Fig. 5.

Charge reversal at R396 and K397 increases the P_o of SK channels in the absence of Ca²⁺. (A). Wild-type SK currents activated with a saturating 10 μM Ca²⁺ in response to a voltage ramp from -80 to 80 mV (Top). Based on the amount of total current at -80 mV and the average single-channel current amplitude at -80 mV (Fig. 2A), this patch has approximately 200 SK channels. The patch was then treated with nominally Ca²⁺-free solution (0 Ca²⁺) and current was recorded at a holding potential of -80 mV (Bottom). The dashed line indicates the expected single-channel current level. The scale bar is the same for single-channel records in A–C. (B). Single-channel recording at -80 mV from a membrane patch containing R396E/K397E channels. In the presence of 200 nM Ca²⁺, the channel opens frequently with no double opening, suggesting a single channel in the patch (Top). This channel occasionally opens in 0 Ca²⁺ (Bottom). (C). Single-channel recording from a patch containing R396E channels at -80 mV in the presence of 200 nM Ca²⁺ (Top). Based on the overall P_o and the presence of double opening, two channels are likely to be present in this patch. R396E channels occasionally open in 0 Ca²⁺ (Bottom). (D). P_o of unliganded R396E/K397E channels was estimated by the ratio between the average current level in 0 Ca²⁺ (Bottom) and the average current in 7.7 μM Ca²⁺ at -80 mV (Top).