Kenjiro Yoshimura

Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity.

Mechanosensitive channel large (MscL) encodes the large conductance mechanosensitive channel of the Escherichia coli inner membrane that protects bacteria from lysis upon osmotic shock. To elucidate the molecular mechanism of MscL gating, we have comprehensively substituted Gly(22) with all other common amino acids. Gly(22) was highlighted in random mutagenesis screens of E. coli MscL (, Proc. Nat. Acad. Sci. USA. 95:11471-11475). By analogy to the recently published MscL structure from Mycobacterium tuberculosis (, Science. 282:2220-2226), Gly(22) is buried within the constriction that closes the pore. Substituting Gly(22) with hydrophilic residues decreased the threshold pressure at which channels opened and uncovered an intermediate subconducting state. In contrast, hydrophobic substitutions increased the threshold pressure. Although hydrophobic substitutions had no effect on growth, similar to the effect of an MscL deletion, channel hyperactivity caused by hydrophilic substitutions correlated with decreased proliferation. These results suggest a model for gating in which Gly(22) moves from a hydrophobic, and through a hydrophilic, environment upon transition from the closed to open conformation.

Chemically Charging the Pore Constriction Opens the Mechanosensitive Channel MscL

MscL is a bacterial mechanosensitive channel that protects the cell from osmotic downshock. We have previously shown that substitution of a residue that resides within the channel pore constriction, MscLs Gly-22, with all other 19 amino acids affects channel gating according to the hydrophobicity of the substitution (). Here, we first make a mild substitution, G22C, and then attach methanethiosulfonate (MTS) reagents to the cysteine under patch clamp. Binding MTS reagents that are positively charged ([2-(trimethylammonium)ethyl] methanethiosulfonate and 2-aminoethyl methanethiosulfonate) or negatively charged (sodium (2-sulfonatoethyl)methanethiosulfonate) causes MscL to gate spontaneously, even when no tension is applied. In contrast, the polar 2-hydroxyethyl methanethiosulfonate halves the threshold, and the hydrophobic methyl methanethiolsulfonate increases the threshold. These observations indicate that residue 22 is in a hydrophobic environment before gating and in a hydrophilic environment during opening to a substate, a finding consistent with our previous study. In addition, we have found that cysteine 22 is accessible to reagents from the cytoplasmic side only when the channel is opened whereas it is accessible from the periplasmic side even in the closed state. These results support the view that exposure of hydrophobic surfaces to a hydrophilic environment during channel opening serves as the barrier to gating.

Molecular and electrophysiological characterization of a mechanosensitive channel expressed in the chloroplasts of Chlamydomonas

MscS is a mechanosensitive channel that is ubiquitous among bacteria. Recent progress in the genome projects has revealed that homologs of MscS are also present in eukaryotes, but whether they operate as ion channels is unknown. In this study we cloned MSC1, a homolog of MscS in Chlamydomonas, and examined its function when expressed in Escherichia coli. Full-length MSC1 was not functional when expressed in E. coli cells. However, removal of the N-terminal signal sequence (ΔN-MSC1) reversed this effect. ΔN-MSC1 was found to open in response to membrane stretch and displayed a preference for anions over cations as permeable ions. ΔN-MSC1 exhibited marked hysteretic behavior in response to ascending and descending stimuli. That is, channel gating occurred in response to significant stimuli but remained open until the stimulus was almost completely removed. Indirect immunofluorescence revealed that MSC1 is present as punctate spots in the cytoplasm and chloroplasts. Moreover, knockdown of MSC1 expression resulted in the abnormal localization of chlorophyll. These findings show that MSC1 is an intracellular mechanosensitive channel and is responsible for the organization of chloroplast in Chlamydomonas.

