Mapping the BK_Ca Channel's “Ca²⁺ Bowl” : Side-chains Essential for Ca²⁺ Sensing

There is controversy over whether Ca²⁺ binds to the BK_Ca channel's intracellular domain or its integral-membrane domain and over whether or not mutations that reduce the channel's Ca²⁺ sensitivity act at the point of Ca²⁺ coordination. One region in the intracellular domain that has been implicated in Ca²⁺ sensing is the “Ca²⁺ bowl”. This region contains many acidic residues, and large Ca²⁺-bowl mutations eliminate Ca²⁺ sensing through what appears to be one type of high-affinity Ca²⁺-binding site. Here, through site-directed mutagenesis we have mapped the residues in the Ca²⁺ bowl that are most important for Ca²⁺ sensing. We find acidic residues, D898 and D900, to be essential, and we find them essential as well for Ca²⁺ binding to a fusion protein that contains a portion of the BK_Ca channel's intracellular domain. Thus, much of our data supports the conclusion that Ca²⁺ binds to the BK_Ca channel's intracellular domain, and they define the Ca²⁺ bowl's essential Ca²⁺-sensing motif. Overall, however, we have found that the relationship between mutations that disrupt Ca²⁺ sensing and those that disrupt Ca²⁺ binding is not as strong as we had expected, a result that raises the possibility that, when examined by gel-overlay, the Ca²⁺ bowl may be in a nonnative conformation.