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.

Linear copolymers of ethylene and acrylonitrile were prepared using palladium complexes bearing phosphine-sulfonate bidentate ligands. Acrylonitrile units located in the linear polyethylene backbones were detected for the first time by 13C NMR spectroscopy. Catalyst systems employing isolated palladium complexes such as 3 showed much higher activity for the copolymerization than the in situ generation procedures, and molecular weight of the copolymers and acrylonitrile incorporation were dependent on the palladium complexes. Obtained linear copolymers of ethylene and acrylonitrile melt at higher temperature than branched copolymers.

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, MscL's 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.

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.

Smartphones are very common devices in daily life that have a built-in tri-axial accelerometer. Similar to previously developed accelerometers, smartphones can be used to assess gait patterns. However, few gait analyses have been performed using smartphones, and their reliability and validity have not been evaluated yet. The purpose of this study was to evaluate the reliability and validity of a smartphone accelerometer. Thirty healthy young adults participated in this study. They walked 20 m at their preferred speeds, and their trunk accelerations were measured using a smartphone and a tri-axial accelerometer that was secured over the L3 spinous process. We developed a gait analysis application and installed it in the smartphone to measure the acceleration. After signal processing, we calculated the gait parameters of each measurement terminal: peak frequency (PF), root mean square (RMS), autocorrelation peak (AC), and coefficient of variance (CV) of the acceleration peak intervals. Remarkable consistency was observed in the test-retest reliability of all the gait parameter results obtained by the smartphone (p<0.001). All the gait parameter results obtained by the smartphone showed statistically significant and considerable correlations with the same parameter results obtained by the tri-axial accelerometer (PF r=0.99, RMS r=0.89, AC r=0.85, CV r=0.82; p<0.01). Our study indicates that the smartphone with gait analysis application used in this study has the capacity to quantify gait parameters with a degree of accuracy that is comparable to that of the tri-axial accelerometer.

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.

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 (⌬N-MSC1) reversed this effect. ⌬N-MSC1 was found to open in response to membrane stretch and displayed a preference for anions over cations as permeable ions. ⌬N-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.MscS ͉ hysteresis ͉ heterogeneous expression ͉ knockdown ͉ green algae M echanosensation is typically involved in auditory perception, touch sensation, proprioception, and gravity perception. The mechanoreceptor potential in sensory cells is mediated by mechanosensitive channels, which are activated by stretching or deformation of the membrane. The patch-clamp technique has revealed that mechanosensitive channels are present in almost every cell type, not just sensory cells. In fact, mechanosensitive channels were first recorded with the patch-clamp method in the muscle cell (1). Mechanosensitive channels in nonsensory cells are believed to be responsible for monitoring cell deformation evoked by contact with surrounding materials and by changes in osmolarity.Bacteria harbor the mechanosensitive channels MscS and MscL, which act to protect against hypoosmotic downshock (2). The presence of MscS homologs in virtually all eubacteria and archaea suggests that MscS has an essential function in prokaryotes, but whether every MscS homolog serves as a mechanosensitive channel is uncertain. For instance, although there are at least six MscS homologs in the Escherichia coli genome, only two of them (MscS and MscK) have been detected by electrophysiological methods (3, 4). Recent genomic projects on eukaryotes have uncovered the presence of MscS homologs in some plants (Arabidopsis and Oryza), protists (Chlamydomonas), and fungi (Schizosaccharomyces). Curiously, MscS homologs have not been found in animals (5, 6). The MscS homologs of Arabidopsis (MSL2 and MSL3) have been observed as one or two foci on the plastid envelope, and their double knockout results in an abnormal size and shape of the plastids (6). This finding suggests that one of the possible functions of the MscS homologs is to control the size and shape of the intr...

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.