Optical Single-Channel Recording via Diffusional Confinement in Membrane Tethers

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 the 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 Ca_V3.2 and compared our results to ensemble electrophysiology and stochastic gating simulations. We detected events corresponding to as little as ∼0.4 fC of transported charge, or only 6-13 free Ca²⁺ ions under typical buffering conditions. Tether-based single-channel recordings are a powerful tool to study dynamics of membrane transport.