Loss-of-Function Mutations at the Rim of the Funnel of Mechanosensitive Channel MscL

MscL is a bacterial mechanosensitive channel that is activated directly by membrane stretch. Although the gene has been cloned and the crystal structure of the closed channel has been defined, how membrane tension causes conformational changes in MscL remains largely unknown. To identify the site where MscL senses membrane tension, we examined the function of the mutants generated by random and scanning mutagenesis. In vitro (patch-clamp) and in vivo (hypoosmotic-shock) experiments showed that when a hydrophilic amino acid replaces one of the hydrophobic residues that are thought to make contact with the membrane lipid near the periplasmic end of the M1 or M2 transmembrane domain, MscL loses the ability to open in response to membrane tension. Hydrophilic (asparagine) substitution of the other residues in the lipid-protein interface did not impair the channels mechanosensitivity. These observations suggest that the disturbance of the hydrophobic interaction between the membrane lipid and the periplasmic rim of the channels funnel impairs the function of MscL.

Chlamydomonas CAV2 encodes a voltage- dependent calcium channel required for the flagellar waveform conversion.

Cilia and flagella can alter their beating patterns through changes in membrane excitation mediated by Ca(2+) influx. The ion channel that generates this Ca(2+) influx and its cellular distribution have not been identified. In this study, we analyzed the Chlamydomonas ppr2 mutant, which is deficient in the production of a flagellar Ca(2+) current and consequently has a defective photophobic response and mechanoshock response. ppr2 had a mutation in CAV2, which encodes a homolog of the alpha(1) subunit of voltage-dependent calcium channels (VDCCs). CAV2 has four domains, each with six transmembrane segments and EEEE loci in the ion-selective filter, which are typical of VDCCs in vertebrates. Interestingly, we found that CAV2 primarily localized toward the distal part of flagella. We provide evidence that CAV2 is transported toward the flagellar tip via intraflagellar transport (IFT) because CAV2 accumulated near the flagellar base when IFT was blocked. The results of this study suggest that the Ca(2+) influx of Chlamydomonas flagella is mediated by the VDCC, CAV2, whose distribution is biased to the distal region of the flagellum.

Phototactic activity in Chlamydomonas 'non-phototactic' mutants deficient in Ca2+-dependent control of flagellar dominance or in inner-arm dynein

In the mechanism underlying the phototactic behavior of Chlamydomonas, Ca2+ has been thought to control the dominance between the two flagella so as to steer the cell to correct directions. A newly isolated mutant, lsp1, that displays weak phototaxis was found to be defective in this Ca2+-dependent shift in flagellar dominance; in demembranated and reactivated cell models, the trans flagellum (the flagellum farthest from the eyespot) beat more strongly than the other (the cis flagellum) in about half of the cells regardless of the Ca2+ concentration between <10-9 M and 10-6 M, a range over which wild-type cell models display switching of flagellar dominance. This is unexpected because ptx1, another mutant that is also deficient in flagellar dominance control, has been reported to lack phototactic ability. We therefore re-examined ptx1 and another reportedly non-phototactic mutant, ida1, which lacks inner arm dynein subspecies f (also called I1). Both were found to retain reduced phototactic abilities. These results indicate that both Ca2+-dependent flagellar dominance control and inner-arm dynein subspecies f are important for phototaxis, but are not absolutely necessary. Analysis of the flagellar beat frequency in lsp1 cell models showed that both of the flagella beat at the frequency of the cis flagellum in wild type. In addition, lsp1 and ptx1 were found to be deficient in determining the sign of phototactic migration. Hence, the Ca2+-dependent flagellar dominance control detected in demembranated cells might be involved in the determination of the sign of phototaxis. The gene responsible for the lsp1 mutation was identified by phenotype rescue experiments and found to have sequences for phosphorylation.

