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• W1657709899 abstract "Mechanosensitive membrane channels in bacteria respond to the mechanical stretching of the membrane. They will open when bacteria are subjected to rapid osmotic down shock. MscS is a bacterial mechanosensitive channel of small conductance. It is a heptameric membrane protein whose transmembrane part, including the gate and its kinetics, has been well characterized. MscS has a large cytoplasmic domain of a cage-like shape that changes its conformation upon gating, but its involvement in gating is not understood. We screened MscS for mutations that cause potassium leak in Escherichia coli strains deficient in potassium transport systems. We did a phenotypic analysis of single and multiple mutants and recorded the single channel activities of some of them. After these analyses, we attributed the effects of a number of mutations to particular functional states of the channel. Our screen revealed that MscS leaks potassium in a desensitized and in an inactivated state. It also appeared that the lower part of TM3 (transmembrane, pore-forming helix) and the cytoplasmic β domain are tightly packed in the inactivated state but are dissociated in the open state. We attribute the TM3-β interaction to stabilization of the inactivated state in MscS and to the control of tight closure of its membrane pore. Mechanosensitive membrane channels in bacteria respond to the mechanical stretching of the membrane. They will open when bacteria are subjected to rapid osmotic down shock. MscS is a bacterial mechanosensitive channel of small conductance. It is a heptameric membrane protein whose transmembrane part, including the gate and its kinetics, has been well characterized. MscS has a large cytoplasmic domain of a cage-like shape that changes its conformation upon gating, but its involvement in gating is not understood. We screened MscS for mutations that cause potassium leak in Escherichia coli strains deficient in potassium transport systems. We did a phenotypic analysis of single and multiple mutants and recorded the single channel activities of some of them. After these analyses, we attributed the effects of a number of mutations to particular functional states of the channel. Our screen revealed that MscS leaks potassium in a desensitized and in an inactivated state. It also appeared that the lower part of TM3 (transmembrane, pore-forming helix) and the cytoplasmic β domain are tightly packed in the inactivated state but are dissociated in the open state. We attribute the TM3-β interaction to stabilization of the inactivated state in MscS and to the control of tight closure of its membrane pore. IntroductionMscS, a bacterial mechanosensitive (MS) 2The abbreviations used are: MSmechanosensitiveIPTGisopropyl 1-thio-β-d-galactopyranosideMTSESsulfonatoethylmethane thiosulfonateMMTSmethylmethanethiosulfonateGOFgain-of-functionLOFloss-of-function. channel of small conductance, is one of the best characterized membrane channels. MscS opens when activated directly by membrane stretch, and the same mechanism activates also MscL, another Escherichia coli MS channel that has higher threshold and bigger conductance than MscS. When open, both channels will jettison osmolytes protecting the bacteria against severe osmotic down shock (1Levina N. Tötemeyer S. Stokes N.R. Louis P. Jones M.A. Booth I.R. EMBO J. 1999; 18: 1730-1737Crossref PubMed Scopus (543) Google Scholar). MscS is a homo-heptamer, and each subunit consists of three membrane-spanning helices (TM1, TM2, and TM3) and a large cytoplasmic domain (Fig. 1A) (2Bass R.B. Strop P. Barclay M. Rees D.C. Science. 2002; 298: 1582-1587Crossref PubMed Scopus (493) Google Scholar). Two crystal structures (2Bass R.B. Strop P. Barclay M. Rees D.C. Science. 2002; 298: 1582-1587Crossref PubMed Scopus (493) Google Scholar, 3Wang W. Black S.S. Edwards M.D. Miller S. Morrison E.L. Bartlett W. Dong C. Naismith J.H. Booth I.R. Science. 2008; 321: 1179-1183Crossref PubMed Scopus (162) Google Scholar) and electron paramagnetic resonance (EPR)-based models of the channel two states, closed and open (4Vásquez V. Sotomayor M. Cortes D.M. Roux B. Schulten K. Perozo E. J. Mol. Biol. 2008; 378: 55-70Crossref PubMed Scopus (70) Google Scholar, 5Vásquez V. Sotomayor M. Cordero-Morales J. Schulten K. Perozo E. Science. 