It has been established that the large conductance Ca2+-activated K+ channel contains two types of high-affinity Ca2+ binding sites, termed the Ca2+ bowl and the RCK1 site. The affinities of these sites, and how they change as the channel opens, is still a subject of some debate. Previous estimates of these affinities have relied on fitting a series of conductance–voltage relations determined over a series of Ca2+ concentrations with models of channel gating that include both voltage sensing and Ca2+ binding. This approach requires that some model of voltage sensing be chosen, and differences in the choice of voltage-sensing model may underlie the different estimates that have been produced. Here, to better determine these affinities we have measured Ca2+ dose–response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca2+ binding site) and for channels that have had one or the other Ca2+ binding site disabled via mutation. To accurately determine these dose–response curves we have used a series of 22 Ca2+ concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude. Our results indicate that at −80 mV the Ca2+ bowl has higher affinity for Ca2+ than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca2+ to the RCK1 site is voltage dependent, whereas at the Ca2+ bowl it is not.

The large-conductance Ca2+-activated potassium (BKCa) channel of smooth muscle is unusually sensitive to Ca2+ as compared with the BKCa channels of brain and skeletal muscle. This is due to the tissue-specific expression of the BKCa auxiliary subunit β1, whose presence dramatically increases both the potency and efficacy of Ca2+ in promoting channel opening. β1 contains no Ca2+ binding sites of its own, and thus the mechanism by which it increases the BKCa channel's Ca2+ sensitivity has been of some interest. Previously, we demonstrated that β1 stabilizes voltage sensor activation, such that activation occurs at more negative voltages with β1 present. This decreases the work that Ca2+ must do to open the channel and thereby increases the channel's apparent Ca2+ affinity without altering the real affinities of the channel's Ca2+ binding sites. To explain the full effect of β1 on the channel's Ca2+ sensitivity, however, we also proposed that there must be effects of β1 on Ca2+ binding. Here, to test this hypothesis, we have used high-resolution Ca2+ dose–response curves together with binding site–specific mutations to measure the effects of β1 on Ca2+ binding. We find that coexpression of β1 alters Ca2+ binding at both of the BKCa channel's two types of high-affinity Ca2+ binding sites, primarily increasing the affinity of the RCK1 sites when the channel is open and decreasing the affinity of the Ca2+ bowl sites when the channel is closed. Both of these modifications increase the difference in affinity between open and closed, such that Ca2+ binding at either site has a larger effect on channel opening when β1 is present.