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Contribution of potential EF hand motifs to the calcium‐dependent gating of a mouse brain large conductance, calcium‐sensitive K + channel
Author(s) -
Braun Andrew P.,
Sy Luisa
Publication year - 2001
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2001.00681.x
Subject(s) - gating , calcium , chemistry , depolarization , voltage dependent calcium channel , biophysics , calcium channel , conductance , binding site , t type calcium channel , membrane potential , calcium activated potassium channel , r type calcium channel , biochemistry , biology , physics , organic chemistry , condensed matter physics
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 α 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, FL D 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 μM 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 (PV D 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 ≈11‐fold (129.5 μM vs. 11.3 μM) 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 α subunit, with sequence similarities to an EF hand motif, functionally contribute to the calcium‐sensitive gating of this channel.