Figure 13.

Extracellular cell surface accumulation of annexin-V Alexa Fluor 488 conjugate in response to increasing cytoplasmic Ca and activation of membrane fusion. In A and B, pipette perfusion of 40 mM Na was used to activate outward exchange current with 2 mM extracellular Ca and 1:100 annexin-V dilution. The pipette solution initially contains 40 mM Li. After recording background for 2 min, lithium is substituted for 40 mM Na via pipette perfusion through a 40-μm inner diameter quartz capillary. Panel A shows the surface membrane fluorescence, and B shows line scans of the cell before (solid line) and 5 min after activating exchange current. (C) Inward exchange current and capacitance responses induced in giant excised BHK patches by applying cytoplasmic solutions with 4 and 200 μM free Ca. The pipette contains 140 mM Na, and the pipette tip is switched between three solutions, as indicated. Capacitance responses are negligible on applying 4 μM free Ca. Two applications of 200 μM free Ca result in a total 300 fF increase of patch capacitance, estimated to be ∼10% of the initial patch membrane capacitance. (D) Extracellular binding of annexin-V Alexa Fluor 488 to the extracellular surface of a giant excised BHK patch upon applying 200 μM free Ca to the cytoplasmic surface. The pipette solution contains 2 mM extracellular Ca with 1:100 annexin-V dilution.