Disruption of a side portal pathway permits closed-state inactivation by BK β subunit N-termini

Cytosolic N-termini of several BK channel β regulatory subunits mediate rapid inactivation. However, in contrast to Kv channels, inactivation does not occur via a simple, open channel block mechanism, but involves two steps, an association step in which ion permeation is maintained (O*), then followed by inactivation (I). To produce inactivation, BK β subunit N-termini enter the central cavity through a lateral entry pathway ("side portal") separating the transmembrane pore-gate-domain and cytosolic gating ring. Comparison of BK conformations reveals an aqueous pathway into the central cavity in the open structure, while in the closed structure three sequential basic residues (R ₃₂₉ K ₃₃₀ K ₃₃₁ ) in S6 occlude central cavity access. We probed the impact of mutations of the RKK motif (RKK3Q, RKK3E, and RKK3V) on inactivation mediated by the β3a N-terminus. All three RKK-mutated constructs differentially reduce depolarization-activated outward current, prolong β3a-mediated tail current upon repolarization, and produce a persistent inward current at potentials down to -240 mV. With depolarization channels are driven into O*-I inactivated states and, upon repolarization, slow tails and persistent inward currents reflect slow changes in O*-I occupancy. However, evaluation of closed state occupancy prior to depolarization and at the end of slow tails reveals that some fraction of closed states at negative potentials corresponds to resting closed states in voltage-independent equilibrium with N-terminal-occluded closed-states. Thus, disruption of the RKK triplet both stabilizes the β3a-N-terminus in its position of inactivation and permits access of that N-terminus to its blocking position in closed states.

The role of BK S6 residues R329K330K331 and E321/E324 in β subunit-mediated inactivation is probed. WT R329K330K331 hinders inactivation in closed states, while RKK mutations stabilize inactivated states even under conditions where channels are otherwise closed. E321/E324 mutations do not permit closed-state inactivation.