Figure 3.

Slow polyamine unbinding from the pore of mutant Kir6.2 channels. Patches expressing Kir6.2[N160D][C166S] were pulsed to +50 mV in (A) 10 μM spermine or (B) 10 μM CGC-11179. With the patch held continuously at +50 mV, the bathing solution was then switched to a polyamine-free solution to observe the time course of dissociation of polyamines from channels in the patch. A voltage step to −50 mV is sufficient to rapidly unblock either spermine or CGC-11179 from the channel and demonstrates that prolonged blockade and holding of the membrane potential at +50 mV does not result in significant channel rundown. The absence of significant blockade in a subsequent pulse to +50 mV demonstrates that most polyamine has diffused away from each patch. Similar experiments were performed on the cysteine-substituted mutants L157C, L164C, and M169C. In the lower panels, the exact details of voltage pulses and timing of solution changes have been omitted but are similar to those illustrated in the top row.