FIGURE 3.
A, histogram of the ΔG for activation and ΔG for inactivation gating (respectively, ΔGact and ΔGinact) of the glycine mutants between positions 592 and 602 in the S4S5 linker in domain II (IIS4S5) of CaV2. 3. ΔGact and ΔGinact were obtained as described under “Experimental Procedures.” The ΔGact of L596G, M600G, and S602G were significantly more negative than CaV2.3 wt at p < 0.001 (one-way analysis of variance). In contrast, the ΔGinact of V594G, S595G, S598G, K601G, and S602G were significantly more positive than CaV2.3 wt at p < 0.01 (one-way analysis of variance). B, gating currents were measured with the cut-open oocyte technique. Normalized gating charge was plotted against test voltages for CaV2.3 wt and L596G. The voltage dependence of the gating charge or V0.5,Q values were obtained from fitting the traces with a single Boltzmann equation as described under “Experimental Procedures.” V0.5,Q values were 6 ± 1 mV (n = 19) for CaV2.3 wt and −24 ± 1 mV (n = 10) for L596G. C, the mean time constants of deactivation (obtained from fitting current traces from monoexponential functions) for CaV2.3 wt and the IIS4S5 glycine mutants (592–602) were plotted against the test potential. Numerical values are found in Table 1. L596G yielded a significantly longer deactivation time constant (p < 0.001) than the CaV2.3 wt at all voltages more positive than −80 mV. D, representative tail currents for CaV2.3 wt and L596G. Currents were activated during 8-ms conditioning depolarizing voltage pulses at 0 mV for the wt and −40 mV for L596G. Deactivation was recorded during subsequent repolarization to test potentials from −120 to 0 mV (10-mV increments) as illustrated on the pulse protocol.