Mechanism of Voltage Sensor Probe Technology and VSP Applications
Figure 1. The VSP FRET pair consists of a mobile, voltage-sensitive acceptor and an outer membrane-bound donor. In resting cells (with a relatively negative internal potential), both members of the FRET pair bind to the outer surface of the cell membrane, resulting in efficient FRET. When the cells are depolarized, however, the donor remains on the outer surface, but the mobile acceptor rapidly translocates to the inner surface of the cell membrane, resulting in diminished FRET. The normalized assay ratio (the ratio of the donor emission to acceptor emission) reports changes in potential and is high in polarized cells and low in depolarized cells.
VSPs can report on any target that changes the membrane potential of the cell, including ligand-gated ion channels, Na, K, Ca, Cl channels and transporters (1–6). VSPs allow development of fluorescence-based high-throughput assays that measure the affinity of reference compounds with excellent correlation to patch-clamp data for voltage-gated NaV channels (7–9) and GABAA ligand-gated channels (10). Representative data seen in Figure 2 demonstrate VSP sensitivity for reporting on voltage-gated sodium channel-mediated depolarization, and on the effects that two compounds (a sodium channel opener and a sodium channel blocker) have on sodium channels.
Figure 2. Identification of NaV agonist and antagonist with a VSP assay.
A. Signal intensity changes for each dye in the FRET pair, and emission ratio changes following sodium addition to cells previously maintained in a sodium-free extracellular buffer. Sodium influx into the cells leads to a dramatic decrease in membrane potential, as reflected in the rapid decrease in oxonol emission. This decrease is accompanied by an increase in coumarin emission, causing a sharp increase in the emission ratio.
B. Depicts two dose-response curves: (1) an ascending curve demonstrating that the cells’ membrane potential significantly decreases when resting cells are exposed to increasing concentrations of a sodium channel opener and (2) a descending curve indicating that the cells’ membrane potential rapidly returns to the resting state when stimulated cells are exposed to increasing concentrations of a sodium channel blocker. This demonstrates the utility of the VSP assay for detecting both openers and blockers of this voltage-gated sodium channel. Using this cell line, a high-throughput screen of 700,000 molecules has been run in a 384-well assay format using the VIPR® II reader to identify novel sodium channel blockers.
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