We studied wild-type (WT) andCav1.3^−/−mouse chromaffin cells (MCCs) with the aim to determine the isoform of L-type Ca²⁺channel (LTCC) and BK channels that underlie the pacemaker current controlling spontaneous firing. Most WT-MCCs (80%) were spontaneously active (1.5 Hz) and highly sensitive to nifedipine and BayK-8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid, methyl ester). Nifedipine blocked the firing, whereas BayK-8644 increased threefold the firing rate. The two dihydropyridines and the BK channel blocker paxilline altered the shape of action potentials (APs), suggesting close coupling of LTCCs to BK channels. WT-MCCs expressed equal fractions of functionally active Cav1.2 and Cav1.3 channels. Cav1.3 channel deficiency decreased the number of normally firing MCCs (30%; 2.0 Hz), suggesting a critical role of these channels on firing, which derived from their slow inactivation rate, sizeable activation at subthreshold potentials, and close coupling to fast inactivating BK channels as determined by using EGTA and BAPTA Ca²⁺buffering. By means of the action potential clamp, in TTX-treated WT-MCCs, we found that the interpulse pacemaker current was always net inward and dominated by LTCCs. Fast inactivating and non-inactivating BK currents sustained mainly the afterhyperpolarization of the short APs (2–3 ms) and only partially the pacemaker current during the long interspike (300–500 ms). Deletion of Cav1.3 channels reduced drastically the inward Ca²⁺current and the corresponding Ca²⁺-activated BK current during spikes. Our data highlight the role of Cav1.3, and to a minor degree of Cav1.2, as subthreshold pacemaker channels in MCCs and open new interesting features about their role in the control of firing and catecholamine secretion at rest and during sustained stimulations matching acute stress.

Long-lasting exposures to brain-derived neurotrophic factor (BDNF) accelerate the functional maturation of GABAergic transmission in embryonic hippocampal neurons, but the molecular bases of this phenomenon are still debated. Evidence in favor of a postsynaptic site of action has been accumulated, but most of the data support a presynaptic site effect. A crucial issue is whether the enhancement of evoked IPSCs (eIPSCs) induced by BDNF is attributable to an increase in any of the elementary parameters controlling neurosecretion, namely the probability of release, the number of release sites, the readily releasable pool (RRP), and the quantal size. Here, using peak-scaled variance analysis of miniature IPSCs, multiple probability fluctuation analysis, and cumulative amplitude analysis of action potential-evoked postsynaptic currents, we show that BDNF increases release probability and vesicle replenishment with little or no effect on the quantal size, the number of release sites, the RRP, and the Ca²⁺dependence of eIPSCs. BDNF treatment changes markedly the distribution of Ca²⁺channels controlling neurotransmitter release. It enhances markedly the contribution of N- and P/Q-type channels, which summed to >100% (“supra-additivity”), and deletes the contribution of R-type channels. BDNF accelerates the switch of presynaptic Ca²⁺channel distribution from “segregated” to “nonuniform” distribution. This maturation effect was accompanied by an uncovered increased control of N-type channels on paired-pulse depression, otherwise dominated by P/Q-type channels in untreated neurons. Nevertheless, BDNF preserved the fast recovery from depression associated with N-type channels. These novel presynaptic BDNF actions derive mostly from an enhanced overlapping and better colocalization of N- and P/Q-type channels to vesicle release sites.

T‐type channels are expressed weakly or not at all in adult rat chromaffin cells (RCCs) and there is contrasting evidence as to whether they play a functional role in catecholamine secretion. Here we show that 3–5 days after application of pCPT‐cAMP, most RCCs grown in serum‐free medium expressed a high density of low‐voltage‐activated T‐type channels without altering the expression and characteristics of high‐voltage‐activated channels. The density of cAMP‐recruited T‐type channels increased with time and displayed the typical biophysical and pharmacological properties of low‐voltage‐activated Ca²⁺ channels: (1) steep voltage‐dependent activation from −50 mV in 10 mm Ca²⁺, (2) slow deactivation but fast and complete inactivation, (3) full inactivation following short conditioning prepulses to −30 mV, (4) effective block of Ca²⁺ influx with 50 μm Ni²⁺, (5) comparable permeability to Ca²⁺ and Ba²⁺, and (6) insensitivity to common Ca²⁺ channel antagonists. The action of exogenous pCPT‐cAMP (200 μm) was prevented by the protein synthesis inhibitor anisomycin and mimicked in most cells by exposure to forskolin and 1‐methyl‐3‐isobutylxanthine (IBMX) or isoprenaline. The protein kinase A (PKA) inhibitor H89 (0.3 μm) and the competitive antagonist of cAMP binding to PKA, Rp‐cAMPS, had weak or no effect on the action of pCPT‐cAMP. In line with this, the selective Epac agonist 8CPT‐2Me‐cAMP nicely mimicked the action of pCPT‐cAMP and isoprenaline, suggesting the existence of a dominant Epac‐dependent recruitment of T‐type channels in RCCs that may originate from the activation of β‐adrenoceptors. Stimulation of β‐adrenoceptors occurs autocrinally in RCCs and thus, the neosynthesis of low‐voltage‐activated channels may represent a new form of ‘chromaffin cell plasticity’, which contributes, by lowering the threshold of action potential firing, to increasing cell excitability and secretory activity during sustained sympathetic stimulation and/or increased catecholamine circulation.