Valentina Carabelli

Loss of Cav1.3 Channels Reveals the Critical Role of L-Type and BK Channel Coupling in Pacemaking Mouse Adrenal Chromaffin Cells

We studied wild-type (WT) and Cav1.3−/− 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.

Chronic hypoxia up‐regulates α1H T‐type channels and low‐threshold catecholamine secretion in rat chromaffin cells

α1H T‐type channels recruited by β1‐adrenergic stimulation in rat chromaffin cells (RCCs) are coupled to fast exocytosis with the same Ca2+ dependence of high‐threshold Ca2+ channels. Here we show that RCCs exposed to chronic hypoxia (CH) for 12–18 h in 3% O2 express comparable densities of functional T‐type channels that depolarize the resting cells and contribute to low‐voltage exocytosis. Following chronic hypoxia, most RCCs exhibited T‐type Ca2+ channels already available at −50 mV with the same gating, pharmacological and molecular features as the α1H isoform. Chronic hypoxia had no effects on cell size and high‐threshold Ca2+ current density and was mimicked by overnight incubation with the iron‐chelating agent desferrioxamine (DFX), suggesting the involvement of hypoxia‐inducible factors (HIFs). T‐type channel recruitment occurred independently of PKA activation and the presence of extracellular Ca2+. Hypoxia‐recruited T‐type channels were partially open at rest (T‐type ‘window‐current’) and contributed to raising the resting potential to more positive values. Their block by 50 μm Ni2+ caused a 5–8 mV hyperpolarization. The secretory response associated with T‐type channels could be detected following mild cell depolarizations, either by capacitance increases induced by step depolarizations or by amperometric current spikes induced by increased [KCl]. In the latter case, exocytotic bursts could be evoked even with 2–4 mm KCl and spike frequency was drastically reduced by 50 μm Ni2+. Chronic hypoxia did not alter the shape of spikes, suggesting that hypoxia‐recruited T‐type channels increase the number of secreted vesicles at low voltages, without altering the mechanism of catecholamine release and the quantal content of released molecules.

Brain-Derived Neurotrophic Factor Enhances GABA Release Probability and Nonuniform Distribution of N- and P/Q-Type Channels on Release Sites of Hippocampal Inhibitory Synapses

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.

BDNF up‐regulates evoked GABAergic transmission in developing hippocampus by potentiating presynaptic N‐ and P/Q‐type Ca2+ channels signalling

Chronic application of brain‐derived neurotrophic factor (BDNF) induces new selective synthesis of non‐L‐type Ca2+ channels (N, P/Q, R) at the soma of cultured hippocampal neurons. As N‐ and P/Q‐channels support neurotransmitter release in the hippocampus, this suggests that BDNF‐treatment may enhance synaptic transmission by increasing the expression of presynaptic Ca2+ channels as well. To address this issue we studied the long‐term effects of BDNF on miniature and stimulus‐evoked GABAergic transmission in rat embryo hippocampal neurons. We found that BDNF increased the frequency of miniature currents (mIPSCs) by ≈40%, with little effects on their amplitude. BDNF nearly doubled the size of evoked postsynaptic currents (eIPSCs) with a marked increase of paired‐pulse depression, which is indicative of a major increase in presynaptic activity. The potentiation of eIPSCs was more relevant during the first two weeks in culture, when GABAergic transmission is depolarizing. BDNF action was mediated by TrkB‐receptors and had no effects on: (i) the amplitude and dose–response of GABA‐evoked IPSCs and (ii) the number of GABAA receptor clusters and the total functioning synapses, suggesting that the neurotrophin unlikely acted postsynaptically. In line with this, BDNF affected the contribution of voltage‐gated Ca2+ channels mediating evoked GABAergic transmission. BDNF drastically increased the fraction of evoked IPSCs supported by N‐ and P/Q‐channels while it decreased the contribution associated with R‐ and L‐types. This selective action resembles the previously observed up‐regulatory effects of BDNF on somatic Ca2+ currents in developing hippocampus, suggesting that potentiation of presynaptic N‐ and P/Q‐channel signalling belongs to a manifold mechanism by which BDNF increases the efficiency of stimulus‐evoked GABAergic transmission.

Exposure to cAMP and β‐adrenergic stimulation recruits CaV3 T‐type channels in rat chromaffin cells through Epac cAMP‐receptor proteins

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.

