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 (BK(Ca) 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 BK(Ca) 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 BK(Ca) 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 microM 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 (PVD(81)EK 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 approximately 11-fold (129.5 microM vs. 11.3 microm) compared to the wild-type channel. 4. We further observed that a bacterially expressed protein encompassing Site 6 of the BK(Ca) channel C-terminus and bovine brain calmodulin were both able to directly bind (45)Ca(2+) following denaturation and polyacrylamide gel electrophoresis (e.g. SDS-PAGE). 5. Our results suggest that two regions within the mammalian BK(Ca) channel alpha subunit, with sequence similarities to an EF hand motif, functionally contribute to the calcium-sensitive gating of this channel.
Braun
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Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada. abraun@ucalgary.ca
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D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D001921:Brain / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D020832:EF Hand Motifs / Q000235:genetics / Q000502:physiology*; D004553:Electric Conductivity; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics / Q000502:physiology; D015221:Potassium Channels / Q000235:genetics / Q000502:physiology*
D002147:Calmodulin; D010446:Peptide Fragments; D015221:Potassium Channels; D002118:Calcium