Homology modeling identifies C-terminal residues that contribute to the Ca2+ sensitivity of a BKCa channel.
Activation of BK(Ca) channels by direct Ca(2+) binding and membrane depolarization occur via independent and additive molecular processes. The "calcium bowl" domain is critically involved in Ca(2+)-dependent gating, and we have hypothesized that a sequence within this domain may resemble an EF hand motif. Using a homology modeling strategy, it was observed that a single Ca(2+) ion may be coordinated by the oxygen-containing side chains of residues within the calcium bowl (i.e., (912)ELVNDTNVQFLD(923)). To examine these predictions directly, alanine-substituted BK(Ca) channel mutants were expressed in HEK 293 cells and the voltage and Ca(2+) dependence of macroscopic currents were examined in inside-out membrane patches. Over the range of 1-10 microM free Ca(2+), single point mutations (i.e., E912A and D923A) produced rightward shifts in the steady-state conductance-voltage relations, whereas the mutants N918A or Q920A had no effect on Ca(2+)-dependent gating. The double mutant E912A/D923A displayed a synergistic shift in Ca(2+)-sensitive gating, as well as altered kinetics of current activation/deactivation. In the presence of 1, 10, and 80 mM cytosolic Mg(2+), this double mutation significantly reduced the Ca(2+)-induced free energy change associated with channel activation. Finally, mutations that altered sensitivity of the holo-channel to Ca(2+) also reduced direct (45)Ca binding to the calcium bowl domain expressed as a bacterial fusion protein. These findings, along with other recent data, are considered in the context of the calcium bowl's high affinity Ca(2+) sensor and the known properties of EF hands.
Sheng
Jian-Zhong
JZ
Department of Pharmacology, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.
Weljie
Aalim
A
Sy
Lusia
L
Ling
Shizhang
S
Vogel
Hans J
HJ
Braun
Andrew P
AP
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000737:chemistry*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D006801:Humans; D007477:Ions; D007700:Kinetics; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D018076:DNA, Complementary; D007477:Ions; C494841:KCNMA1 protein, human; C465449:Kcnma1 protein, mouse; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D011993:Recombinant Fusion Proteins; D008274:Magnesium; D000409:Alanine; D002118:Calcium