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 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. This work establishes tether-based single-channel recordings as a powerful tool to study dynamics of membrane transport.