Translocation of C1-GFP domains to the surface membrane of BHK cells by short-chain DAG8 (A), phorbol ester (B), and carbachol (C), followed by redistribution to the cytoplasm in all three cases by activation of Ca influx (i.e., outward NCX1 current). (A) Application of 20 μM C8 diacylglycerol brings the majority of C1 domain to the surface membrane with a time constant of ∼45 s. Upon activating reverse exchange current, domains return quantitatively to the cytoplasm with a time constant of ∼35 s. (B) Application of 0.2 μM phorbol ester (PMA) brings C1 domains to the cell surface with a time constant of ∼30 s. During activation of exchange current, C1 domains reach a substantially higher concentration in the cytoplasm than in the basal state with a time constant of ∼30 s, and C1 domains begin to move back to membrane immediately upon terminating Ca influx. (C) C1-GFP domain records together with cell capacitance and membrane current in a BHK cell expressing NCX1 and hM1 receptors. With application of carbachol (0.3 mM) cytoplasmic fluorescence decreases by 60% with a time constant of ∼20 s, and cell capacitance increases modestly. Upon activation of Ca influx, cell capacitance increases by ∼8 pF (30%) in less than 10 s, and C1 domains accumulate again in the cytoplasm with time constant of ∼10 s. Upon deactivating exchange current, C1-GFP domains move partially back to the surface membrane.