Voltage-Dependent Motions Reported from the N-Terminal Region in Human Slo1 BKCa Channels: S0 and Voltage Sensor Operation

Abstract

The transmembrane region of large-conductance, voltage- and Ca2+-activated K+ (BKCa) channel α subunits (Slo1) possesses a unique topological feature when compared with those of other voltage-activated proteins: S0, an additional transmembrane segment that renders their short N-terminus extracellular (Wallner et al., 1996, PNAS). S0 mediates the interaction between pore-forming α and auxilliary β subunits, which facilitate channel activation and, in the case of β2 and β3, induce fast inactivation. Recent findings have proposed that S0 is flanked by transmembrane voltage-sensing domains S2-S4 (Liu et al., 2008, JGP). We used cut-open oocyte voltage-clamp fluorometry to explore the role of S0 in the function of the BK voltage sensor. By substituting unique Cysteines at positions 17-19 (20 thought to be at the extracellular tip of S0) of hSlo1 M4, and labeling them with the environment-sensitive fluorophore TMRM, we have resolved voltage-dependent conformational rearrangements, with Vhalf = −79 ±2.9 mV and z = 0.84 ±0.046 e0. Intriguingly, channel activation in these Cysteine mutants exhibited a facilitated voltage dependence of ionic conductance (∈″Vhalf = −30 mV) compared to pseudo-WT channels. We provide direct evidence that the N-terminus influences the operation of the voltage-sensing S2 and S4 transmembrane domains: mutation R20A induced pronounced shifts in the activations of voltage-sensing segments S2 and S4 (∈″Vhalf = 46 mV for S2, 66mV for S4) and a 35% reduction in the effective charge (z) of both. These results strongly indicate that the N-terminal region plays a significant role in the voltage sensor operation of human BKCa channels.