David Vandael

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.

Excitation of histaminergic tuberomamillary neurons by thyrotropin-releasing hormone.

The histaminergic tuberomamillary nucleus (TMN) controls arousal and attention, and the firing of TMN neurons is state-dependent, active during waking, silent during sleep. Thyrotropin-releasing hormone (TRH) promotes arousal and combats sleepiness associated with narcolepsy. Single-cell reverse-transcription-PCR demonstrated variable expression of the two known TRH receptors in the majority of TMN neurons. TRH increased the firing rate of most (ca 70%) TMN neurons. This excitation was abolished by the TRH receptor antagonist chlordiazepoxide (CDZ; 50 μm). In the presence of tetrodotoxin (TTX), TRH depolarized TMN neurons without obvious change of their input resistance. This effect reversed at the potential typical for nonselective cation channels. The potassium channel blockers barium and cesium did not influence the TRH-induced depolarization. TRH effects were antagonized by inhibitors of the Na+/Ca2+ exchanger, KB-R7943 and benzamil. The frequency of GABAergic spontaneous IPSCs was either increased (TTX-insensitive) or decreased [TTX-sensitive spontaneous IPSCs (sIPSCs)] by TRH, indicating a heterogeneous modulation of GABAergic inputs by TRH. Facilitation but not depression of sIPSC frequency by TRH was missing in the presence of the κ-opioid receptor antagonist nor-binaltorphimine. Montirelin (TRH analog, 1 mg/kg, i.p.) induced waking in wild-type mice but not in histidine decarboxylase knock-out mice lacking histamine. Inhibition of histamine synthesis by (S)-α-fluoromethylhistidine blocked the arousal effect of montirelin in wild-type mice. We conclude that direct receptor-mediated excitation of rodent TMN neurons by TRH demands activation of nonselective cation channels as well as electrogenic Na+/Ca2+ exchange. Our findings indicate a key role of the brain histamine system in TRH-induced arousal.

Calcium channel types contributing to chromaffin cell excitability, exocytosis and endocytosis

Voltage gated Ca(2+) channels are effective voltage sensors of plasma membrane which convert cell depolarizations into Ca(2+) signaling. The chromaffin cells of the adrenal medulla utilize a large number of Ca(2+) channel types to drive the Ca(2+)-dependent release of catecholamines into blood circulation, during normal or stress-induced conditions. Some of the Ca(2+) channels expressed in chromaffin cells (L, N, P/Q, R and T), however, do not control only vesicle fusion and catecholamine release. They also subserve a variety of key activities which are vital for the physiological and pathological functioning of the cell, like: (i) shaping the action potentials of electrical oscillations driven either spontaneously or by ACh stimulation, (ii) controlling the action potential frequency of tonic or bursts firing, (iii) regulating the compensatory and excess endocytosis following robust exocytosis and (iv) driving the remodeling of Ca(2+) signaling which occurs during stressors stimulation. Here, we will briefly review the well-established properties of voltage-gated Ca(2+) channels accumulated over the past three decades focusing on the most recent discoveries on the role that L- (Cav1.2, Cav1.3) and T-type (Cav3.2) channels play in the control of excitability, exocytosis and endocytosis of chromaffin cells in normal and stress-mimicking conditions.

Ca(V)1.3-Driven SK Channel Activation Regulates Pacemaking and Spike Frequency Adaptation in Mouse Chromaffin Cells