2008; 321: 1210-1214Crossref PubMed Scopus (145) Google Scholar), as well as several molecular dynamics studies (6Sotomayor M. Schulten K. Biophys. J. 2004; 87: 3050-3065Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 7Anishkin A. Sukharev S. Biophys. J. 2004; 86: 2883-2895Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 8Sotomayor M. van der Straaten T.A. Ravaioli U. Schulten K. Biophys. J. 2006; 90: 3496-3510Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 9Sotomayor M. Vásquez V. Perozo E. Schulten K. Biophys. J. 2007; 92: 886-902Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 10Spronk S.A. Elmore D.E. Dougherty D.A. Biophys. J. 2006; 90: 3555-3569Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 11Akitake B. Anishkin A. Liu N. Sukharev S. Nat. Struct. Mol. Biol. 2007; 14: 1141-1149Crossref PubMed Scopus (86) Google Scholar, 12Anishkin A. Akitake B. Sukharev S. Biophys. J. 2008; 94: 1252-1266Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 13Anishkin A. Kamaraju K. Sukharev S. J. Gen. Physiol. 2008; 132: 67-83Crossref PubMed Scopus (48) Google Scholar) have been published. The first resolved crystal structure was initially thought to represent the open state (2Bass R.B. Strop P. Barclay M. Rees D.C. Science. 2002; 298: 1582-1587Crossref PubMed Scopus (493) Google Scholar), but recent molecular dynamics studies have suggested that it actually shows an inactivated state (7Anishkin A. Sukharev S. Biophys. J. 2004; 86: 2883-2895Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 12Anishkin A. Akitake B. Sukharev S. Biophys. J. 2008; 94: 1252-1266Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 13Anishkin A. Kamaraju K. Sukharev S. J. Gen. Physiol. 2008; 132: 67-83Crossref PubMed Scopus (48) Google Scholar, 14Belyy V. Anishkin A. Kamaraju K. Liu N. Sukharev S. Nat. Struct. Mol. Biol. 2010; 17: 451-458Crossref PubMed Scopus (60) Google Scholar). The other structure was solved for the A106V mutant that has been shown to stabilize the open state (15Edwards M.D. Li Y. Kim S. Miller S. Bartlett W. Black S. Dennison S. Iscla I. Blount P. Bowie J.U. Booth I.R. Nat. Struct. Mol. Biol. 2005; 12: 113-119Crossref PubMed Scopus (105) Google Scholar), and it is believed to represent an open or partially open state (3Wang W. Black S.S. Edwards M.D. Miller S. Morrison E.L. Bartlett W. Dong C. Naismith J.H. Booth I.R. Science. 2008; 321: 1179-1183Crossref PubMed Scopus (162) Google Scholar). The structures, experimental data, and modeling studies have led to the models of the closed (12Anishkin A. Akitake B. Sukharev S. Biophys. J. 2008; 94: 1252-1266Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and open (13Anishkin A. Kamaraju K. Sukharev S. J. Gen. Physiol. 2008; 132: 67-83Crossref PubMed Scopus (48) Google Scholar) states and pointed toward channel rearrangements upon gating (16Miller S. Edwards M.D. Ozdemir C. Booth I.R. J. Biol. Chem. 2003; 278: 32246-32250Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 17Edwards M.D. Booth I.R. Miller S. Curr. Opin. Microbiol. 2004; 7: 163-167Crossref PubMed Scopus (44) Google Scholar, 18Akitake B. Anishkin A. Sukharev S. J. Gen. Physiol. 2005; 125: 143-154Crossref PubMed Scopus (102) Google Scholar, 19Grajkowski W. Kubalski A. Koprowski P. Biophys. J. 2005; 88: 3050-3059Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 20Nomura T. Sokabe M. Yoshimura K. Biophys. J. 2008; 94: 1638-1645Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 21Machiyama H. Tatsumi H. Sokabe M. Biophys. J. 2009; 97: 1048-1057Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The emerging model is that TM3, the channel pore-lining helix, buckles and straightens at glycines 113 and 121. This action happens during transitions between the closed, open, desensitized, and inactivated states (11Akitake B. Anishkin A. Liu N. Sukharev S. Nat. Struct. Mol. Biol. 2007; 14: 1141-1149Crossref PubMed Scopus (86) Google Scholar). Mutation of these glycines to alanines (in G113A/G121A mutant) allows the channel to open but jams it in the open state even after pressure release (11Akitake B. Anishkin A. Liu N. Sukharev S. Nat. Struct. Mol. Biol. 2007; 14: 1141-1149Crossref PubMed Scopus (86) Google Scholar). A few experimental tests have been conducted to support the model; however, the conclusions drawn from them were restricted to the transmembrane part of the channel. The MscS large cytoplasmic chamber was previously shown to change its conformation upon gating (19Grajkowski W. Kubalski A. Koprowski P. Biophys. J. 2005; 88: 3050-3059Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 21Machiyama H. Tatsumi H. Sokabe M. Biophys. J. 2009; 97: 1048-1057Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 22Koprowski P. Kubalski A. J. Biol. Chem. 2003; 278: 11237-11245Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 23Miller S. Edwards M.D. Ozdemir C. Booth I.R. J. Biol. Chem. 2003; 278: 32246-32250Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar); however, it is not understood nor is it modeled how this cage-like shaped domain is involved in gating. To obtain a comprehensive understanding of MscS gating, an approach that provides a broader search in a model-independent manner is required.Until now, bacterial MS channels have been functionally characterized in vivo mostly by isolation of so-called “gain-of-function” (GOF) and “loss-of-function” (LOF) mutants. GOF mutants inhibited cell growth because they opened at a lower threshold of activation. On the other hand, LOF mutants were less effective in protecting cells against osmotic down shocks because of impaired opening. Multiple mutants of MscL exhibiting GOF phenotype have been isolated from the random mutant library (24Ou X. Blount P. Hoffman R.J. Kung C. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 11471-11475Crossref PubMed Scopus (167) Google Scholar). The isolation was possible by screening for clones that block bacterial growth when overexpressed. This allowed mapping the channel gate even before its crystal structure was known. Similar approaches applied to MscS have been less successful because only one GOF mutant was found (V40D) (25Okada K. Moe P.C. Blount P. J. Biol. Chem. 2002; 277: 27682-27688Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Analysis of LOF mutants of MscL isolated through random and scanning mutagenesis with mutations in the periplasmic rim of its funnel (26Yoshimura K. Nomura T. Sokabe M. Biophys. J. 2004; 86: 2113-2120Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) indicated the importance of the lipid-protein interaction. Lipid-protein interactions have been also invoked as crucial in channel function in the study in which externally exposed amino acids from TM1/2 of MscS were mutated to arginines. In this study, few LOF mutants were identified (27Nomura T. Sokabe M. Yoshimura K. Biophys. J. 2006; 91: 2874-2881Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar).We used a genetic complementation approach in Escherichia coli strains deficient in potassium transport systems (e.g. LB2003 or TK2446). These strains cannot grow on a media with low (1–10 mm) potassium (28Dosch D.C. Helmer G.L. Sutton S.H. Salvacion F.F. Epstein W. J. Bacteriol. 1991; 173: 687-696Crossref PubMed Google Scholar), but expression of potassium channels or transporters can restore their growth (29Parfenova L.V. Crane B.M. Rothberg B.S. J. Biol. Chem. 2006; 281: 21131-21138Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). This strategy was successful for isolation of potassium channels and transporters (30Sun S. Gan J.H. Paynter J.J. Tucker S.J. Physiol. Genomics. 2006; 26: 1-7Crossref PubMed Scopus (16) Google Scholar) as well as the mutations that activate potassium channels (31Irizarry S.N. Kutluay E. Drews G. Hart S.J. Heginbotham L. Biochemistry. 2002; 41: 13653-13662Crossref PubMed Scopus (56) Google Scholar, 32Paynter J.J. Sarkies P. Andres-Enguix I. Tucker S.J. Channels. 2008; 2: 413-418Crossref PubMed Scopus (14) Google Scholar). A similar approach but utilizing the Saccharomyces cerevisiae K+ transport-deficient mutant was also used to study functional substitutions in the Kir2.1 inwardly rectifying potassium channel (33Minor Jr., D.L. Masseling S.J. Jan Y.N. Jan L.Y. Cell. 1999; 96: 879-891Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The assay also worked for detecting nonphysiological pathways of potassium influx and for isolation of potassium-conducting mutants of transport proteins that are not potassium-selective. In this way single genomic mutants of MscL and ProP were isolated (34Buurman E.T. McLaggan D. Naprstek J. Epstein W. J. Bacteriol. 