Ca2+-binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca2+-dependent activation

Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca2+-activated K+ channel (BK channel) contains Ca2+-binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca2+ binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG- and His(6)-tagged protein in Escherichia coli, We purified Dslo-C280 in soluble form and used a Ca-45(2+)-overlay protein blot assay to detect Ca2+ binding. Dslo-C280 exhibits specific binding of Ca-45(2+) in comparison with various control proteins and known EF-hand Ca2+-binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the Ca-bowl motif are changed to Asn, reduces Ca-45(2+)-binding activity by 56%, By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca2+ sensitivity for activation and a shift of approximate to +80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca2+ and a reduction in apparent Ca2+ affinity, suggesting the loss of one Ca2+-binding site per monomer, These results demonstrate a functional correlation between Ca2+ binding to a specific region of the BK protein and Ca2+-dependent activation, thus providing a biochemical approach to study this process.