Homology Modeling Identifies C-terminal Residues That Contribute to the Ca²⁺ Sensitivity of a BK_Ca Channel

¹ University of Calgary

^* To whom correspondence should be addressed. E-mail: abraun{at}ucalgary.ca.

Submitted on March 27, 2005
Revised on April 19, 2005
Accepted on 27 July 2005

	Abstract

Activation of BKCa channels by direct Ca2+ binding and membrane depolarization occur via independent and additive molecular processes. The 'calcium bowl' domain is critically involved in Ca2+-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 Ca2+ ion may be coordinated by the oxygen-containing side chains of residues within the calcium bowl (i.e. 912ELVNDTNVQFLD923). To examine these predictions directly, alanine-substituted BKCa channel mutants were expressed in HEK 293 cells and the voltage and Ca2+ dependence of macroscopic currents were examined in inside-out membrane patches. Over the range of 1-10 M free Ca2+, single point mutations (i.e. E912A and D923A) produced right-ward shifts in the steady-state conductance-voltage relations, whereas the mutants N918A or Q920A had no effect on Ca2+-dependent gating. The double mutant E912A/D923A displayed a synergistic shift in Ca2+-sensitive gating, as well as altered kinetics of current activation/deactivation. In the presence of 1, 10 and 80 mM cytosolic Mg2+, this double mutation significantly reduced the Ca2+-induced free energy change associated with channel activation. Finally, mutations that altered sensitivity of the holo-channel to Ca2+ also reduced direct 45Ca binding to the calcium bowl domain expressed as bacterial fusion proteins. These findings, along with other recent data, are considered in the context of the calcium bowl's high affinity Ca2+ sensor and the known properties of EF hands.

Key Words: EF hand, calcium binding, calcium-activated K+ channel, electrophysiology, structure

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