2004; 186: 4238-4245Crossref PubMed Scopus (40) Google Scholar), and seven mutants of MscK, a MscS paralog, were tested (35Li C. Edwards M.D. Jeong H. Jeong H. Roth J. Booth I.R. Mol. Microbiol. 2007; 64: 560-574Crossref PubMed Scopus (20) Google Scholar). This was possible due to the high inward potassium electrochemical gradient, so any “leaky” protein could provide a route for potassium influx. This strategy may therefore result in isolation of mutants with a variety of functional changes in protein. We have been able to isolate a plethora of mutations that cause MscS to leak potassium. After phenotypic analysis, we have also been able to assign the role of several of them to the particular functional state of the channel.Our data presented here indicate the following: (i) the lower part of TM3 (TM3b) and the β domain are tightly packed in the closed and desensitized/inactivated states; (ii) they dissociate upon opening suggesting that MscS leaks potassium in the desensitized/inactivated state, and (iii) this supports the previous proposal that inactivation from the open state involves uncoupling of TM1/2 from TM3 and identifies residues important for the TM1/2-TM3 interaction. The list of mutants isolated in the screen provides mutant candidates for further detailed studies by means of electrophysiology, EPR, and crystallography.RESULTSWe used a genetic complementation approach in the E. coli strains deficient in potassium transport (TK2446 and LB2003), which cannot grow on a media with low potassium concentration (28Dosch D.C. Helmer G.L. Sutton S.H. Salvacion F.F. Epstein W. J. Bacteriol. 1991; 173: 687-696Crossref PubMed Google Scholar). Their growth, however, could be restored by expression of an exogenous channel that allowed for an inwardly directed potassium flow (31Irizarry S.N. Kutluay E. Drews G. Hart S.J. Heginbotham L. Biochemistry. 2002; 41: 13653-13662Crossref PubMed Scopus (56) Google Scholar). We constructed a random mutant library of MscS in the pTRC99A plasmid. This library was transformed into the E. coli TK2446 and LB2003 strains, and ∼107 clones were plated on low potassium. We isolated clones that grew on 1 and 3 mm potassium, with a low concentration of IPTG (10 and 50 μm) that produced a weak induction of protein expression. We opted for weak induction because we expected that some mutants might have a GOF phenotype. These mutants could inhibit growth if overproduced. We realized that there is an optimal level of channel activity required to rescue growth (32Paynter J.J. Sarkies P. Andres-Enguix I. Tucker S.J. Channels. 2008; 2: 413-418Crossref PubMed Scopus (14) Google Scholar), and the level of protein expression should be much lower than that previously used for isolation of GOF mutants (24Ou X. Blount P. Hoffman R.J. Kung C. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 11471-11475Crossref PubMed Scopus (167) Google Scholar). Therefore, the screens were performed under low induction such that even potentially toxic mutations could be metabolically tolerated and retrieved. Our expectation was that leaky mutants, meaning channels that complement potassium uptake deficiency, would emerge from the screen.Catalog of the Mutants and Their Location on the MscS StructureWe isolated 77 unique mutants that grew on low potassium (supplemental Table S1). Among them were five single mutants, and the rest were the multiple ones bearing up to six mutations. Most of the mutations, mainly those with charged and to a lesser extent polar substitutions of hydrophobic amino acids, were found within TM1–3 helices of the channel (Fig. 1, B and C). The list of mutations (supplemental Table S2) clearly shows that mutants with mutations in TM3 were isolated most frequently. While analyzing the results of the screen, the question we are addressing is as follows. How does a single substitution of a particular amino acid contribute to the potassium leak? Because the majority of the mutants had multiple mutations, a straightforward answer would be difficult. In multiple mutants, one or more of the mutations could be responsible