Inactivation of Bk Channels Mediated by the Nh(2) Terminus of the β3b Auxiliary Subunit Involves a Two-Step Mechanism: Possible Separation of Binding and Blockade

A family of auxiliary β subunits coassemble with Slo α subunit to form Ca(2)+-regulated, voltage-activated BK-type K(+) channels. The β subunits play an important role in regulating the functional properties of the resulting channel protein, including apparent Ca(2)+ dependence and inactivation. The β3b auxiliary subunit, when coexpressed with the Slo α subunit, results in a particularly rapid (∼1 ms), but incomplete inactivation, mediated by the cytosolic NH(2) terminus of the β3b subunit (Xia et al. 2000). Here, we evaluate whether a simple block of the open channel by the NH(2)-terminal domain accounts for the inactivation mechanism. Analysis of the onset of block, recovery from block, time-dependent changes in the shape of instantaneous current-voltage curves, and properties of deactivation tails suggest that a simple, one step blocking reaction is insufficient to explain the observed currents. Rather, blockade can be largely accounted for by a two-step blocking mechanism ( [Figure: see text] ) in which preblocked open states (O*(n)) precede blocked states (I(n)). The transitions between O* and I are exceedingly rapid accounting for an almost instantaneous block or unblock of open channels observed with changes in potential. However, the macroscopic current relaxations are determined primarily by slower transitions between O and O*. We propose that the O to O* transition corresponds to binding of the NH(2)-terminal inactivation domain to a receptor site. Blockade of current subsequently reflects either additional movement of the NH(2)-terminal domain into a position that hinders ion permeation or a gating transition to a closed state induced by binding of the NH(2) terminus.