Ca transients associated with exocytosis cause dramatic changes of the surface membrane, as reported by electrogenic and optical membrane probes. Standard solutions with no ATP or polyamines. (A) Effects on DPA signals. 2 µM DPA was applied for 4 s and removed before and after activating reverse Na/Ca exchange in the absence of cytoplasmic ATP. Cm increases by 60%, and the DPA-capacitive signal increases by a factor of 4. The DPA current is unchanged, whereas the rate of rise of the capacitive DPA signal (dCm/dt, measured in pF/S) is increased by 70%. DSP, 3.3. (B) Effects on C6TPP signals. 300 µM C6TPP is applied and removed six times. Ca influx associated with a 40% increase of membrane area causes a 2.4-fold increase of C6TPP currents, causes decay phases of the currents, and induces C6TPP to develop a capacitive signal component. These changes are consistent with decreased dissociation rates from the membrane, similar to the effects of DPA. DSF, 670. (C) Effects on ANEPPDHQ optical signals. Ca transients with membrane fusion cause large shifts in fluorescence spectra of ANEPPDHQ (8 µM) binding. The large relative increase of emission above 640 nm (gray line) is similar to changes described for cholesterol depletion and presumably increased membrane disorder (Jin et al., 2006). Electrophysiological parameters were recorded in parallel with time-lapse confocal imaging at emission bandwidths of 500–580 nm (black) and 640 nmLP (gray).
