Mol. Cell. Biol. 1998; 18: 7353-7359Crossref PubMed Google Scholar). Cns1 has been shown to associate with both Hsp90 and Hsp70; however, due to the presence of a single TPR domain, no ternary complexes can be formed (37Hainzl O. Wegele H. Richter K. Buchner J. J. Biol. Chem. 2004; 279: 23267-23273Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The function of Cns1 remains elusive. Hsp90 is embedded into several control mechanisms that influence its activity. As mentioned above, the ATPase activity is intrinsically decelerated due to slow conformational changes of the lid segment within the N-domains. In addition to this, the ATPase activity of Hsp90 is regulated by several cofactors. Another set of cofactors modulates substrate processing without changing the ATPase activity. Finally, the activity of Hsp90 is also regulated by post-translational modifications. Several of the cofactors of Hsp90 modulate its ATPase activity by interacting preferentially with a specific conformation of Hsp90. p23/Sba1 binds to the ATPase domain and stabilizes the N-terminally dimerized conformation at the late stage of the ATPase cycle (Fig. 1) (28Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar, 38Chadli A. Bouhouche I. Sullivan W. Stensgard B. McMahon N. Catelli M.G. Toft D.O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12524-12529Crossref PubMed Scopus (135) Google Scholar, 39Siligardi G. Hu B. Panaretou B. Piper P.W. Pearl L.H. Prodromou C. J. Biol. Chem. 2004; 279: 51989-51998Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). This positions p23/Sba1 to be part of the Hsp90 complex at the moment of hydrolysis and appears to be the reason for the decrease in the ATP turnover rate of Hsp90 in the presence of p23/Sba1. A second site of cofactor interaction resides in the M-domain, to which Aha1, the only known activator of the Hsp90 ATPase, binds. This interaction stimulates the weak ATPase activity of Hsp90 by >10-fold (40Panaretou B. Siligardi G. Meyer P. Maloney A. Sullivan J.K. Singh S. Millson S.H. Clarke P.A. Naaby-Hansen S. Stein R. Cramer R. Mollapour M. Workman P. Piper P.W. Pearl L.H. Prodromou C. Mol. Cell. 2002; 10: 1307-1318Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). The stimulatory interaction of Aha1 with the M-domain of Hsp90 suggests a participation of this domain in the rate-limiting step of the ATPase reaction. Structural studies that Aha1 binding the M-domain the active and the domain to a conformation the closed for ATP hydrolysis 1, step 3) L.H. Prodromou C. Annu. Rev. Biochem. 2006; 75: 271-294Crossref PubMed Scopus (875) Google Scholar, P. Prodromou C. C. Hu B. Roe S.M. Vaughan C.K. I. Panaretou B. Piper P.W. Pearl L.H. EMBO J. 2004; PubMed Scopus Google Scholar). Another for the ATPase activity of Hsp90 is by the co-chaperone in binds to the C-terminal end of Hsp90 via its TPR In is a second binding site in the N- or M-domain K. P. Hainzl O. Reinstein J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). to this second site to the ATPase of Hsp90 K. P. Hainzl O. Reinstein J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, C. Siligardi G. O'Brien R. Woolfson D.N. L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; 18: PubMed Scopus Google Scholar). Interestingly, ATP binding is not K. P. Hainzl O. Reinstein J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). As in the of p23/Sba1, selects a specific conformation of Hsp90 for suggests that binding the N-terminal dimerization and association of the N- and (Fig. 1, step 3) K. P. Hainzl O. Reinstein J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of ATP hydrolysis is not is This is the of a The open state by is the state for as outlined above. Cdc37 also inhibits the ATPase activity of Hsp90 (31Siligardi G. Panaretou B. Meyer P. Singh S. Woolfson D.N. Piper P.W. Pearl L.H. Prodromou C. J. Biol. Chem. 2002; 277: 20151-20159Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, C. Siligardi G. O'Brien R. Woolfson D.N. L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; 18: PubMed Scopus Google Scholar). of the respective mechanisms are still a in which Cdc37 binds to the ATP lid in the N-terminal site of Hsp90 and the N-terminal dimerization by as a dimer in between the two S.M. M.M. Meyer P. Vaughan C.K. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Because Cdc37 is in the loading of Hsp90 with kinases, it is with the state of Hsp90 that Cdc37 also Hsp90 in an open The of the ATPase activity this state to for an of these cofactors the of the conformational changes of Hsp90 that can be as substrate processing steps to the specific of certain binding to Hsp90 in the of p23/Sba1, its A further of regulation that has increasing is of Hsp90, such as K. D. M.G. S. K. S. Toft D. L. N. Neckers L. Mol. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar), G. R. R. G. A. Nature. 1998; PubMed Scopus Google Scholar), and K. T. J. 28: PubMed Google Scholar, J.J. Toft D.O. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.J. D.A. Sullivan W.P. Toft D.O. PubMed Scopus Google Scholar). These lead to in the maturation of Hsp90 substrates Neckers L. 2007; PubMed Scopus Google Scholar). some of the amino acid positions have been identified by of the it is a to a of the modifications. For an between human Hsp90 and its substrate was G. R. R. G. A. Nature. 1998; PubMed Scopus Google Scholar, A. L. C. D. M.A. C. I. J. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the one activity on the chaperone activity of on the other the human Hsp90 at is part of the C-terminal As a the Hsp90 ATPase activity is A. L. C. D. M.A. C. I. J. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), which inhibits in turn the of activity by Hsp90. This might facilitate a regulation of in a A. L. C. D. M.A. C. I. J. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). For a similar scenario which result in the of Hsp90, lead to a interaction with and maturation of several of its substrate proteins, such as and the receptor J.J. S. M. Toft D.O. W.B. Mol. Cell. 18: Full Text Full Text PDF PubMed Scopus Google Scholar, M.G. Neckers L. J. Natl. Cancer 2002; PubMed Scopus Google Scholar, R. L. P. M. M. J. L. Smith C. J. R. P. K. Cancer Res. 2003; Google Scholar, Y. J.J. W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). As a an in degradation of some Hsp90 substrates was was identified as the Hsp90 J.J. S. M. Toft D.O. W.B. Mol. Cell. 18: Full Text Full Text PDF PubMed Scopus Google Scholar). In Hsp90 is at at two one was identified as (in human K. D. M.G. S. K. S. Toft D. L. N. Neckers L. Mol. