Figure 4.

Function of background constructs and single-Cys mutants. Macroscopic currents conducted by (A) wild-type (WT) α, (B) pseudo-wild type (pWT) α with the native extracellular Cys mutated to Ala (i.e., C14A, C141A), and (C) pWT α with the HRV consensus site inserted between S0–S1 transmembrane segments (pWT-HRV α). Each construct was expressed in HEK293 cells, and the currents shown were in response to step depolarizations to 0, 40, and 80 mV, in 10 μM Ca²⁺. (D) For WT, pWT, and pWT-HRV in inside-out macropatches, normalized conductance is plotted against the activating voltage. The data were from the maximum of the tail currents obtained after stepping the voltage from the plotted voltage to −80 mV in nominally zero (open symbols) or 10 μM (filled symbols) Ca²⁺. The data were fit by 1/{1 + exp[−zF(V − V₅₀)/RT)] }. (E) For each of the single-Cys mutants indicated on the abscissa, V₅₀ was determined as in D in nominally 0 and 10 μM Ca²⁺. From the mean V₅₀ (n > 3) was subtracted the mean V₅₀ for pWT-HRV to obtain ΔV₅₀. (F) For each single-Cys mutant, the time course of activation at 240 mV was fit with an exponential function to estimate the rate constant for activation; the mean rate constants (n > 4) were divided by the mean rate constant for activation of pWT-HRV, and the log of this ratio is plotted. Similarly, the mean rate constants for deactivation at −80 mV were determined and divided by the mean deactivation rate constant for pWT-HRV. The log of this ratio is shown. The errors in the differences and ratios are propagated from the standard errors of the means of the arguments, and the errors in the logs are from the approximation log(x ± err) = log(x) + log(1 ± err/x) ≈ log(x) ± 0.434(err/x), where err is the standard error of the mean x.