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

Jian-Zhong Sheng ^*, Aalim Weljie , Lusia Sy ^*, Shizhang Ling ^*, Hans J. Vogel  and Andrew P. Braun ^*

Departments of ^* Pharmacology and Therapeutics and Biological Sciences, University of Calgary, Calgary, Alberta, Canada

Correspondence: Address reprint requests to Andrew P. Braun, Dept. of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, 3330 Hospital Dr., NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-8861; Fax: 403-270-2211; E-mail: abraun{at}ucalgary.ca.

Activation of BK_Ca 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 BK_Ca 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 µM free Ca²⁺, 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 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 a bacterial fusion protein. 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.

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