for the phenotype, although the others could be neutral. It is possible that multiple mutations contributed additively/synergistically to the potassium leak phenotype, or alternatively, the rate of mutagenesis was too high for isolation of single mutants. To narrow down these possibilities and determine which of the mutations is responsible for the phenotype, we selected 4 single and 14 multiple mutants (with 2–5 mutations each) for detailed analysis. We chose multiple mutants in such a way as to include mutations that were scattered over diverse regions of the channel except for mutations in 26 amino acids in the unresolved N-terminal part of MscS. The mutations were studied in single mutants that were isolated in the screen or constructed (Table 1) or as a combination of substitutions in double, triple, and quadruple mutants as found in multiple mutants isolated in the screen (supplemental Table S3). It yielded a set of 66 mutants for further analysis.TABLE 1Phenotypic analysis of the single mutant set Open table in a new tab Each mutant (in strain TK2446) was tested for growth in various potassium concentrations as follows: from 1 to 10 mm in 1 mm increments and in 15 mm with 50 μm IPTG to determine permeability for K+. An additional growth test in 115 mm potassium (nonlimiting concentration) with 1 or 2 mm IPTG was performed to identify mutants with impaired growth in these conditions. Those were recognized as exhibiting a GOF phenotype, which reflects increased channel opening at resting membrane tension. In addition, each mutant was tested in strain MJF465 (ΔmscKΔmscLΔmscS) for survival of osmotic down shock. We used two protocols in the tests performed as follows: one under low expression (where there was no exogenous induction, and expression was due to promoter leak) and the other under high expression (induction by 1 mm IPTG). These survival tests enabled us to identify mutations that interfered with gating of the channel and resulted in channels that lost the ability to protect the cell from down shocks (LOF phenotype). Growth and survival tests allowed for precise detection of how a particular mutation may be involved in potassium leak (Table 1, supplemental Table S3, and supplemental Figs. S1 and S2). GOF and LOF phenotypes can give additional information on the role and involvement of particular amino acids during MscS gating. All collected data on the set of 66 mutants are included in supplemental Table S3.To be clear and concise, we will present and discuss in detail our observations on all 35 single mutants taken from the set of 66 mutants. This group of mutants includes 31 mutants that we constructed and 4 isolated directly in the screen. To gain a better insight into the correlation between location of mutation and its impact on the channel behavior, we mapped the mutations on the MscS high resolution crystal structure (Fig. 2, A–C). We also grouped single mutants according to their associated GOF or LOF phenotype. In addition, we recognized a group of single mutants with “no GOF, no LOF” phenotype, which included mutants with channels leaking potassium but whose growth and osmotic down shock survival remained unchanged.FIGURE 2Localization of mutations from the potassium-leaking single mutants within the MscS structure (Protein Data Bank code 2oau) in reference to their associated phenotypes such as cell growth and contribution to down shock protection. A, GOF mutations (excessive, growth-retarding leak); B, LOF mutations (impaired protection against down shocks), two clusters highlighted by ovals; C, localization of both GOF (green space fill) and LOF (orange space fill) mutations on the transmembrane part of the heptamer (residues 27–127) as viewed from the periplasmic side of the channel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)As detailed below, from the mutants revealed by the screen we analyzed 35 single mutants. Among them there were 7 GOF mutants (3 were described earlier (15Edwards M.D. Li Y. Kim S. Miller S. Bartlett W. Black S. Dennison S. Iscla I. Blount P. Bowie J.U. Booth I.R. Nat. Struct. Mol. Biol. 2005; 12: 113-119Crossref PubMed Scopus (105) Google Scholar, 37Miller S. Bartlett W. Chandrasekaran S. Simpson S. Edwards M. Booth I.R. EMBO J. 2003; 22: 36-46Crossref