Intersubunit metal bridges can form between A364C and Q476C in the closed state. (A) Representative current traces were recorded in the absence (black) or presence (red) of 1 µM Cd²⁺ from excised inside-out patches expressing one of the tandem dimers (364C476C_364C476C, WT_364C476C, or 364C_476C). Currents were elicited by voltage steps to −120 mV from a holding potential of 0 mV, followed by a voltage step to +50 mV. Currents from a 2-s (364C476C_364C476C) or 1-s (WT_364C476C and 364C_476C) hyperpolarizing pulse in the presence of Cd²⁺ are superimposed on the control current traces to highlight the slowing of the activation kinetics by Cd²⁺. (B) Effects of Cd²⁺ on the average rise time of currents produced by the intersubunit (364C_476C) and intrasubunit (WT_364C476C) tandem dimers. (C) The time course of activation was fit to a double-exponential function, and the time constants for the two components, along with the relative proportion of the slow component, are shown. The slowing of activation kinetics was mainly caused by an increase in the proportion of the slow time constant (tau).