Low-Threshold Exocytosis Induced by cAMP-Recruited CaV3.2 (α1H) Channels in Rat Chromaffin Cells

Abstract

We have studied the functional role of CaV3 channels in triggering fast exocytosis in rat chromaffin cells (RCCs). CaV3 T-type channels were selectively recruited by chronic exposures to cAMP (3 days) via an exchange protein directly activated by cAMP (Epac)-mediated pathway. Here we show that cAMP-treated cells had increased secretory responses, which could be evoked even at very low depolarizations (-50, -40 mV). Potentiation of exocytosis in cAMP-treated cells did not occur in the presence of 50 microM Ni2+, which selectively blocks T-type currents in RCCs. This suggests that the "low-threshold exocytosis" induced by cAMP is due to increased Ca2+ influx through cAMP-recruited T-type channels, rather than to an enhanced secretion downstream of Ca2+ entry, as previously reported for short-term cAMP treatments (20 min). Newly recruited T-type channels increase the fast secretory response at low voltages without altering the size of the immediately releasable pool. They also preserve the Ca2+ dependence of exocytosis, the initial speed of vesicle depletion, and the mean quantal size of single secretory events. All this indicates that cAMP-recruited CaV3 channels enhance the secretory activity of RCCs at low voltages by coupling to the secretory apparatus with a Ca2+ efficacy similar to that of already existing high-threshold Ca2+ channels. Finally, using RT-PCRs we found that the fast inactivating low-threshold Ca2+ current component recruited by cAMP is selectively associated to the alpha1H (CaV3.2) channel isoform.

Fast exocytosis mediated by T- and L-type channels in chromaffin cells: distinct voltage-dependence but similar Ca2+-dependence

“…Here, by reanalyzing and summing-up the results of two recent papers Giancippoli et al 2006), we show that in chromaYn cells the two channels (T-and L-type) although lacking a "synprint" region, appear equally eVective in controlling neurotransmitter release despite their diVerent voltagedependence, activation-inactivation gating and degree of expression. A rigorous analysis of the biophysical parameters associated with the excitation-secretion coupling of the two channels shows close similarities.…”

“…To do this, one needs to determine the size of the immediately releasable pool (IRP), the rate of vesicle release, the probability of release and the Ca 2+ -dependence of secretion when the two channels are selectively activated Giancippoli et al 2006). According to the model described by Voets (2000), secretory granules in chromaYn cells are distributed into three diVerent pools, with their own size, kinetics of release and fusion competence.…”

“…e I-V curves for T and L-type channels obtained using ramp commands from ¡60 to +60 mV (see Cesetti et al 2003 andGiancippoli et al 2006 for details). Notice the presence of a single peak for the recording of L-type current (grey trace) and the dual peak when measuring the T-type current (black trace).…”

“…In the latter case, the peak at more positive voltage is due to contamination of R-type currents insensitive to -toxins and nifedipine. Adapted from Carabelli et al (2003), Giancippoli et al (2006) and from unpublished data taken from the work of Cesetti et al (2003) 123 increases ( C 1 and C 2 ) and are requested to carry the same quantity of Ca 2+ charge. In this way, from the amplitude of C 2 one can estimate the percentage of vesicle depletion that occurs during the Wrst pulse ( C 1 ) and with simple calculation one can determine the size of IRP and the probability of release (P).…”

“…This slower exocytotic process is primarily controlled by Ca 2+ inXux through multiple isoforms of high-voltage activated Ca 2+ channels, which contribute to secretion proportionally to their density of expression and gating properties, with no speciWc requirements of Ca 2+ channels colocalization to the release sites (Kim et al 1995;Engisch and Nowycky 1996;Klingauf and Neher 1997;Lukyanetz and Neher 1999;Ulate et al 2000;Carabelli et al 2003;Chan et al 2005;Giancippoli et al 2006;Polo-Parada et al 2006). Alternatively, other groups suggest a preferential role of some channel subtype in close proximity to the secretory sites (Lopez et al 1994;Artalejo et al 1994;Lara et al 1998;O'Farrel and Marley 2000;Albillos et al 2000) and a further role of mitochondria in sequestering Ca 2+ entering through Ca 2+ channels (Ales et al 2005).…”