PubMed Scopus (95) Google Scholar)); 12 LOF (5 were found earlier (14Belyy V. Anishkin A. Kamaraju K. Liu N. Sukharev S. Nat. Struct. Mol. Biol. 2010; 17: 451-458Crossref PubMed Scopus (60) Google Scholar, 27Nomura T. Sokabe M. Yoshimura K. Biophys. J. 2006; 91: 2874-2881Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar)) and 16 no GOF, no LOF mutants (3 were described earlier (11Akitake B. Anishkin A. Liu N. Sukharev S. Nat. Struct. Mol. Biol. 2007; 14: 1141-1149Crossref PubMed Scopus (86) Google Scholar, 15Edwards M.D. Li Y. Kim S. Miller S. Bartlett W. Black S. Dennison S. Iscla I. Blount P. Bowie J.U. Booth I.R. Nat. Struct. Mol. Biol. 2005; 12: 113-119Crossref PubMed Scopus (105) Google Scholar)).Mutants with Substitutions in the Pore-facing Surface of TM3a Exhibit GOF PhenotypeThe screen revealed that polar substitutions of hydrophobic amino acids clustered in the upper pore-exposed part of TM3a resulted in GOF phenotype. These mutations include I97N, A98S, and A101S/A101D (Table 1 and Fig. 2A). The latter one was found only in multiple mutants and resulted in an extremely strong GOF phenotype, which prevented its analysis as a single mutation (supplemental Tables S1–S3). There was an A94D mutation also found in this cluster; however, it did not evoke GOF (Table 1). Serine substitutions of alanines 94 and 98 were introduced previously and shown to lower the threshold of the MscS activation (15Edwards M.D. Li Y. Kim S. Miller S. Bartlett W. Black S. Dennison S. Iscla I. Blount P. Bowie J.U. Booth I.R. Nat. Struct. Mol. Biol. 2005; 12: 113-119Crossref PubMed Scopus (105) Google Scholar). G104S and A106T mutants with mutations in the middle part of the pore-exposed surface of TM3a did not exhibit any obvious growth phenotype. They grew only on a very high potassium concentration (Table 1). The gate mutations (L109P/L109Q) were isolated in multiple mutants; however, we were unable to analyze them as single mutants due to extreme GOF phenotype.Based on previous findings and the results presented here, we think that, in general, this category of mutations pinpoints regions and residues affecting the channel opening. As a consequence, the channels carrying mutations in the pore region have a lower threshold of opening and a higher probability of opening in vivo than the wild type MscS.Mutations at the TM1/2-TM3 Interface Lead to LOF PhenotypeWe found mutants leaking potassium but showing LOF in osmotic shock experiments. The mutations leading to this phenotype were as follows: V65D, A85T, L86N, and G90A on TM2; V96D on TM3a; and L115Q, A119D, and G121D on TM3b (Fig. 2B). A85T does not complement potassium transport deficiency in concentrations up to 15 mm [K+] (Table 1) but dramatically enhances potassium leak in the double mutant A85T/G121D (supplemental Table S3).Interestingly, all but one of these mutations point toward the TM1/TM2-TM3 interface, which is in contrast to GOF mutations that point toward the pore (Fig. 2C). The only LOF-inducing single mutation found beyond this interface was N167Y/N167I localized in the β domain (Fig. 2B). The mutations in TM1/2 and TM3 form two clusters (Fig. 2B). One (upper) cluster is found in the helical turn region around Gly-90, which is a hinge for TM1/2 swinging motion upon the channel transition between open and inactivated states (2Bass R.B. Strop P. Barclay M. Rees D.C. Science. 2002; 298: 1582-1587Crossref PubMed Scopus (493) Google Scholar). The mutation G90S might decrease hinge flexibility and/or increase hinge angle leading to the detaching of the TM1/2 paddle from the TM3 barrel. This possibility would lead to an idea that the resting conformation of mutated channels may resemble an inactivated state of the wild type channel. The other mutations in this region are V96D, A85T, and L86N. Mutations in the 85 and 86 positions were previously found to result in LOF phenotype, and this was interpreted as an effect of improper lipid-protein interaction (27Nomura T. Sokabe M. Yoshimura K. Biophys. J. 2006; 91: 2874-2881Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Our data presented in this paper and other recently published results (14Belyy V. Anishkin A. Kamaraju K. Liu N. Sukharev S. Nat. Struct. Mol. Biol. 2010; 17: 451-458Crossref PubMed Scopus (60) Google Scholar) suggest that in potassium-leaking mutants, LOF phenotype is rather an effect of broken or loosened protein-protein interactions.The lower cluster of mutations is located in the crevice between the TM1/2 paddle and the outer (facing the paddle) surface of TM3b (Fig. 2B) and includes V65D on TM2 as well as L115Q and A119D on TM3b. These two structures interact in a modeled MscS closed conformation (12Anishkin A. Akitake B. Sukharev S. Biophys. J. 2008; 94: 1252-1266Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and form a hydrophobic contact between Val-65, Phe-68, and Leu-69 on TM2 and Leu-111 and Leu-115 on TM3 (38Anishkin A. Sukharev S. J. Biol. Chem. 2009; 284: 19153-19157Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The interaction of the TM1/2 paddle and TM3 is also critical for force transmission from the membrane to the gate and activation of the channel (14Belyy V. Anishkin A. Kamaraju K. Liu N. Sukharev S. Nat. Struct. Mol. Biol. 2010; 17: 451-458Crossref PubMed Scopus (60) Google Scholar). Any perturbation of the contact between interacting residues leads in fact to the inactivated conformation in which the channel is pressure-insensitive (14Belyy V. Anishkin A. Kamaraju K. Liu N. Sukharev S. Nat. Struct. Mol. Biol. 2010; 17: 451-458Crossref PubMed Scopus (60) Google Scholar).To support the notion that channels with this class of mutations (leaking potassium and showing LOF) reside in vivo in an inactivated state, we chose V65D, L115Q, and A119D mutants for the electrophysiological experiments. We detected channel activity of the V65D mutant in the strain MJF465; however, the number of channels was significantly lower than in the case of the wild type MscS expressed under the same conditions (0–5 V65D channels versus 50–100 wild type channels), and the channel showed fast inactivating, flickery behavior indicating a tendency for closure (supplemental Fig. S3). While testing the L115Q mutant in the MJF465 strain (ΔmscKΔmscLΔmscS), we were unable to detect any mechanosensitive channel activity that could be attributed to MscS channels (data not shown). This observation is consistent with a very strong LOF phenotype (Table 1) that may be an effect of a persistent inactivation of the channel. The L115Q mutant, similarly to A119D, was very proficient in potassium leak (growth on 1 mm K+), but unlike A119D, it was completely deficient in protection against osmotic down shocks (Table 1). The mutant A119D in TM2 showed very similar activities to those recorded from V65D when expressed in the strain MJF465 (supplemental Fig. S3). To measure a relative threshold of channel activation, A119D was expressed in the strain MJF429 (ΔmscKΔmscS). Its activities (if any) overlapped with the activities of MscL whose threshold of activation is significantly higher than that of the wild type MscS (data not shown). This indicates that the mutant channel has a higher activation threshold and points to impaired force transmission between the TM1/2 paddle and the TM3 gate. The level of mechanosensitive activity of the LOF mutants correlates with the severity of LOF phenotype but not with the level of MscS expression. The level of expression of the L115Q mutant is comparable with the level of expression of A119D, but A119D shows channel activity and L115Q does not (supplemental Fig. S4).The mutants found in this screen confirm recent findings that serine substitutions of the residues in the TM1/2-TM3 crevices (Val-65, Phe-68, Leu-111, and Leu-115) result in channels exhibiting partial LOF phenotype (with L115S giving the strongest LOF phenotype), are inactivated without opening (silent inactivation), and show high thresholds of activation, fast inactivation, and flickery behavior (14Bel" @default.
• W1657709899 created "2016-06-24" @default.
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• W1657709899 creator A5033638574 @default.
• W1657709899 creator A5067612021 @default.
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• W1657709899 date "2011-01-01" @default.
• W1657709899 modified "2023-10-18" @default.
• W1657709899 title "Genetic Screen for Potassium Leaky Small Mechanosensitive Channels (MscS) in Escherichia coli" @default.
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