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      Energetic and Spatial Parameters for Gating of the Bacterial Large Conductance Mechanosensitive Channel, MscL

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          Abstract

          MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient ( P) to vary the tension ( T). The tension was calculated 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 dependence on T, with a midpoint ( T 1/2) of 11.8 dyn/cm. The maximal slope sensitivity of P o/ P c was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was ΔE = 18.6 k B T. If the mechanosensitivity arises from tension acting on a change of in-plane area (Δ A), the free energy, TΔ A, would correspond to Δ A = 6.5 nm 2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest ΔA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm 2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to Δ A. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.

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          Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective.

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            Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events.

            We present here a maximal likelihood algorithm for estimating single-channel kinetic parameters from idealized patch-clamp data. The algorithm takes into account missed events caused by limited time resolution of the recording system. Assuming a fixed dead time, we derive an explicit expression for the corrected transition rate matrix by generalizing the theory of Roux and Sauve (1985, Biophys. J. 48:149-158) to the case of multiple conductance levels. We use a variable metric optimizer with analytical derivatives for rapidly maximizing the likelihood. The algorithm is applicable to data containing substates and multiple identical or nonidentical channels. It allows multiple data sets obtained under different experimental conditions, e.g., concentration, voltage, and force, to be fit simultaneously. It also permits a variety of constraints on rate constants and provides standard errors for all estimates of model parameters. The algorithm has been tested extensively on a variety of kinetic models with both simulated and experimental data. It is very efficient and robust; rate constants for a multistate model can often be extracted in a processing time of approximately 1 min, largely independent of the starting values.
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              Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle.

              The membrane of tissue-cultured chick pectoral muscle contains an ionic channel which is activated by membrane stretch. Nicotinic channels and Ca2+-activated K+ channels are not affected by stretch. In 150 mM-external K+ and 150 mM-internal Na+ the channel has a conductance of 70 pS, linear current-voltage relationship between -50 and -140 mV and a reversal potential of +30 mV. Kinetic analysis of single-channel records indicates that there are one open (O) and three closed (C) states. The data can be fitted by the reaction scheme: C1-C2-C3-O. Only the rate constant that governs the C1-C2 transition (k1,2) is stretch-sensitive. None of the rates are voltage-sensitive. The rate constant k1,2 varies with the square of the tension as k1, 2 = k0 X e alpha T2, where alpha is a constant describing the sensitivity to stretch and T is the tension. A typical value of alpha is 0.08 (dyn cm-1)-2. Following exposure to cytochalasin B the channel becomes more sensitive to stretch. The stretch-sensitivity constant, alpha, increases from 0.08 to 2.4 (dyn cm-1)-2. The probability of the channel being open is strongly dependent upon the extracellular K+ concentration. With a suction of 2 cmHg the probability increases from 0.004 in normal saline (5 mM-K+) to 0.26 in 150 mM-K+. The channel appears to gather force from a large area of membrane (greater than 3 X 10(5) A2), probably by a cytochalasin-resistant cytoskeletal network.
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                Author and article information

                Journal
                J Gen Physiol
                The Journal of General Physiology
                The Rockefeller University Press
                0022-1295
                1540-7748
                1 April 1999
                : 113
                : 4
                : 525-540
                Affiliations
                From the [* ]Department of Biology, University of Maryland, College Park, Maryland 20742; []Department of Physiology and Biophysical Sciences, State University of New York, Buffalo, New York 14214; and [§ ]Laboratory of Molecular Biology and Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706
                Author notes

                Address correspondence to Wade J. Sigurdson, Ph.D., Dept. Physiology and Biophysics, 320 Cary Hall, SUNY at Buffalo, Buffalo, NY 14214. Fax: 716-829-2028; E-mail: wjs@ 123456buffalo.edu

                Article
                10.1085/jgp.113.4.525
                2217166
                10102934
                28055514-e2ce-4a46-be0a-24e0080ba72a
                Copyright @ 1999
                History
                : 17 June 1998
                : 3 February 1999
                Categories
                Article

                Anatomy & Physiology
                mechanical,membrane,tension,stretch,kinetics
                Anatomy & Physiology
                mechanical, membrane, tension, stretch, kinetics

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