Low pHo boosts burst firing and catecholamine release by blocking TASK-1 and BK channels while preserving Cav1 channels in mouse chromaffin cells.

KEY POINTS: Mouse chromaffin cells (MCCs) generate spontaneous burst-firing that causes large increases of Ca2+ -dependent catecholamine release, and is thus a key mechanism for regulating the functions of MCCs. With the aim to uncover a physiological role for burst-firing we investigated the effects of acidosis on MCC activity. Lowering the extracellular pH (pHo ) from 7.4 to 6.6 induces cell depolarizations of 10-15 mV that generate bursts of ∼330 ms at 1-2 Hz and a 7.4-fold increase of cumulative catecholamine-release. Burst-firing originates from the inhibition of the pH-sensitive TASK-1-channels and a 60% reduction of BK-channel conductance at pHo 6.6. Blockers of the two channels (A1899 and paxilline) mimic the effects of pHo 6.6, and this is reverted by the Cav1 channel blocker nifedipine. MCCs act as pH-sensors. At low pHo , they depolarize, undergo burst-firing and increase catecholamine-secretion, generating an effective physiological response that may compensate for the acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue. ABSTRACT: Mouse chromaffin cells (MCCs) generate action potential (AP) firing that regulates the Ca2+ -dependent release of catecholamines (CAs). Recent findings indicate that MCCs possess a variety of spontaneous firing modes that span from the common 'tonic-irregular' to the less frequent 'burst' firing. This latter is evident in a small fraction of MCCs but occurs regularly when Nav1.3/1.7 channels are made less available or when the Slo1ß2-subunit responsible for BK channel inactivation is deleted. Burst firing causes large increases of Ca2+ -entry and potentiates CA release by ∼3.5-fold and thus may be a key mechanism for regulating MCC function. With the aim to uncover a physiological role for burst-firing we investigated the effects of acidosis on MCC activity. Lowering the extracellular pH (pHo ) from 7.4 to 7.0 and 6.6 induces cell depolarizations of 10-15 mV that generate repeated bursts. Bursts at pHo 6.6 lasted ∼330 ms, occurred at 1-2 Hz and caused an ∼7-fold increase of CA cumulative release. Burst firing originates from the inhibition of the pH-sensitive TASK-1/TASK-3 channels and from a 40% BK channel conductance reduction at pHo 7.0. The same pHo had little or no effect on Nav, Cav, Kv and SK channels that support AP firing in MCCs. Burst firing of pHo 6.6 could be mimicked by mixtures of the TASK-1 blocker A1899 (300 nm) and BK blocker paxilline (300 nm) and could be prevented by blocking L-type channels by adding 3 µm nifedipine. Mixtures of the two blockers raised cumulative CA-secretion even more than low pHo (∼12-fold), showing that the action of protons on vesicle release is mainly a result of the ionic conductance changes that increase Ca2+ -entry during bursts. Our data provide direct evidence suggesting that MCCs respond to low pHo with sustained depolarization, burst firing and enhanced CA-secretion, thus mimicking the physiological response of CCs to acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue.

Cell-type-specific tuning of Cav1.3 Ca(2+)-channels by a C-terminal automodulatory domain.

Cav1.3 L-type Ca(2+)-channel function is regulated by a C-terminal automodulatory domain (CTM). It affects channel binding of calmodulin and thereby tunes channel activity by interfering with Ca(2+)- and voltage-dependent gating. Alternative splicing generates short C-terminal channel variants lacking the CTM resulting in enhanced Ca(2+)-dependent inactivation and stronger voltage-sensitivity upon heterologous expression. However, the role of this modulatory domain for channel function in its native environment is unkown. To determine its functional significance in vivo, we interrupted the CTM with a hemagglutinin tag in mutant mice (Cav1.3DCRD(HA/HA)). Using these mice we provide biochemical evidence for the existence of long (CTM-containing) and short (CTM-deficient) Cav1.3 α1-subunits in brain. The long (HA-labeled) Cav1.3 isoform was present in all ribbon synapses of cochlear inner hair cells. CTM-elimination impaired Ca(2+)-dependent inactivation of Ca(2+)-currents in hair cells but increased it in chromaffin cells, resulting in hyperpolarized resting potentials and reduced pacemaking. CTM disruption did not affect hearing thresholds. We show that the modulatory function of the CTM is affected by its native environment in different cells and thus occurs in a cell-type specific manner in vivo. It stabilizes gating properties of Cav1.3 channels required for normal electrical excitability.

Cav1.3 Channels as Key Regulators of Neuron-Like Firings and Catecholamine Release in Chromaffin Cells.

Neuronal and neuroendocrine L-type calcium channels (Cav1.2, Cav1.3) open readily at relatively low membrane potentials and allow Ca(2+) to enter the cells near resting potentials. In this way, Cav1.2 and Cav1.3 shape the action potential waveform, contribute to gene expression, synaptic plasticity, neuronal differentiation, hormone secretion and pacemaker activity. In the chromaffin cells (CCs) of the adrenal medulla, Cav1.3 is highly expressed and is shown to support most of the pacemaking current that sustains action potential (AP) firings and part of the catecholamine secretion. Cav1.3 forms Ca(2+)-nanodomains with the fast inactivating BK channels and drives the resting SK currents. These latter set the inter-spike interval duration between consecutive spikes during spontaneous firing and the rate of spike adaptation during sustained depolarizations. Cav1.3 plays also a primary role in the switch from "tonic" to "burst" firing that occurs in mouse CCs when either the availability of voltage-gated Na channels (Nav) is reduced or the ß2 subunit featuring the fast inactivating BK channels is deleted. Here, we discuss the functional role of these "neuron-like" firing modes in CCs and how Cav1.3 contributes to them. The open issue is to understand how these novel firing patterns are adapted to regulate the quantity of circulating catecholamines during resting condition or in response to acute and chronic stress.

