Sustained Exocytosis after Action Potential-Like Stimulation at Low Frequencies in Mouse Chromaffin Cells Depends on a Dynamin-Dependent Fast Endocytotic Process

Abstract

Under basal conditions the action potential firing rate of adrenal chromaffin cells is lower than 0.5 Hz. The maintenance of the secretory response at such frequencies requires a continuous replenishment of releasable vesicles. However, the mechanism that allows such vesicle replenishment remains unclear. Here, using membrane capacitance measurements on mouse chromaffin cells, we studied the mechanism of replenishment of a group of vesicles released by a single action potential-like stimulus (APls). The exocytosis triggered by APls (ETAP) represents a fraction (40%) of the immediately releasable pool, a group of vesicles highly coupled to voltage dependent calcium channels. ETAP was replenished with a time constant of 0.73 ± 0.11 s, fast enough to maintain synchronous exocytosis at 0.2–0.5 Hz stimulation. Regarding the mechanism involved in rapid ETAP replenishment, we found that it depends on the ready releasable pool; indeed depletion of this vesicle pool significantly delays ETAP replenishment. On the other hand, ETAP replenishment also correlates with a dynamin-dependent fast endocytosis process (τ = 0.53 ± 0.01 s). In this regard, disruption of dynamin function markedly inhibits the fast endocytosis and delays ETAP replenishment, but also significantly decreases the synchronous exocytosis during repetitive APls stimulation at low frequencies (0.2 and 0.5 Hz). Considering these findings, we propose a model in where both the transfer of vesicles from ready releasable pool and fast endocytosis allow rapid ETAP replenishment during low stimulation frequencies.

Ca2+‐independent and voltage‐dependent exocytosis in mouse chromaffin cells

“…Figure A represents a typical recording of CIVDE, where the following decay was fitted with a single exponential function with a time constant 0.9 seconds (white line). The averaged time constant obtained from these experiments was 0.56 ± 0.12 seconds (n = 14), which is similar to the one estimated previously for fast endocytosis induced after brief depolarizations . It is important to mention that this decay in capacitance has a similar amplitude than the preceding exocytosis (Figure B), suggesting that it is a compensatory endocytotic process.…”

“…For practical reasons we applied a voltage template composed by a 2.5 ms ascendant voltage ramp from −80 to + 50 mV, followed by a 2.5 ms descendant ramp that returns to holding potential (see methods), which is a modification of the template applied by Chan and collaborators . We demonstrated before that this action potential‐like stimulus (AP ls ) induces similar I Ca2+ and C m changes than the real action potentials measured in our cells . Figure A shows typical C m recordings obtained in cells stimulated with individual AP ls in presence of 5 mM Ca 2+ (black), 5 mM Ca 2+ plus 100 µM Cd 2+ (red), and Ca 2+ ‐free external solution (blue).…”

“…In some cells, immediately after the application of a depolarization pulse, we noted the presence of a brief transient capacitance change, probably associated to sodium channels gating . In our preparation this transient became negligible 50 ms after the end of depolarization . We avoided any influence of this fast capacitance transient by measuring the capacitance starting 100 ms after the end of the depolarization …”

“…To evaluate if the Cm decay after CIVDE is associated to a dynamin‐dependent endocytosis, we dialysed an anti‐dynamin monoclonal antibody (Anti‐Dyn) through patch pipette for 7 minutes after the establishment of whole‐cell configuration . Then, as in previous experiments, we applied a 100 ms depolarizing pulse on cells bathed in Ca +2 ‐free solution to induce CIVDE.…”

“…Our data suggest that this exocytotic process is coupled to the activation of P/Q‐type VDCC. Chromaffin cells at rest conditions fires action potentials at low frequencies, where Ca 2+ does not accumulate between stimuli and exocytosis would be limited to vesicles closely coupled to VDCC . So, it is likely that the importance of CIVDE resides in its highly efficient stimulus‐exocytosis coupling and high rate of recovery, which can allows its participation in secretion during action potentials at low rates.…”