Distance-Resolving Voltage Clamp Fluorometry (drVCF) Quantifies Intramolecular Transitions in the Human BK and CI-VSP Voltage Sensors Under Physiologically-Relevant Conditions

Abstract

The BK channel voltage-sensing domain (VSD) exhibits important differences from the canonical VSD model: an additional helix (S0), a decentralized distribution of voltage-sensing charges throughout S2-S3-S4 and cooperative S2/S4 activation transitions. To understand the voltage-evoked conformational rearrangements of this unique voltage sensor, and address the unmet need of quantifying short-range intramolecular distances in conducting channels under physiological conditions, we have been developing a new theoretical and experimental framework: distance-resolving Voltage Clamp Fluorometry (drVCF).drVCF exploits state-dependent collisional quenching of site-specifically conjugated tetramethylrhodamine fluorophores of different length by tryptophan. Firstly, voltage-evoked, Trp-dependent fluorescence deflections of each label are acquired from the VSD of conducting human BK channels expressed in Xenopus oocytes using VCF-enabled cut-open oocyte voltage clamp. The labeling site/tryptophan distance in the resting and active states is determined by fitting VCF data to quenching probability density over distance, generated by fluorophore molecular dynamics simulations. Simultaneous fitting of data from multiple fluorophores results in well-constrained solutions. Confidence intervals are established using bootstrap resampling.At rest, S0, S1 and S2 are approximately equidistant from S4: S0-S4 mean=12.5A; 95% CI [12.4-12.8A]; S1-S4=11.9A [10.4-12.9A]; S2-S4=11.5A [8.8-12.3A]. Upon VSD activation, S4 diverges from S0 (22.0A [21.4-23.1A]) and S1 (18.4A [17.7-20.0A]), while the S2-S4 distance exceeds 24A. Using this information, we constructed a structural model for the BK voltage sensor in the resting and active states. Applying drVCF in Ci-VSP, we resolved that positions 137 (S1) and 212 (S4) are 19-24A apart in the active state, as in the atomic structure (22A: Li et al., 2014; PDB#4G7V). Thus, the new drVCF approach complements FRET-based techniques for distance measurements of short-range transitions under physiologically-relevant conditions.