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Department of Biology, University of Maryland College Park, Maryland
Correspondence: Address reprint requests to Sergei Sukharev, University of Maryland, Dept. of Biology, Bldg. 144, College Park, MD 20742. Tel.: 301-405-6923; Fax: 301-314-9358; E-mail: ss311{at}umail.umd.edu.
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 
30% of the open time and reduce the mean integral current by 6%, essentially independent of tension or Po. 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 Po, and treatment of dose-response curves in the two-state approximation. The apparent energy and area differences between the states 
E and A, extracted from 29 independent dose-response curves, varied in a linearly correlated manner whereas the midpoint tension stayed at 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 E and A. The slope of initial parts of activation curves, with a few channels being active, gave estimates of E = 51 ± 13 kT and A = 20.4 ± 4.8 nm2, the latter being consistent with structural models of MscL, which predict A = 23 nm2.
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