Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca2+

BK channels (Slo1) are widely distributed V channels that control Ca2+-dependent processes and cellular excitability. Their activation by intracellular Ca2+ (Ca-i(2+)) is highly cooperative, with Hill coefficients of typically 2-5. To investigate the cooperativity contributed by each of the four a subunits that form the BK channel,we studied single channels comprised of mixtures of functional subunits and subunits with a mutation to disrupt a key site (Ca-bowl) required for activation by low concentrations of Ca-i(2+). As the number of functional subunits increased, we found a stepwise increase in the Hill coefficient of 0.3-0.8 per functional subunit and a stepwise decrease in the Ca-i(2+) required for half activation (K-d). These results show directly that BK channels can open with 0, 1, 2, 3, or 4 functional Ca-bowls, and that each subunit with a functional Ca-bowl contributes a stepwise increase to both the cooperativity of activation and the apparent Ca2+ affinity. A model with 0-4 high-affinity allosteric activators and four low-affinity allosteric activators was examined. In this model, Ca2+ bindings were independent of one another and the cooperativity arose from the joint action of the allosteric activators on the open-closed equilibrium. Although this model described well the major features of the experimental data, some differences between the observed and predicted results indicated that additional factors not included in the model also contribute to the cooperativity.