Mouse chromaffin cells (MCCs) fire spontaneous action potentials (APs) at rest. Cav1.3 L-type calcium channels sustain the pacemaker current, and their loss results in depolarized resting potentials (Vrest), spike broadening, and remarkable switches into depolarization block after BayK 8644 application. A functional coupling between Cav1.3 and BK channels has been reported but cannot fully account for the aforementioned observations. Here, using Cav1.3−/− mice, we investigated the role of Cav1.3 on SK channel activation and how this functional coupling affects the firing patterns induced by sustained current injections. MCCs express SK1–3 channels whose tonic currents are responsible for the slow irregular firing observed at rest. Percentage of frequency increase induced by apamin was found inversely correlated to basal firing frequency. Upon stimulation, MCCs build-up Cav1.3-dependent SK currents during the interspike intervals that lead to a notable degree of spike frequency adaptation (SFA). The major contribution of Cav1.3 to the subthreshold Ca2+ charge during an AP-train rather than a specific molecular coupling to SK channels accounts for the reduced SFA of Cav1.3−/− MCCs. Low adaptation ratios due to reduced SK activation associated with Cav1.3 deficiency prevent the efficient recovery of NaV channels from inactivation. This promotes a rapid decline of AP amplitudes and facilitates early onset of depolarization block following prolonged stimulation. Thus, besides serving as pacemaker, Cav1.3 slows down MCC firing by activating SK channels that maintain NaV channel availability high enough to preserve stable AP waveforms, even upon high-frequency stimulation of chromaffin cells during stress responses.

CaV1.3 as pacemaker channels in adrenal chromaffin cells: Specific role on exo- and endocytosis?

Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca2+-dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (CaV1.2 and CaV1.3), CaV1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using CaV1 .3-/- KO mice and the AP-clamp it has been possible to resolve the time course of CaV1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells CaV1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca2+ currents and activate BK channels confers to CaV1.3 the unique feature of driving Ca2+ loading during long interspike intervals and, possibly, to control the Ca2+-dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.

Reduced availability of voltage-gated sodium channels by depolarization or blockade by tetrodotoxin boosts burst firing and catecholamine release in mouse chromaffin cells

Mouse chromaffin cells (MCCs) of the adrenal medulla possess fast‐inactivating Nav channels whose availability alters spontaneous action potential firing patterns and the Ca2+‐dependent secretion of catecholamines. Here, we report MCCs expressing large densities of neuronal fast‐inactivating Nav1.3 and Nav1.7 channels that carry little or no subthreshold pacemaker currents and can be slowly inactivated by 50% upon slight membrane depolarization. Reducing Nav1.3/Nav1.7 availability by tetrodotoxin or by sustained depolarization near rest leads to a switch from tonic to burst‐firing patterns that give rise to elevated Ca2+‐influx and increased catecholamine release. Spontaneous burst firing is also evident in a small percentage of control MCCs. Our results establish that burst firing comprises an intrinsic firing mode of MCCs that boosts their output. This occurs particularly when Nav channel availability is reduced by sustained splanchnic nerve stimulation or prolonged cell depolarizations induced by acidosis, hyperkalaemia and increased muscarine levels.

Cav1.3 and Cav1.2 channels of adrenal chromaffin cells: emerging views on cAMP/cGMP-mediated phosphorylation and role in pacemaking.

Voltage-gated Ca²⁺ channels (VGCCs) are voltage sensors that convert membrane depolarizations into Ca²⁺ signals. In the chromaffin cells of the adrenal medulla, the Ca²⁺ signals driven by VGCCs regulate catecholamine secretion, vesicle retrievals, action potential shape and firing frequency. Among the VGCC-types expressed in these cells (N-, L-, P/Q-, R- and T-types), the two L-type isoforms, Ca(v)1.2 and Ca(v)1.3, control key activities due to their particular activation-inactivation gating and high-density of expression in rodents and humans. The two isoforms are also effectively modulated by G protein-coupled receptor pathways delimited in membrane micro-domains and by the cAMP/PKA and NO/cGMP/PKG phosphorylation pathways which induce prominent Ca²⁺ current changes if opposingly regulated. The two L-type isoforms shape the action potential and directly participate to vesicle exocytosis and endocytosis. The low-threshold of activation and slow rate of inactivation of Ca(v)1.3 confer to this channel the unique property of carrying sufficient inward current at subthreshold potentials able to activate BK and SK channels which set the resting potential, the action potential shape, the cell firing mode and the degree of spike frequency adaptation during spontaneous firing or sustained depolarizations. These properties help chromaffin cells to optimally adapt when switching from normal to stress-mimicking conditions. Here, we will review past and recent findings on cAMP- and cGMP-mediated modulations of Ca(v)1.2 and Ca(v)1.3 and the role that these channels play in the control of chromaffin cell firing. This article is part of a Special Issue entitled: Calcium channels.

