Role of an Intersubunit Ca2+ Bridge in Structure and Function of BK Channels

Abstract

Large conductance Ca2+-activated K+ (BK) channels are found in a variety of tissues and serve a range of functions, including control of action potential duration and smooth muscle contractility. Ca2+-dependent gating of BK channels is controlled by conformational changes of the “gating ring,” formed by the C-terminal domains (CTD) of its four pore-forming subunits, each comprised of two regulators of conductance of K+ (RCK) domains, RCK1 and RCK2. Residues D895-D897 comprise a well-established high-affinity Ca2+ binding site within RCK2 known as the “Ca2+ bowl”, and the crystal structure of the Ca2+ bound gating ring suggests that Ca2+ coordination at this site may also involve formation of bridge to residue N449 in the RCK1 domain of the adjacent subunit. Here we implemented a combination electrophysiology, biochemistry, and X-ray crystallography to interrogate the role of the putative Ca2+ bridge in BK channel activation. In excised patch recordings from transfected HEK cells, N449A mutant BK channels displayed a +50 mV shift in V1/2 compared to wild-type, suggesting that the N449 side chain plays a role in gating. We then overexpressed the wild-type and mutant BK-CTD in sf9 cells and analyzed the Ca2+-dependence of tetramerization by size exclusion chromatography. The wild-type BK-CTD formed tetramers in Ca2+-containing solutions with a biphasic dose-response relation, defined by K1/2 values of 25 μM and 11.7 mM. The N449A mutation greatly reduced the high-affinity effect of Ca2+, whereas the low affinity effect of Ca2+ on tetramerization remained (K1/2 =12.6 mM). Preliminary X-ray crystallographic data suggests that the N449A mutation does not effect the overall structure of the CTD. Together, these experiments point to a functional role for an intersubunit Ca2+ bridge between N449 and the Ca2+ bowl in BK channel activation.