Fig. 6. Parallel recording of nucleotide and peptide release from individual granules. (A,B) Confocal images of a section of the footprint of a voltage-clamped cell expressing both IAPP-pHluorin and P2X₂-mRFP. The images were recorded at the indicated times relative the onset of the IAPP-pHluorin flash. Exocytosis was elicited by intracellular dialysis of the cell with a buffer in which [Ca²⁺]_i was set at 2 µM. Note that the highlighted granules increase their fluorescence during the displayed sequence. In A, the signal from the granule is rapidly lost, while in B it remains elevated for several seconds. (C,D) Image sequences of a cell co-transfected with IAPP-pHluorin and P2X₂-mRFP1 and stimulated as in A and B. The entire cell was imaged in the IAPP-pHluorin channel (see Materials and Methods), and whole-cell current spikes due to activation of the P2X₂ receptors were recorded in parallel. Examples of a short-lived fluorescence transient in C, and a long-lasting event in D. (E,F) Time course of the average fluorescence intensity in the ROIs indicated by the white circle in C and D (top) and inward membrane currents associated with the events (lower). Note slow decay of fluorescence in F. (G) Histogram of the decay constants of 56 events as in C and D. The shaded area indicates decay constants greater than 350 milliseconds. (H) Cross-correlation histogram of the times of fluorescence peaks vs the times of current peaks in three experiments similar to that shown in C and D.