Figure 8. Zinc accessibility experiments with V440C–I1287C channels.

(a) Inward sodium current from V440C–I1287C channels in response to 40-ms test pulses to −20 mV was recorded before and after multiple treatments of 10 μM zinc for 2 s each while the channels were held closed. Ten seconds elapsed between each test pulse. The red trace indicates the final test pulse after the cumulative exposure time. The decrease in current is comparable to that seen for WSW in both closed- and open-state experiments. (b) The effect of zinc application onto open channels was assessed by giving a test pulse to −20 mV for 20 ms before zinc treatment (black trace), a 5-s recovery at −120 mV, a 100-ms exposure to 10 μM zinc while the channels were held open at −20 mV (orange line), a 40-s washout and recovery period at −120 mV, followed by another test pulse to −20 mV for 20 ms (red trace). No further decrease in current is observed after a second round of zinc treatment. Sodium current recorded during the first exposure to zinc shows no sign of inhibition, suggesting that zinc does not form a bridge with V440C and I1287C while channels are open, but does so after entering the pore and channel closure. The data are representative of three separate experiments.