Closure of open channels is slowed by millimolar extracellular Ca²⁺. (A) Representative time courses of macroscopic current decay upon sudden removal of intracellular Ca²⁺, with ∼4 µM (left trace) and 1 mM (right trace) free Ca²⁺ in the pipette solution. Smooth lines are single-exponential fits, with time constants shown. Inset shows mean ± SEM decay time constants for the above two conditions. (B) Cartoon interpretation of channel closing kinetics when intracellular Ca²⁺ is washed away. (top) In the absence of extracellular Ca²⁺ the activating sites rapidly lose Ca²⁺ yielding unliganded channels that close fast (Fig. 5 D, solid arrow). (bottom) In the presence of extracellular Ca²⁺ the activating sites, which are located in a deep vestibule near the pore entrance, remain liganded because of Ca²⁺ ions entering through the open pore. Thus, channels close at the slow rate characteristic of fully liganded channels (Fig. 5 D, dotted arrow). Once channels have closed, the activating sites, which are located intracellularly of the gate, are cut away from Ca²⁺, hence channels remain shut. Note a few occasional reopening events in the right current trace in A, which typically follow brief closures (e.g., blue arrow), suggesting that some fraction of very brief closed events is too short to allow dissociation of Ca²⁺ from the activating sites. Such occasional reopenings might explain why in the presence of 1 mM of extracellular Ca²⁺ and 4.4 µM [Ca²⁺]_i the macroscopic current relaxations yield a slightly smaller estimate of closing rate than the steady-state data (Fig. 5 C, white circle vs. diamond for 4.4 µM [Ca²⁺]_i).