Homology Modeling Identifies C-Terminal Residues that Contribute to the Ca2+ Sensitivity of a BKCa Channel

摘要: Abstract Activation of BK Ca channels by direct 2+ binding and membrane depolarization occur via independent additive molecular processes. The "calcium bowl" domain is critically involved in -dependent gating, we have hypothesized that a sequence within this may resemble an EF hand motif. Using homology modeling strategy, it was observed single ion be coordinated the oxygen-containing side chains residues calcium bowl (i.e., 912 ELVNDTNVQFLD 923 ). To examine these predictions directly, alanine-substituted channel mutants were expressed HEK 293 cells voltage dependence macroscopic currents examined inside-out patches. Over range 1–10 μ M free , point mutations E912A D923A) produced rightward shifts steady-state conductance-voltage relations, whereas N918A or Q920A had no effect on gating. double mutant E912A/D923A displayed synergistic shift -sensitive as well altered kinetics current activation/deactivation. In presence 1, 10, 80mM cytosolic Mg mutation significantly reduced -induced energy change associated with activation. Finally, sensitivity holo-channel to also 45 bacterial fusion protein. These findings, along other recent data, are considered context bowl's high affinity sensor known properties hands.