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the to interact with its substrate proteins, the binding of ATP by Hsp90 was also shown to be upon of Hsp90 Y. J.J. W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. E. D. H. 2003; PubMed Scopus (85) Google Scholar). of Hsp90 has been known for K. T. J. 28: PubMed Google Scholar). it was found that at of Hsp90 Mol. Cell. Biol. PubMed Scopus Google Scholar, J.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). For Hsp90 proteins, the of Hsp90 molecules on three D.A. J.J. Biochem. PubMed Scopus Google Scholar). The Hsp90 is J. Biol. Chem. Full Text PDF PubMed Google Scholar). shock an Hsp90 turnover M. O. PubMed Scopus Google Scholar). known in the N-domain of Hsp90, no specific can be to a to Hsp90 protein in yeast the homolog in Neckers L. 2007; PubMed Scopus Google Scholar). Interestingly, one of the cofactors with Hsp90 via a TPR domain is PP5/Ppt1, a The yeast homolog Hsp90 in a specific manner only bound to Hsp90 Wegele H. Buchner J. EMBO J. 2006; PubMed Scopus Google Scholar). of Hsp90 is for its in as in a the maturation of several substrate proteins was found to be Wegele H. Buchner J. EMBO J. 2006; PubMed Scopus Google Scholar). It be a highly to Hsp90 regulation at the of post-translational in as this the to control over the Hsp90 chaperone The is more than as co-chaperones of Hsp90 are regulated by such as as well Hessling M. F. Buchner J. Smith D.F. Mol. Endocrinol. 2007; PubMed Scopus Google Scholar, Y. E. Mol. Cell. Biochem. PubMed Scopus Google Scholar). In this context, a long open is the of substrate of Hsp90. with other chaperones, a large number of substrate proteins are known due to their that of the respective of the identified Hsp90 substrate proteins into two as and and signaling The with regulatory proteins apparently Hsp90 to influence such as Lindquist S. Science. PubMed Scopus Google Scholar, C. Lindquist S. Nature. 2002; PubMed Scopus Google Scholar), mitochondrial J. T. J. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar), and the of R. M. R. J. Genes Dev. 2007; PubMed Scopus Google Scholar). the known substrate proteins are different in several as to which are for interaction with Hsp90. A is This protein in two a and a forms are with just a amino acid although is in its is largely of Hsp90 Y. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar). A first step the basis for the in the Hsp90 of and is the that the of the two forms with being a highly protein S.F. S. A. M. Buchner J. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). of kinases have further to the notion that it is not a specific binding rather the of the protein that seems to be for binding W. G. P. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus (45) Google Scholar). this is the general scheme remains to be as is evidence that a specific in the might participate in the interaction with Hsp90 K. K. S. T. K. Y. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). of the Hsp90 is other chaperones such as Hsp70 and for which we have a clear of this For Hsp90, experimental evidence for binding in three domains of the protein T. Weikl T. Buchner J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1495-1499Crossref PubMed Scopus (229) Google Scholar, C. Hartl F.U. 1997; PubMed Scopus Google Scholar, C.K. U. F. M.M. Prodromou C. Pearl L.H. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). However, have to on substrate of full-length Hsp90 structures of open and closed is a major in this (4Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 5Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (683) Google Scholar, 6Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). In to chaperones and from of Hsp90 to R. Inouye M. Trends Biochem. Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), it was that the substrate protein may be by the Hsp90 dimer. This notion is not in with the in which is not to a substrate protein between the two Hsp90 The first of the structure of an complex from and processing of Hsp90 in complex with the and the co-chaperone In this the substrate is bound in a highly to the M-domain and the N-domain of one the co-chaperone Cdc37 resides between the C.K. U. F. M.M. Prodromou C. Pearl L.H. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). this association is the for kinases and whether this can be for other substrate proteins to be the of ATP turnover and associated conformational changes of Hsp90 to be solved now, other of the Hsp90 machinery are not to These include substrate turnover and for of A of in and in be required to these we a more of the of this molecular machine, we can to specific substrate proteins, cofactors, and mechanisms of regulation and post-translational into the and for on the

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl(-) concentrations in the kidney and in other epithelia. We tested whether Cl(-) alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl(-) with SO4(2-), H2PO4(-), or SCN(-) produced a large increase in ENaC current, indicating that extracellular Cl(-) inhibits ENaC. Extracellular Cl(-) also inhibited ENaC in Na+-transporting epithelia. The anion selectivity sequence was SCN(-) < SO4(2-) < H2PO4(-) < F(-) < I(-) < Cl(-) < Br(-). Crystallization of ASIC1a revealed a Cl(-) binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (alpha(H418A) and beta(R388A)) disrupted the response to Cl(-), suggesting that Cl(-) might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl(-). The response to Cl(-) was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl(-). Cl(-) regulated ENaC activity in part through enhanced Na+ self-inhibition, a process by which extracellular Na+ inhibits ENaC. Together, the data indicate that extracellular Cl(-) regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl(-) might modulate epithelial Na+ absorption.

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.