Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by t ension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified Msc L reconstituted in liposomes, we recorded single channel currents and varie d the pressure gradient (P) to vary the tension (T). The tension was calcul ated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (P-o) has a steep sigmoidal de pendence on T, with a midpoint (T-1/2) of 11.8 dyn/cm. The maximal slope se nsitivity of P-o/P-c was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distr ibution, the energy difference between the closed and fully open states in the unstressed membrane was Delta E = 18.6 k(B) T. If the mechanosensitivit y arises from tension acting on a change of in-plane area (Delta A), the fr ee energy, T Delta A, would correspond to Delta A = 6.5 nm(2). MscL is not a binary channel, but has four conducting states and a closed state. Most t ransition rates are independent of tension, but the rate-limiting step to o pening is the transition between the closed state and the lowest conductanc e substate. This transition thus involves the greatest Delta A. When summed over all transitions, the in-plane area change from closed to fully open w as 6 nm(2), agreeing with the value obtained in the two-state analysis. Ass uming a cylindrical channel, the dimensions of the (fully open) pore were c omparable to Delta A. Thus, the tension dependence of channel gating is pri marily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to in volve conformational changes internal to the channel that don't involve cha nges in area.