Xenbase Image ID: 128391

Figure 1. Limiting slope estimates the effective gating charge. (A) Alignment of the S4 region (teal box) of Hv channels from C. intestinalis (Ci-Hv), Xenopus (XenHv), Mus musculus mouse (mHv), Homo sapiens (hHv), Gallus gallus (ChHv), and Danio rerio (DanHv) and S4 from Kv1.2, Shaker K, and domain IV of Nav1.4 channels. The arrow shows residues R1454 in Nav1.4 and R368 in Shaker that have been shown to move completely across the membrane during channel activation (Yang et al., 1996; Baker et al., 1998). Red letters indicate positively charged residues in S4. (B) Simulated conductance versus voltage, G(V), curve (black circles) for a three-state, two-subunit Hodgkin-Huxley type model with a gating charge of z = 2 e0 per subunit. The G(V) was fit with a Boltzmann curve, G(V) = G0/(1 + exp(−z(V − V1/2)/kT)) (zfit = 2.43 e0; dashed red line), and a Boltzmann curve to the second (zfit = 2.0 e0, resulting in a ztotal(fit) = 2 × zfit = 4.0 e0; red line) and third power (zfit = 1.87 e0, resulting in a ztotal(fit) = 3 × zfit = 5.6 e0; teal dashed line). (C) Simulated data and fits from B shown in lin-log plot (colored as in B). Also the best fit to the limiting slope at negative voltages is shown (log G(V) = constant + zlimitV/kT; zlimit(fit) = 3.8 e0; teal straight line). (D) Currents from excised patches containing WT Ci-Hv1 channels (black line) or from patches taken from uninjected oocytes (teal line) in response to slow voltage ramps (1.5 mV/s) from −60 to 0 mV and then back to −60 mV. (E) The calculated conductance (I/(V − Erev)) during the up and down ramp (data from D). Also shown is the calculated conductance from a patch from an uninjected oocyte (open circles). The currents were leak subtracted in response to ramps between −100 and −80 mV. The reversal potential Erev was estimated to −85 mV in this experiment (pHi = 5.5 and pHo = 7). Note that the conductance estimated during the up ramp overlaps with the conductance during the down ramp, showing that the channels are at equilibrium at all voltages. This shows that the speed of the ramp is slow enough to correctly estimate the conductance. (F and G) Conductance, G, measured as in D from excised patches containing WT Ci-Hv1 channels in response to slow voltage ramps (1.5 mV/s) from −60 to 50 mV. The G(V) was fit with a Boltzmann curve, G(V) = G0/(1 + exp(−z(V − V1/2)/kT)) (zfit = 3.5 e0; dashed red line), and a Boltzmann curve to the second power, G(V) = G0/(1 + exp(−z(V − V1/2)/kT))2 (zfit = 3.0 e0, resulting in a ztotal(fit) = 2 × zfit = 6.0 e0; solid red line). (G) Data and fits from F shown in lin-log plot (colored as in F). Also the best fit to the limiting slope at negative voltages is shown (log G(V) = constant + zlimitV/kT; teal straight line). zlimit(fit) = 5.3 e0. Image published in: Gonzalez C et al. (2013) © 2013 Gonzalez et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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