Gating the bacterial mechanosensitive channel MscL in vivo

YggB and MscL are the major mechanosensitive channels in Escherichia coli, and each can rescue the double knockout mutant from osmotic downshock. However, the role of MscL in wild-type bacteria is in question, not only because cells without MscL survive severe osmotic downshocks, but because 1.8 times more suction is required to gate MscL than YggB under patch clamp. Here, we extend previous evidence [Ajouz, B., Berrier, C., Garrigues, A., Besnard, M. & Ghazi, A. (1998) J. Biol. Chem. 273, 26670–26674] to show that downshock gates MscL in vivo even in the presence of YggB. We have made this determination by engineering a channel we can structurally modify in vivo (Leu-19→Cys MscL). MscLs with charges in their constrictions are known to open easily and transiently to substates and stop cell growth. In this study, we use downshock to stretch this region open to allow attachment of a charged thiosulfonate reagent MTSET+, thereby creating a toxic channel. Therefore, channel opening can be monitored by loss of colony forming units. By this measure, we find that an ≈800 mmol/kg downshock from 1,200 mmol/kg medium opens Leu-19→Cys MscL in the presence of YggB, but a downshock of only ≈400 mmol/kg is required in the absence of YggB. In parallel, Leu-19→Cys MscL, stretched open by large sustained suction in the presence of MTSET+ in voltage-clamped patches, subsequently flickers open with little suction. These observations show that MscL opening is triggered by a specific downshock, even in the presence of YggB, that YggB buffers MscL gating in vivo, and that residue 19 becomes exposed upon channel opening.

Mechanoreception in motile flagella of Chlamydomonas

Ciliates and flagellates temporarily swim backwards on collision by generating a mechanoreceptor potential. Although this potential has been shown to be associated with cilia in Paramecium, the molecular entity of the mechanoreceptor has remained unknown. Here we show that Chlamydomonas cells express TRP11, a member of the TRP (transient receptor potential) subfamily V, in the proximal region of the flagella, and that suppression of TRP11 expression results in loss of the avoiding reaction. The results indicate that Chlamydomonas flagella exhibit mechanosensitivity, despite constant motility, by localizing the mechanoreceptor in the proximal region, where active bending is restricted.

Gating-associated conformational changes in the mechanosensitive channel MscL

Bacterial cells avoid lysis in response to hypoosmotic shock through the opening of the mechanosensitive channel MscL. Upon channel opening, MscL is thought to expand in the plane of the membrane and form a large pore with an estimated diameter of 3–4 nm. Here, we set out to analyze the closed and open structure of cell-free MscL. To this end, we characterized the function and structure of wild-type MscL and a mutant form of the protein (G22N MscL) that spontaneously adopts an open substate. Patch-clamp analysis of MscL that had been reconstituted into liposomes revealed that wild-type MscL was activated only by mechanical stimuli, whereas G22N MscL displayed spontaneous opening to the open substate. In accord with these results, Ca2+ influx into G22N MscL-containing liposomes occurred in the absence of mechanical stimulation. The electrophoretic migration of chemically cross-linked G22N MscL was slower than that of cross-linked wild-type MscL, suggesting that G22N MscL is in an expanded form. Finally, electron microscopy using low-angle rotary shadowing revealed the presence of a pore at the center of G22N MscL. No pore could be detected in wild-type MscL. However, wild-type MscL possessed a protrusion at one end, which was absent in G22N MscL. The deletion of carboxyl-terminal 27 residues resulted in the loss of protrusion and proper multimerization. The structures of wild-type and G22N MscL reveal that the opening of MscL is accompanied by the dissociation of a carboxyl-terminal protrusion and pore formation.

Mechanosensitivity of ion channels based on protein -lipid interactions

Ion channels form a group of membrane proteins that pass ions through a pore beyond the energy barrier of the lipid bilayer. The structure of the transmembrane segment of membrane proteins is influenced by the charges and the hydrophobicity of the surrounding lipids and the pressure on its surface. A mechanosensitive channel is specifically designed to change its conformation in response to changes in the membrane pressure (tension). However, mechanosensitive channels are not the only group that is sensitive to the physical environment of the membrane: voltage-gated channels are also amenable to the lipid environment. In this article, we review the structure and gating mechanisms of the mechanosensitive channels and voltage-gated channels and discuss how their functions are affected by the physical properties of the lipid bilayer.