The contribution of voltage clamp fluorometry to the understanding of channel and transporter mechanisms

Abstract

Key advances in single particle cryo-EM methods in the past decade have ushered in a resolution revolution in modern biology. The structures of many ion channels and transporters that were previously recalcitrant to crystallography have now been solved. Yet, despite having atomistic models of many complexes, some in multiple conformations, it has been challenging to glean mechanistic insight from these structures. To some extent this reflects our inability to unambiguously assign a given structure to a particular physiological state. One approach that may allow us to bridge this gap between structure and function is voltage clamp fluorometry (VCF). Using this technique, dynamic conformational changes can be measured while simultaneously monitoring the functional state of the channel or transporter. Many of the important papers that have used VCF to probe the gating mechanisms of channels and transporters have been published in the Journal of General Physiology In this review, we provide an overview of the development of VCF and discuss some of the key problems that have been addressed using this approach. We end with a brief discussion of the outlook for this technique in the era of high-resolution structures.

Figure 1.

VCF highlights the unique role of the sodium channel’s domain IV voltage sensor. (A) Membrane topology of a voltage-gated sodium ion channel. The location of the fluorescent probes on each of the S4s are highlighted using colored stars. (B) Comparison of the fluorescence response from each of the four domains of the sodium channel with ionic currents (black) in response to a depolarizing voltage pulse. The fluorescence response from the S4s of domains I, II, and III correlate with the activation of sodium channel. The domain IV fluorescence signal was inverted to highlight the tight correlation between sodium channel inactivation and domain IV activation kinetics. Note that these fluorescence kinetics were remarkably consistent over multiple positions in the same S4 segment. Ionic currents were obtained in the absence of external sodium and therefore represent efflux of internal potassium ions through the sodium channel. Adopted from Chanda and Bezanilla (2002).