Localized Secretion of ATP and Opioids Revealed through Single Ca2+ Channel Modulation in Bovine Chromaffin Cells

In bovine chromaffin cells, the Ca2+ channels involved in exocytosis are effectively inhibited by ATP and opioids that are coreleased with catecholamines during cell activity. This autocrine loop causes a delay in Ca2+ channel activation that is quickly removed by preceding depolarizations. Changes in Ca2+ channel gating by secreted products thus make it possible to correlate Ca2+ channel activity to secretory events. Here, using cell-attached patch recordings, we found a remarkable correlation between delayed Ca2+ channel openings and neurotransmitter secretion induced by either local or whole-cell Ba2+ stimulation. The action is specific for N- and P/Q-type channels and largely prevented by PTX and mixtures of purinergic and opioid receptor antagonists. Overall, our data provide evidence that exocytosis, viewed through the autocrine inhibition of non-L-type channels, is detectable in membrane patches of approximately 1 microm2 distributed over 30%-40% of the total cell surface, while Ca2+ channels and autoreceptors are uniformly distributed over most of the cell membrane.

Voltage-dependent modulation of single N-Type Ca2+ channel kinetics by receptor agonists in IMR32 cells

The voltage-dependent inhibition of single N-type Ca(2+) channels by noradrenaline (NA) and the delta-opioid agonist D-Pen(2)-D-Pen (5)-enkephalin (DPDPE) was investigated in cell-attached patches of human neuroblastoma IMR32 cells with 100 mM Ba(2+) and 5 microM nifedipine to block L-type channels. In 70% of patches, addition of 20 microM NA + 1 microM DPDPE delayed markedly the first channel openings, causing a four- to fivefold increase of the first latency at +20 mV. The two agonists or NA alone decreased also by 35% the open probability (P(o)), prolonged partially the mean closed time, and increased the number of null sweeps. In contrast, NA + DPDPE had little action on the single-channel conductance (19 versus 19.2 pS) and minor effects on the mean open time. Similarly to macroscopic Ba(2+) currents, the ensemble currents were fast activating at control but slowly activating and depressed with the two agonists. Inhibition of single N-type channels was effectively removed (facilitated) by short and large depolarizations. Facilitatory pre-pulses increased P(o) significantly and decreased fourfold the first latency. Ensemble currents were small and slowly activating before pre-pulses and became threefold larger and fast decaying after facilitation. Our data suggest that slowdown of Ca(2+) channel activation by transmitters is mostly due to delayed transitions from a modified to a normal (facilitated) gating mode. This single-channel gating modulation could be well simulated by a Monte Carlo method using previously proposed kinetic models predicting marked prolongation of first channel openings.

Nitric oxide inhibits neuroendocrine CaV1 L-channel gating via cGMP-dependent protein kinase in cell-attached patches of bovine chromaffin cells

Nitric oxide (NO) regulates the release of catecholamines from the adrenal medulla but the molecular targets of its action are not yet well identified. Here we show that the NO donor sodium nitroprusside (SNP, 200 μM) causes a marked depression of the single CaV1 L‐channel activity in cell‐attached patches of bovine chromaffin cells. SNP action was complete within 3‐5 min of cell superfusion. In multichannel patches the open probability (NPo) decreased by ∼60 % between 0 and +20 mV. Averaged currents over a number of traces were proportionally reduced and showed no drastic changes to their time course. In single‐channel patches the open probability (Po) at +10 mV decreased by the same amount as that of multichannel patches (∼61 %). Such a reduction was mainly associated with an increased probability of null sweeps and a prolongation of mean shut times, while first latency, mean open time and single‐channel conductance were not significantly affected. Addition of the NO scavenger carboxy‐PTIO or cell treatment with the guanylate cyclase inhibitor ODQ prevented the SNP‐induced inhibition. 8‐Bromo‐cyclicGMP (8‐Br‐cGMP; 400 μM) mimicked the action of the NO donor and the protein kinase G blocker KT‐5823 prevented this effect. The depressive action of SNP was preserved after blocking the cAMP‐dependent up‐regulatory pathway with the protein kinase A inhibitor H89. Similarly, the inhibitory action of 8‐Br‐cGMP proceeded regardless of the elevation of cAMP levels, suggesting that cGMP/PKG and cAMP/PKA act independently on L‐channel gating. The inhibitory action of 8‐Br‐cGMP was also independent of the G protein‐induced inhibition of L‐channels mediated by purinergic and opiodergic autoreceptors. Since Ca2+ channels contribute critically to both the local production of NO and catecholamine release, the NO/PKG‐mediated inhibition of neuroendocrine L‐channels described here may represent an important autocrine signalling mechanism for controlling the rate of neurotransmitter release from adrenal glands.

