Gating of the Large Mechanosensitive Channel In Situ: Estimation of the Spatial Scale of the Transition from Channel Population Responses

Abstract

Physical expansion associated with the opening of a tension-sensitive channel has the same meaning as gating charge for a voltage-gated channel. Despite increasing evidence for the open-state conformation of MscL, the energetic description of its complex gating remains incomplete. The previously estimated in-plane expansion of MscL is considerably smaller than predicted by molecular models. To resolve this discrepancy, we conducted a systematic study of currents and dose-response curves for wild-type MscL in Escherichia coli giant spheroplasts. Using the all-point histogram method and calibrating tension against the threshold for the small mechanosensitive channel (MscS) in each patch, we found that the distribution of channels among the subconducting states is significantly less dependent on tension than the distribution between the closed and conducting states. At -20 mV, all substates together occupy approximately 30% of the open time and reduce the mean integral current by approximately 6%, essentially independent of tension or P(o). This is consistent with the gating scheme in which the major rate-limiting step is the transition between the closed state and a low-conducting substate, and validates both the use of the integral current as a measure of P(o), and treatment of dose-response curves in the two-state approximation. The apparent energy and area differences between the states deltaE and deltaA, extracted from 29 independent dose-response curves, varied in a linearly correlated manner whereas the midpoint tension stayed at approximately 10.4 mN/m. Statistical modeling suggests slight variability of gating parameters among channels in each patch, causing a strong reduction and correlated spread of apparent deltaE and deltaA. The slope of initial parts of activation curves, with a few channels being active, gave estimates of deltaE = 51 +/- 13 kT and deltaA = 20.4 +/- 4.8 nm(2), the latter being consistent with structural models of MscL, which predict deltaA = 23 nm(2).

Lactococcus lactis Uses MscL as Its Principal Mechanosensitive Channel

“…Open dwell times for MscL Ll 6H were short compared with MscL Ec and most other MscL homologues studied to date, giving rise to a "flickery" appearance in channel recordings. The activation thresholds of both L. lactis channels were comparable with those of the E. coli homologues, as were the channel substates observed for MscL Ll (43). We also showed that MscL Ll could be activated by modifying cysteine residues with MTSET (G20C or V21C) at the constriction site of the pore, demonstrating that MscL from L. lactis functions very much like MscL from E. coli.…”

Atomistic insight into lipid translocation by a TMEM16 scramblase

“…This deformation thins the membrane by 36%, thus providing a shorter pathway for a hydrophilic headgroup to cross. The computed membrane distortion energy value for nhTMEM16 is 8-20 times higher than theoretical and experimental values for gramicidin (25,35) and rhodopsin (36) and about twice as high as values estimated for the membrane associated gating energy of the mechanosensitive channel of large conductance (MscL) (37,38). Because the leaflet-to-leaflet distance is still large (18.3Å), thinning is not likely the sole mechanism.…”

Feeling the hidden mechanical forces in lipid bilayer is an original sense

“…This expansion in area (ΔA) requires 220 pN to stretch its 22-nm perimeter, doing 2 × 10 −23 J (50 kT) work. Half of the channels are open at 10-12 pN/nm, not far from the lytic tension of lipid bilayer (22,23). The ΔA estimated from activities approximates the structural estimate from crystallography (24) and electron paramagnetic resonance spectroscopy (25).…”