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. 2017 Oct 25;6:e30203. doi: 10.7554/eLife.30203

Figure 4. PI(4,5)P2 uncaging potentiates exocytosis in adrenal chromaffin cells, which depends on the lipid head group but does not alter depolarization-induced currents.

(a) Physiological stimulation paradigm to investigate the effect of PI(4,5)P2 uncaging on exocytosis. Cells were loaded with compounds 1a,b or 2a,b prior to experiments. After a pre-pulse of depolarizing voltage steps, cells were either subjected to UV uncaging (PI(4,5)P2 uncaging group) or not (control group). The effect of PI(4,5)P2 uncaging was investigated in a subsequent test pulse. The pre-pulse and the test pulse consisted of six brief (10 ms) and four longer (100 ms) depolarizations to allow Ca2+ influx and induce exocytosis (Voets et al., 1999). (b,ci) Whole-cell membrane capacitance measurements during the pre- and the test pulse were performed to quantify exocytosis (average traces are shown). (b) Uncaging a PI(4,5)P2 variant featuring a non-natural short-chain fatty acid composition (1a,b in Figure 1a) increased exocytosis during the test pulse. (ci) Uncaging of PI(4,5)P2 with the natural fatty acid composition (SAG, compound 2a,b, Figure 1a,b) had similar effects. (cii) Depolarization-induced cumulative currents (charges, Q, which mostly originate from Ca2+-currents) were similar between both groups for all 10 depolarization steps of pre- and test pulse. Insert: average currents during the first 100 ms depolarization, dashed line indicates baseline. Scale bar in the insert: 0.5 nA and 50 ms. See Figure 4—figure supplement 2 for corresponding analysis of compound 1a,b. Number of cells (n): n = 27 (wild type control, loaded with 1a,b), n = 26 (wild type PI(4,5)P2 uncaging, loaded with 1a,b); n = 23 (wild type control, loaded with 2a,b), n = 23 (wild type PI(4,5)P2 uncaging, loaded with 2a,b).

Figure 4.

Figure 4—figure supplement 1. Incubation with cg-PI(4,5)P2 does not affect exocytosis.

Figure 4—figure supplement 1.

Chromaffin cells were incubated with either 0.02% pluronic (Control) or 0.02% pluronic +25 µM cg-PI(4,5)P2 (+cg-PI(4,5)P2) for 30 min as described earlier. Reliable uptake of cg- PI(4,5)P2 was verified by the coumarin fluorescence at the end of the recording session (only for the +cg-PI(4,5)P2 group). (a) Average capacitance traces of exocytosis (left panel) elicited during the pre-pulse (a) and test pulse (b) stimulations of cells incubated with pluronic (Control) (dark red) or pluronic +cg-PI(4,5)P2 (light blue) and quantification (mean ±S.E.M) (right panel). The cells were not subjected to UV-light between the pre-pulse and test pulse. The release was identical from both groups during the pre-pulse and test pulse stimulation. Secretion from both groups recovered to comparable magnitudes during the test pulse as estimated by the CM (membrane capacitance) recovery (test pulse ΔCM_4 s/pre-pulse ΔCM_4 s) indicating that release was not influenced by the loading of cg-PI(4,5)P2. The calcium charges were calculated by integrating the currents during the depolarization pulses. Charges ratio were calculated by dividing the sum of charges in test pulse by the sum of charges in the pre-pulse. All the parameters were statistically tested using Student’s two-tailed t-test: pre-pulse: (IRP) p=0.9060, (RRP-IRP) p=0.9469, (total-RRP) p=0.4634, (calcium charges) p=0.8425; test pulse: (IRP) p=0.4078, (RRP-IRP) p=0.5360, (total-RRP) p=0.6249, (calcium charges) p=0.9344, (CM recovery) p=0.7082, (charges ratio) p=0.6618. Scale bars 25 fF/1 s. Number of cells (n) in electrophysiological recordings: n = 24 (Control), n = 21 (+cg-PI(4,5)P2).

Figure 4—figure supplement 2. Uncaging of PI(4,5)P2 DOG (compound 1a,b in Figure 1a) does not alter depolarization-induced currents.

Figure 4—figure supplement 2.

Depolarization-induced cumulative currents (charges mostly originating from Ca2+-currents) corresponding to the capacitance traces depicted in Figure 4b during the pre-pulse (left) and the test pulse (right). Uncaging of the PI(4,5)P2 analog (compound 1a,b) did not lead to increased depolarization induced charges. Number of cells (n): n = 27 (wild type control, loaded with 1a,b), n = 26 (wild type PI(4,5)P2 uncaging, loaded with 1a,b).

Figure 4—figure supplement 3. Uncaging of PI(4,5)P2 does not cause an increase of intracellular [Ca2+] but enhances the rate of single vesicle fusion events.

Figure 4—figure supplement 3.

All cells were loaded with compound 2a,b, (Figure 1) and analyzed following a depolarization pre-pulse protocol as depicted in Figure 4a (not shown). (a) Measurements (once every second) of intracellular Ca2+ concentrations. A possible effect of PI(4,5)P2 uncaging was investigated. In the left panel no UV light for uncaging was applied (control, grey). Ca2+ levels are elevated due to the preceding pre-pulse and show relaxation behavior towards baseline levels. In the middle panel, a possible effect PI(4,5)P2 uncaging on intracellular Ca2+ concentration was investigated (PI(4,5)P2 uncaging group, blue). The timing of the flash is indicated by the vertical red dotted line. No increase of intracellular Ca2+ was observed upon uncaging. In the right panel, the ratios of the Ca2+ concentrations after uncaging divided by the Ca2+ levels prior to the flash were investigated in a cell-wise manner. No effect of PI(4,5)P2 uncaging was seen. (b) PI(4,5)P2 uncaging increases the rate of single vesicle fusion events monitored by amperometry, indicating an effect of PI(4,5)P2 uncaging on release in the absence of an effect on [Ca2+]. Amperometric current (continuous trace; left ordinate axis) to detect catecholamine release from single vesicle fusion events (spikes) and mean spike frequency (points with error bars depicting SEM; right ordinate axis) during a recording at a holding potential of Vm = −70 mV. In the left panel no UV light was applied (control, grey). In the middle panel PI(4,5)P2 was uncaged and the average frequency of vesicle fusion events was increased (PI(4,5)P2 uncaging group, blue). Please note that the transient coinciding with the uncaging flash is due to an artifact induced by the photoelectric effect when the light hits the amperometric fiber). The right panel depicts the ratio of single vesicle fusion frequencies in the 2.5 s bin after uncaging divided by the frequency in the 2.5 s bin preceding the flash. Opposite behaviors for both groups are seen. (c) Frequencies of single vesicle fusion events transiently increase following PI(4,5)P2 uncaging. Same data as in panel (b), but with extended view of event frequencies following the first two bins depicted above (note the extended time axis). The event frequency in both groups was investigated in 2.5 s long bins. The red vertical dashed line indicates the timing of the UV flash given in the uncaging group. All data are shown as mean ±SEM. Number of cells, n = 20 (control, loaded with cg-PI(4,5)P2 but not subjected to UV), n = 23 (PI(4,5)P2 uncaging). P values denote result of two-tailed Student’s t-test.