Abstract
Single-channel recordings revolutionized our understanding of voltage-gated ion channels by allowing observation of behaviors that are obscured by large ensemble averages. Yet despite its power, single-channel recordings only allow indirect inference about the motions of the voltage sensor since only transitions between "open" and "closed" states of the channel can be seen; most transitions between states remain hidden. For this reason, direct measurement of the motion of a single voltage sensor has long been a goal to understand the details of voltage sensing; unfortunately, at present the elementary charge transition is below experimental resolution. We report here observation of fluorescence from single voltage sensors conjugated to fluorescent proteins. These recordings respond to voltage and are able to recapitulate macroscopic recordings when averaged together. The protein we used is the "ArcLight" voltage sensor (Jin, L. et al. Neuron, 2012.), along with derivatives thereof. This sensor consists of the voltage sensing domain from Ci-VSP coupled to a GFP derivative, and it shows robust changes in fluorescence in response to voltage. Our recordings are taken from oocyte membranes using total internal reflection microscopy at a frame rate of 500 hertz and at a temperature of approximately 13 degrees Celsius. This combination of low temperature and fast acquisition allows detection of residencies of the protein at distinct fluorescence levels with stochastic movement between these levels being biased by voltage. Presumably these distinct fluorescence levels correspond to distinct states of the voltage sensor. The transitions between these states can be analyzed and modeled, producing a novel picture of how the voltage sensor moves and how these movements are influenced by membrane potential. Support: NIH GM030376.
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