Comparative Mechanisms Of Activation Of The Slo1 BK Channel By Ca2+ And H+ Mediated By The RCK1 Domain

Abstract

Large-conductance Ca2± and voltage-gated K+ (Slo1 BK) channels are allosterically activated by depolarization and intracellular Ca2+. High-affinity activation of the channel by Ca2+ involves two sites, the Ca2+ bowl sensor and the RCK1 sensor, the latter of which is also required for the stimulatory action of intracellular H+ (Hou et al., Nat Struct Mol Biol. 15, 403, 2008). We investigated the comparative effects of Ca2+ and H+ on activation of the Slo1 BK channel mediated by the RCK1 sensor using a Ca2+ bowl-defective mutant expressed in HEK cells. Decreasing pHi from 7.5 to 6.2 shifted the voltage-conductance (GV) curve to the left by ∼50 mV. The shift in GV by H+ was, however, only ∼40% of that caused by a saturating concentration of Ca2+ in the mutant. Single-channel measurements at negative voltages where voltage sensor activation is negligible verified that 200 uM Ca2+ drastically increased open probability, corresponding to the allosteric coupling factor C = ∼4 in the model of Horrigan and Aldrich (J Gen Physiol. 120, 267, 2002). In contrast, at such negative voltages, H+ at pH = 6.2 was much less effective in increasing open probability, leading to the estimated C value = ∼1.3, placing H+ between Ca2+ and Mg2+ in the ability to promote opening in the absence of voltage sensor activation. Likewise, at the respective saturation concentrations, H+ was less effective than Ca2+ at regulating channel kinetics. Our electrophysiological measurements and simulations collectively suggest that H+ is a partial agonist of the RCK1 high-affinity Ca2+ sensor and that a small change in the relative position of His and Asp residues in the sensor, ∼0.1 nm, may underlie the activation of the channel by H+. Supported by NIH.