Optical single-channel recording via diffusional confinement in membrane tethers

Abstract

Single-channel electrophysiology probes ion channel gating, but how can one probe membrane transport when the single-unit current is undetectable? We pulled membrane tethers from live cells to isolate individual transmembrane proteins. The tether constrained diffusion of transported substrate to the tether axis, leading to [~]1000-fold enhancement of substrate concentration and observation time compared to planar membranes. Fluorescent reporters inside the tether revealed individual transport events. We imaged unitary Ca2+ transport events in tethers containing the low-conductance T-type Ca2+ channel CaV3.2, and compared our results to ensemble electrophysiology and stochastic gating simulations. This work establishes tether-based single-channel recordings as a powerful tool to study dynamics of membrane transport. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/652649v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1ed0ac1org.highwire.dtl.DTLVardef@15d1b6eorg.highwire.dtl.DTLVardef@1b3ff91org.highwire.dtl.DTLVardef@4f99e0_HPS_FORMAT_FIGEXP M_FIG C_FIG