Divalent Cation-Dependent Motion of the BK Channel Gating-Ring Reported by State Dependent FRET

Abstract

Large conductance voltage- and calcium-dependent potassium channels (BK) are important controllers of cell excitability. The channel's open probability is regulated by changes in transmembrane voltage, intracellular calcium and magnesium concentrations1. The voltage sensor resides within the transmembrane region of the channel. Divalent cation binding is sensed by the intracellular C-terminal “gating ring”, formed by eight Regulator of Conductance for K+ (RCK) domains2. Using patch-clamp fluorometry we have recently shown that remarkable large rearrangements of the gating ring occur upon Ca2+ binding in intact BK channels3, much larger than those predicted by existing X-ray structures of the isolated gating ring 2. Using the same technical approach, we have now explored the conformational changes of the gating ring in intact channels induced by other divalent cations (Mg2+ and Ba2+ 4,5) in relation with channel function. Our results show that Mg2+ and Ba2+ binding to the high affinity Ca2+ binding site (Calcium Bowl) also induce structural rearrangements of the gating ring, although to a lesser extent than those observed after Ca2+ binding. All the same, these rearrangements are not strictly coupled to the opening of the pore. Structural changes of the gating ring induced by Ca2+, Mg2+ and Ba2+ show different magnitude and voltage-dependence. These results indicate the existence of a complex movement of the gating ring induced by the binding of divalent cations to the calcium bowl.