Pyrimidine-2,4,6-triones are a new class of voltage-gated L-type Ca2+ channel activators

Cav1.2 and Cav1.3 are the main L-type Ca2+ channel subtypes in the brain. Cav1.3 channels have recently been implicated in the pathogenesis of Parkinson’s disease. Therefore, Cav1.3-selective blockers are developed as promising neuroprotective drugs. We studied the pharmacological properties of a pyrimidine-2,4,6-trione derivative (1-(3-chlorophenethyl)-3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione, Cp8) recently reported as the first highly selective Cav1.3 blocker. Here we show, in contrast to this previous study, that Cp8 reproducibly increases inward Ca2+ currents of Cav1.3 and Cav1.2 channels expressed in tsA-201 cells by slowing activation, inactivation and enhancement of tail currents. Similar effects are also observed for native Cav1.3 and Cav1.2 channels in mouse chromaffin cells, while non-L-type currents are unaffected. Evidence for a weak and non-selective inhibition of Cav1.3 and Cav1.2 currents is only observed in a minority of cells using Ba2+ as charge carrier. Therefore, our data identify pyrimidine-2,4,6-triones as Ca2+ channel activators.

Are Cav1.3 pacemaker channels in chromaffin cells? Possible bias from resting cell conditions and DHP blockers usage

Mouse and rat chromaffin cells (MCCs, RCCs) fire spontaneously at rest and their activity is mainly supported by the two L-type Ca2+ channels expressed in these cells (Cav1.2 and Cav1.3). Using Cav1.3-/- KO MCCs we have shown that Cav1.3 possess all the prerequisites for carrying subthreshold currents that sustain low frequency cell firing near resting (0.5 to 2 Hz at -50 mV)1: low-threshold and steep voltage dependence of activation, slow and incomplete inactivation during pulses of several hundreds of milliseconds. Cav1.2 contributes also to pacemaking MCCs and possibly even Na+ channels may participate in the firing of a small percentage of cells. We now show that at potentials near resting (–50 mV), Cav1.3 carries equal amounts of Ca2+ current to Cav1.2 but activates at 9 mV more negative potentials. MCCs express only TTX-sensitive Nav1 channels that activate at 24 mV more positive potentials than Cav1.3 and are fully inactivating. Their blockade prevents the firing only in a small percentage of cells (13%). This suggests that the order of importance with regard to pacemaking MCCs is: Cav1.3, Cav1.2 and Nav1. The above conclusions, however, rely on the proper use of DHPs, whose blocking potency is strongly holding potential dependent. We also show that small increases of KCl concentration steadily depolarize the MCCs causing abnormally increased firing frequencies, lowered and broadened AP waveforms and an increased facility of switching “non-firing” into “firing” cells that may lead to erroneous conclusions about the role of Cav1.3 and Cav1.2 as pacemaker channels in MCCs.2

Altered excitability of cultured chromaffin cells following exposure to multi-walled carbon nanotubes

Abstract We studied the effects of multi-walled carbon nanotubes (MWCNTs) on the electrophysiological properties of cultured mouse chromaffin cells, a model of spontaneously firing cells. The exposure of chromaffin cells to MWCNTs at increasing concentrations (30–263 μg/ml) for 24 h reduced, in a dose-dependent way, both the cell membrane input resistance and the number of spontaneously active cells (from 80–52%). Active cells that survived from the toxic effects of MWCNTs exhibited more positive resting potentials, higher firing frequencies and unaltered voltage-gated Ca2+, Na+ and K+ current amplitudes. MWCNTs slowed down the inactivation kinetics of Ca2+-dependent BK channels. These electrophysiological effects were accompanied by MWCNTs internalization, as confirmed by transmission electron microscopy, indicating that most of the toxic effects derive from a dose-dependent MWCNTs-cell interaction that damages the spontaneous cell activity.