Exceeding physiological limits of the cell membrane potential compromises structural integrity, enabling the passage of normally impermeant solutes and disrupting cell function. Electropermeabilization has been studied extensively at the cellular scale, but not at the individual membrane lesion level. We employed fast total internal reflection fluorescence (TIRF) imaging of Ca2+ entry transients to discern individual lesions in a hyperpolarized cell membrane and characterize their focality, thresholds, electrical conductance, and the lifecycle. A diffuse and momentary membrane permeabilization without a distinct pore formation was observed already at a -100 mV threshold. Polarizing down to -200 mV created focal pores with a low 50- to 300-pS conductance, which disappeared instantly once the hyperpolarization was removed. Charging to – 240 mV created high-conductance (> 1 nS) pores which persisted for seconds even at zero membrane potential. With (…)
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