Voltage-independent autocrine modulation of L-type channels mediated by ATP, opioids and catecholamines in rat chromaffin cells

The inhibition of L‐type channels induced by either bath application of ATP, opioids and catecholamines or by endogenously released neurotransmitters was investigated in rat chromaffin cells with whole‐cell recordings (5 mm Ba2+). In both cases, the L‐type current, isolated pharmacologically using ω‐toxin peptides and potentiated by Bay K 8644, was inhibited by ∼ 50% with nearly no changes to the activation–inactivation kinetics. Inhibition was voltage independent at a wide range of potentials (–20 to +50 mV) and insensitive to depolarizing prepulses (+100 mV, 50 ms). Onset and offset of the inhibition were fast (time constants: τon ∼ 0.9 s, τoff ∼ 3.6 s), indicating a rapid mechanism of channel modulation. Whether induced exogenously or from the released granules content in conditions of stopped cell superfusion, the neurotransmitter action was reversible and largely prevented by either intracellular GDP‐β‐S, cell treatment with pertussis toxin or simultaneous application of P2y,2x δ/μ‐opioidergic and α/β‐adrenergic antagonists. This suggests the existence of converging modulatory pathways by which autoreceptors‐activated G‐proteins reduce the activity of L‐type channels through fast interactions. The autocrine inhibition of L‐type currents, which was absent in superfused isolated cells, was effective on cell clusters, suggesting that L‐type channels may be potently inhibited by cell exocytosis under physiological conditions resembling the intact adrenal glands.

Ca2+ and Na+ permeability of high‐threshold Ca2+ channels and their volt age‐dependent block by Mg2+ ions in chick sensory neurones

1 The Mg2+ block of Na+ and Ca2+ currents through high‐voltage activated (HVA; L‐ and N‐type) Ca2+ channels was studied in chick dorsal root ganglion neurones. 2 In low extracellular [Ca2+] (< 10−8 M) and with Nao+ and Csi+ as the main charge carriers (120 mm), HVA Na+ currents started to activate at −40 mV, reached inward peak values near 0 mV and reversed at about +40 mV. 3 Addition of 30–500 μm Mg2+ to the bath caused a strong depression of inward Na+ currents that was voltage and dose dependent (KD=39 μm in 120 mm Na+ at −10 mV). The block was maximal at negative potentials (< −70 mV) and decreased with increasing positive potentials, suggesting that Mg2+ cannot escape to the cell interior. 4 Block of Ca2+ currents by Mg2+ was also voltage dependent, but by three orders of magnitude less potent than with Na+ currents (KD= 24 mm in 2 mm Ca2+ at −30 mV). The high concentration of Mg2+ caused a prominent voltage shift of channel gating kinetics induced by surface charge screening effects. To compensate for this, Mg2+ block of inward Ca2+ currents was estimated from the instantaneous I–V relationships on return from very positive potentials (+100 mV). 5 Inward Na+ and Ca2+ tail currents following depolarization to +90 mV were markedly depressed, suggesting that channels cleared of Mg2+ ions during strong depolarization are quickly re‐blocked on return to negative potentials. The kinetics of re‐block by Mg2+ was too fast (< 100 μs) to be resolved by our recording apparatus. This implies a rate of entry for Mg2+ > 1.45 × 108 M− s−1 when Na+ is the permeating ion and a rate approximately 3 orders of magnitude smaller for Ca2+. 6 Mg2+ unblock of HVA Na+ currents at +100 mV was independent of the size of outward currents, whether Na+, Cs+ or NMG+ were the main internal cations. 7 Consistent with the idea of a high‐affinity binding site for Ca2+ inside the channel, micromolar amounts of Ca2+ caused a strong depression of Na+ currents between −40 and 0 mV, which was effectively relieved with more positive as well as with negative potentials (KD= 0.7μm in 120 mm Na+ at −20 mV). In this case, the kinetics of re‐block could be resolved and gave rates of entry and exit for Ca2+ of 1.4 × 108 M−1 s−1 and 2.95 × 102 s−1, respectively. 8 The strong voltage dependence and weak current dependence of HVA channel block by divalent cations and the markedly different KD values of Na+ and Ca2+ current block by Mg2+ can be well described by a previously proposed model for Ca2+ channel permeation based on interactions between the permeating ion and the negative charges forming the high‐affinity binding site for Ca2+ inside the pore ( Lux, Carbone & Zucker, 1990 ).