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

Abstract

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 voltagedependent 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 a 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 insideout 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 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 (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 ~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 a subunit, with sequence similarities to an EF hand motif, functionally contribute to the calcium-sensitive gating of this channel.

Large conductance Ca2+-activated K+ (BK) channel: Activation by Ca2+ and voltage

“…1C) was able to reduce the Ca 2+ -binding affinity by about 60%. This result demonstrates a direct correlation between Ca 2+ binding and Ca 2+ sensitivity for channel activation (see also Braun and Sy, 2001). The D5N5 mutant expressed in cells exhibits a lower Ca 2+ sensitivity for activation and reduces the dSlo channel's Hill coefficient 2-fold, as if one binding site per monomer is lost in the D5N5 mutant.…”

“…A third, low affinity mechanism (discussed below), can be put into action by Ca 2+ , Sr 2+ , Cd 2+ , Mn 2+ , Ni 2+ , and Co 2+ . In the absence of binding assays like those performed with BK carboxyl terminus fusion proteins (Bian et al, 2001;Braun and Sy, 2001), there exists the possibility, however, that all the results are due to coincidental allosteric effects on Ca 2+ binding to a region that lies outside the RCK region. Independent of these considerations, the unique divalent cation selectivity of the different BK regulatory mechanisms supports the existence of at least three distinct divalent cation binding sites and that the sites act independently of each other.…”

“…1A, C). Partial deletion or point mutations of the aspartates contained in the calcium bowl produced BK channel that were less sensitive to Ca 2+ -at the same Ca 2+ concentration, the calcium bowl mutant conductance-voltage (G-V) curves were right-shifted compared to the wildtype BK channel G-V curve (Schreiber and Salkoff, 1997;Bian et al, 2001;Braun and Sy, 2001). If the Ca 2+ bowl is disrupted by deleting crucial aspartates, this high Ca 2+ affinity regulatory site is lost, but the mutant and the wild-type BK showed the same sensitivity to Cd 2+ ions.…”

Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca 2+

“…These coefficients suggest that a minimum of 2-5 Ca 2ϩ must be bound to allosteric activators to maximally activate the channel. Consistent with this necessity, BK channels are tetramers (12), with each of the four pore-forming ␣ subunits having at least two Ca 2ϩ -binding sites (13)(14)(15)(16)(17)(18). One of these sites is a high-affinity (Ͻ10 M) Ca 2ϩ -binding site and the other is a low-affinity (Ͼ100 M) site that binds Ca 2ϩ and Mg 2ϩ (17,18).…”

“…Disruption of the Ca-bowl by mutations in the Ca-bowl or by replacement of the tail of the channel that includes the Ca-bowl with a tail from Slo3, a Ca 2ϩ insensitive BK-like channel, greatly decreases the Ca 2ϩ sensitivity (11,14,15,19) and also decreases the Hill coefficient for Ca 2ϩ activation from 4.5 to 1.3 at ϩ50 mV (11). Fragments of the C terminus that include the Ca-bowl (the last Ϸ230 or Ϸ280 aa residues of the channel) bind Ca 2ϩ (11,16). The location of the low-affinity nonselective site is not clear, but it is closer to the core of the channel than the Ca-bowl (17).…”