Contribution of potential EF hand motifs to the calcium-dependent gating of a mouse brain large conductance, calcium-sensitive K+ channel

1. The large conductance, calcium-sensitive K+ channel (BKCa channel) is a unique member of the K+-selective ion channel family in that activation is dependent upon both direct calcium binding and membrane depolarization. Calcium binding acts to dynamically shift voltage-dependent gating in a negative or left-ward direction, thereby adjusting channel opening to changes in cellular membrane potential. 2. We hypothesized that the intrinsic calcium-binding site within the BKCa channel alpha subunit may contain an EF hand motif, the most common, naturally occurring calcium binding structure. Following identification of six potential sites, we introduced a single amino acid substitution (D/E to N/Q or A) at the equivalent of the -z position of a bona fide EF hand that would be predicted to lower calcium binding affinity at each of the six sites. 3. Using macroscopic current recordings of wild-type and mutant BKCa channels in excised inside-out membrane patches from HEK 293 cells, we observed that a single point mutation in the C-terminus (Site 6, FLD(923)QD to N), adjacent to the 'calcium bowl' described by Salkoff and colleagues, shifted calcium-sensitive gating right-ward by 50-65 mV over the range of 2-12 muM free calcium, but had little effect on voltage-dependent gating in the absence of calcium. Combining this mutation at Site 6 with a similar mutation at Site 1 ((PVDEK)-E-81 to N) in the N-terminus produced a greater shift (70-90 mV) in calcium-sensitive gating over the same range of calcium. We calculated that these combined mutations decreased the apparent calcium binding affinity similar to 11-fold (129.5 muM vs. 11.3 muM) compared to the wild-type channel. 4. We further observed that a bacterially expressed protein encompassing Site 6 of the BKCa, channel C-terminus and bovine brain calmodulin were both able to directly bind Ca-45(2+) following denaturation and polyacrylamide gel electrophoresis (e.g. SDS-PAGE). 5. Our results suggest that two regions within the mammalian BKCa channel alpha subunit, with sequence similarities to an EF hand motif, functionally contribute to the calcium-sensitive gating of this channel.