Photoinactivation of the V0 a1-I or V1 A subunits impairs chromaffin cell catecholamine release in different ways. Chromaffin cells coexpressing either the V0 a1-I or the V1 A subunits bearing a Flag-TC tag and GFP were successively incubated with the FlAsH-EDT2 probe that specifically binds to the TC motif and 1 mM BAL. Photoinactivation was obtained by a 1-min illumination. Catecholamine release was evoked by a 10-s application of 100 mM KCl onto the recorded cell and measured by carbon fiber amperometry. Control release was measured from untransfected cells in the same culture dish or from cells that express GFP alone (both submitted to the FlAsH-EDT2 and BAL treatments and the flash of light). For comparison, catecholamine release from untransfected cells that were only treated by 0.4 µM bafilomycin A1 was measured in parallel. (A) Typical amperometric recordings from a control cell (control); a cell expressing the V0 recombinant a1-I subunit, 5 min after the photoinactivating flash (Flag-TC-a1-I 5′); cells expressing the V1 recombinant A subunit, 5 or 30 min after the flash (Flag-TC-A 5′ and 30’, respectively); and untransfected cells after 5 or 30 min of bafilomycin A1 application (bafilomycin 5′ and 30’, respectively). (B) Number of amperometric spikes detected per cell in the various conditions (means ± SD from 25 to 55 cells). Control cells, 5 or 30 min after the flash (control 5′ and 30’, respectively); cells expressing the V0 a1-I subunit with the Flag-TC tag in N- or C-terminal position, 5 min after the flash (TC-a1-I and a1-I-TC); cells expressing the V1 Flag-TC-A subunit, 5 and 30 min after the flash (TC-A 5′ and TC-A 30’, respectively); and cells bafilomycin A1 treated for 5 or 30 min (bafilo 5′ and 30’). (C) Scheme showing the different parameters of the amperometric response that were measured. A foot current was detected for ∼20–30% of amperometric spikes, regardless the conditions tested. (D) Main characteristics of amperometric spikes in the different conditions tested (as in B). (E) Characteristics of foot currents in the different conditions tested (as in B). Data are means ± SEM (except for B). *, P < 0.05; ***, P < 0.001.
