Probing the Dynamic Structure of the BK Ca Voltage Sensor: Relative Motion of Segments S0 & S4 During Activation

Abstract

Voltage- and Ca-activated, Large-conductance Potassium (BKCa) channel α subunits (Slo1) possess the transmembrane helix S0 at their N-terminus, which is absent in other members of the voltage-gated channel superfamily. S0 mediates the interaction between pore-forming α and modulatory β subunits, while it is thought to pack closely to voltage-sensing segment S4 (Liu et al., 2010). We previously reported that conformational rearrangements relevant to voltage-dependent activation occur in the proximity of the BKCa N-terminus, by using voltage clamp fluorometry (Pantazis et al., BPS meeting 2010). In this work, we have identified the principal molecular source of the fluorescence signal reported from the extracellular flank of S0 (positions 17, 18 or 19 labeled with the fluorophore TMRM) in W203, at the extracellular tip of S4: Substitution of W203 with a Valine reduced the amplitude of voltage-dependent ΔF/F reported from the extracellular portion of S0 by ≈90%. Accordingly, we demonstrate that the tryptophan side-chain is able to efficiently quench TMRM fluorescence with a Stern-Volmer constant KSV = 41.3 M−1. Considering this data, we suggest a model for the dynamic structure of the BKCa voltage sensor domain: at rest, S0 is in collisional proximity with S4, so that TMRM labeling S0 is quenched by W203. Upon depolarization, a relative motion between the two segments increases the distance between them, relieving TMRM quenching. Regarding the relevance of the relative motion between S0 and S4 to channel activation, we propose that S0 acts as a pivot, against which the voltage-sensitive S4 moves to actuate channel gating. We speculate that the modulation of the relative positions of S0 and S4 by β subunits could mechanistically explain their effects on voltage-dependent activation.