Takeshi Nomura

The bacterial mechanosensitive channel of large conductance MscL is activated exclusively by increased tension in the membrane bilayer. Despite many proposed models for MscL opening, its precise mechano-gating mechanism, particularly how the received force at the tension sensor transmits to the gate remains incomplete. Previous studies have shown that along with amphipathic N-terminus located near the cytoplasmic surface of the membrane, Phe78 residue near the outer surface also acts as a "tension sensor," while Gly22 is a central constituent of the "hydrophobic gate." Present study focused on elucidating the force transmission mechanism from the sensor Phe78 in the outer transmembrane helix (TM2) to the gate in the inner transmembrane helix (TM1) of MscL by applying the patch clamp and molecular dynamics (MD) simulations to the wild type MscL channel and its single mutants at the sensor (F78N), the gate (G22N) and their combination (G22N/F78N) double mutant. F78N MscL resulted in a severe loss-of-function, while G22N MscL caused a gain-of-function channel exhibiting spontaneous openings at the resting membrane tension. We initially speculated that the spontaneous opening in G22N mutant might occur without tension acting on Phe78 residue. To test this hypothesis, we examined the (G22N/F78N) double mutant, which unexpectedly exhibited neither spontaneous activity nor activity by a relatively high membrane tension. To understand the underlying mechanism, we conducted MD simulations and analyzed the force transduction pathway. Results showed that the mutation at the tension sensor (F78N) in TM2 caused decreased interaction of this residue not only with lipids, but also with a group of amino acids (Ile32-Leu36-Ile40) in the neighboring TM1 helix, which resulted in an inefficient force transmission to the gate-constituting amino acids on TM1. This change also induced a slight tilting of TM1 towards the membrane plane and decreased the size of the channel pore at the gate, which seems to be the major mechanism for the inhibition of spontaneous opening of the double mutant channel. More importantly, the newly identified interaction between the TM2 (Phe78) and adjacent TM1 (Ile32-Leu36-Ile40) helices seems to be an essential force transmitting mechanism for the stretch-dependent activation of MscL given that substitution of any one of these four amino acids with Asn resulted in severe loss-of-function MscL as reported in our previous work.

Ion channel data recorded using the patch clamp technique are low-pass filtered to remove high-frequency noise. Almanjahie et al. (Eur Biophys J 44:545-556, 2015) based statistical analysis of such data on a hidden Markov model (HMM) with a moving average adjustment for the filter but without correlated noise, and used the EM algorithm for parameter estimation. In this paper, we extend their model to include correlated noise, using signal processing methods and deconvolution to pre-whiten the noise. The resulting data can be modelled as a standard HMM and parameter estimates are again obtained using the EM algorithm. We evaluate this approach using simulated data and also apply it to real data obtained from the mechanosensitive channel of large conductance (MscL) in Escherichia coli. Estimates of mean conductances are comparable to literature values. The key advantages of this method are that it is much simpler and computationally considerably more efficient than currently used HMM methods that include filtering and correlated noise.

Tension in cell membranes is closely related to various cellular events, including cell movement and morphogenesis. Therefore, modulation of membrane tension can be a new approach for manipulating cellular events. Here, we show that an amphipathic peptide derived from the influenza M2 protein (M2[45-62]) yields lamellipodia at multiple sites in the cell. Effect of M2[45-62] on cell membrane tension was evaluated by optical tweezer. The membrane tension sensor protein FBP17 was involved in M2[45-62]-driven lamellipodium formation. Lysine-to-arginine substitution in M2[45-62] further enhanced its activity of lamellipodium formation. M2[45-62] had an ability to reduce cell motility, evaluated by scratch wound migration and transwell migration assays. An increase in neurite outgrowth was also observed after treatment with M2[45-62]. The above results suggest the potential of M2[45-62] to modulate cell movement and morphology by modulating cell membrane tension.

The large conductance mechanosensitive channel (MscL), acts as an osmoprotective emergency valve in bacteria by opening a large, water- filled pore in response to changes in membrane tension. In its closed configuration, the last 36 residues at the C- terminus form a bundle of five a- helices co- linear with the five- fold axis of symmetry. Here, we examined the structural dynamics of the C- terminus of EcMscL using site- directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy. These experiments were complemented with computational modelling including molecular dynamics (MD) simulations and finite element (FE) modelling. Our results show that under physiological conditions, the C- terminus is indeed an a- helical bundle, located near the five- fold symmetry axis of the molecule. Both experiments and computational modelling demonstrate that only the top part of the C- terminal domain (from the residue A110 to E118) dissociates during the channel gating, while the rest of the C- terminus stays assembled. This result is consistent with the view that the C- terminus functions as a molecular sieve and stabilizer of the oligomeric MscL structure as previously suggested.

The detailed single-channel gating kinetics of mouse pannexin 1 (mPanx1) remains unknown, although mPanx1 is reported to be a voltage-activated anion-selective channel. We investigated characteristics of single-channel conductances and opening and closing rates of mPanx1 using patch-clamp techniques. The unitary current of mPanx1 shows outward rectification with single-channel conductances of similar to 20 pS for inward currents and similar to 80 pS for outward currents. The channel open time for outward currents (Cl- influx) increases linearly as the amplitude of single channel currents increases, while the open time for inward currents (Cl- efflux) is constant irrespective of changes in the current amplitude, as if the direction and amplitude of the unitary current regulates the open time. This is supported by further observations that replacement of extracellular Cl- with gluconate(-) diminishes the inward tail current (Cl- efflux) at a membrane potential of -100 mV due to the lowered outward current (gluconate(-) influx) at membrane potential of 100 mV. These results suggest that the direction and rate of charge-carrier movement regulate the open time of mPanx1, and that the previously reported voltage-dependence of Panx1 channel gating is not directly mediated by the membrane potential but rather by the direction and amplitude of currents through the channel.