Slow spermine unbinding coincides with the steeply voltage-dependent phase of spermine block. (A) The voltage dependence of spermine block in MTSET-modified Kir2.1*-176C channels is biphasic with a steep range (i) and a shallow voltage range (ii; at voltages below −50 mV). (B) Representative kinetics of blocker unbinding after prepulses to the steeply voltage-dependent range of block (i; −40 to 0 mV) or to voltages in the range of shallow voltage-dependent block (ii). Slow kinetics of spermine unblock develop as a progressively larger fraction of channels are blocked by spermine in the steeply voltage-dependent binding site.