Using CaV-Type Dependent BK Channel Behavior to Probe BK-CaV Channel Coupling and Submembrane [Ca2+] Dynamics

Abstract

Voltage- and Ca2+-gated K+ (BK) channels and voltage-gated Ca2+ (CaV) channels assemble into macromolecular complexes such that Ca2+ entering through CaV channels during membrane depolarization activates BK channels. To understand the [Ca2+] dynamics underlying BK channel activation, we used the cut-open Xenopus oocyte preparation to record BK channel currents activated by Ca2+ entering the cell through co-expressed CaV1.2 or CaV2.2 channels at an imposed buffering condition of 5 mM EGTA. We found that CaV1.2 and CaV2.2 channels exhibit distinctly different functional coupling to BK channels: CaV2.2 activated BK channels more potently than CaV1.2, as evidenced by a larger magnitude and faster activating IBK (τact = 0.20 ±0.02 ms and τact = 0.29 ±0.04 ms, respectively). Moreover, when IBK amplitude was reduced by reducing the number of available CaV channels (by imposing different degrees of CaV channel inactivation), the IBK activation kinetics in the presence of CaV2.2 remained unchanged but slowed in the presence of CaV1.2 channels. These results strongly suggest that each BK channel is functionally coupled to one CaV2.2 channel but to several CaV1.2 channels. We have also determined the PO and activation kinetics of BK channels using inside-out patch clamp recordings in different known fixed [Ca2+]. Based on these properties and measured Ca2+ currents for each CaV type, we are fitting a 3-dimensional diffusion-reaction model combined with a kinetic BK channel model to the IBK data. Ultimately, we will adjust the parameters of this model to fit comparable recordings from the more physiologically-relevant synapses formed in Xenopus nerve-muscle cell co-cultures.