Excitement about inhibitory presynaptic terminals.

Based on extrapolation from excitatory synapses, it is often assumed that depletion of the releasable pool of synaptic vesicles is the main factor underlying depression at inhibitory synapses. In this issue of Neuron, using subcellular patch-clamp recording from inhibitory presynaptic terminals, Kawaguchi and Sakaba (2015) show that at Purkinje cell-deep cerebellar nuclei neuron synapses, changes in presynaptic action potential waveform substantially contribute to synaptic depression.

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

KEY POINTS: Mouse chromaffin cells (MCCs) of the adrenal medulla possess fast-inactivating Nav channels whose availability alters spontaneous action potential firing patterns and the Ca(2+)-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 Ca(2+)-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. ABSTRACT: Action potential (AP) firing in mouse chromaffin cells (MCCs) is mainly sustained by Cav1.3 L-type channels that drive BK and SK currents and regulate the pacemaking cycle. As secretory units, CCs optimally recruit Ca(2+) channels when stimulated, a process potentially dependent on the modulation of the AP waveform. Our previous work has shown that a critical determinant of AP shape is voltage-gated sodium channel (Nav) channel availability. Here, we studied the contribution of Nav channels to firing patterns and AP shapes at rest (-50 mV) and upon stimulation (-40 mV). Using quantitative RT-PCR and immunoblotting, we show that MCCs mainly express tetrodotoxin (TTX)-sensitive, fast-inactivating Nav1.3 and Nav1.7 channels that carry little or no Na(+) current during slow ramp depolarizations. Time constants and the percentage of recovery from fast inactivation and slow entry into closed-state inactivation are similar to that of brain Nav1.3 and Nav1.7 channels. The fraction of available Nav channels is reduced by half after 10 mV depolarization from -50 to -40 mV. This leads to low amplitude spikes and a reduction in repolarizing K(+) currents inverting the net current from outward to inward during the after-hyperpolarization. When Nav channel availability is reduced by up to 20% of total, either by TTX block or steady depolarization, a switch from tonic to burst firing is observed. The spontaneous occurrence of high frequency bursts is rare under control conditions (14% of cells) but leads to major Ca(2+)-entry and increased catecholamine release. Thus, Nav1.3/Nav1.7 channel availability sets the AP shape, burst-firing initiation and regulates catecholamine secretion in MCCs. Nav channel inactivation becomes important during periods of high activity, mimicking stress responses.

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 Ca(2+) 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 Ca(2+) 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 Ba(2+) as charge carrier. Therefore, our data identify pyrimidine-2,4,6-triones as Ca(2+) channel activators.

T-type channel-mediated neurotransmitter release.

Besides controlling a wide variety of cell functions, T-type channels have been shown to regulate neurotransmitter release in peripheral and central synapses and neuroendocrine cells. Growing evidence over the last 10 years suggests a key role of Cav3.2 and Cav3.1 channels in controlling basal neurosecretion near resting conditions and sustained release during mild stimulations. In some cases, the contribution of low-voltage-activated (LVA) channels is not directly evident but requires either the activation of coupled presynaptic receptors, block of ion channels, or chelation of metal ions. Concerning the coupling to the secretory machinery, T-type channels appear loosely coupled to neurotransmitter and hormone release. In neurons, Cav3.2 and Cav3.1 channels mainly control the asynchronous appearance of "minis" [miniature inhibitory postsynaptic currents (mIPSCs) and miniature excitatory postsynaptic currents (mEPSCs)]. The same loose coupling is evident from membrane capacity and amperometric recordings in chromaffin cells and melanotropes where the low-threshold-driven exocytosis possesses the same linear Ca(2+) dependence of the other voltage-gated Ca(2+) channels (Cav1 and Cav2) that is strongly attenuated by slow calcium buffers. The intriguing issue is that, despite not expressing a consensus "synprint" site, Cav3.2 channels do interact with syntaxin 1A and SNAP-25 and, thus, may form nanodomains with secretory vesicles that can be regulated at low voltages. In this review, we discuss all the past and recent issues related to T-type channel-secretion coupling in neurons and neuroendocrine cells.

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. Ca(v)1.3 L-type calcium channels sustain the pacemaker current, and their loss results in depolarized resting potentials (V(rest)), spike broadening, and remarkable switches into depolarization block after BayK 8644 application. A functional coupling between Ca(v)1.3 and BK channels has been reported but cannot fully account for the aforementioned observations. Here, using Ca(v)1.3(-/-) mice, we investigated the role of Ca(v)1.3 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 Ca(v)1.3-dependent SK currents during the interspike intervals that lead to a notable degree of spike frequency adaptation (SFA). The major contribution of Ca(v)1.3 to the subthreshold Ca(2+) charge during an AP-train rather than a specific molecular coupling to SK channels accounts for the reduced SFA of Ca(v)1.3(-/-) MCCs. Low adaptation ratios due to reduced SK activation associated with Ca(v)1.3 deficiency prevent the efficient recovery of Na(V) 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, Ca(v)1.3 slows down MCC firing by activating SK channels that maintain Na(V) channel availability high enough to preserve stable AP waveforms, even upon high-frequency stimulation of chromaffin cells during stress responses.

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

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 Ca(2+), Na(+) and K+ current amplitudes. MWCNTs slowed down the inactivation kinetics of Ca(2+)-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.

Structural and functional differences between L-type calcium channels: crucial issues for future selective targeting.

Within the family of voltage-gated calcium channels (VGCCs), L-type channels (L-VGCCs) represent a well-established therapeutic target for calcium channel blockers, which are widely used to treat hypertension and myocardial ischemia. L-VGCCs outside the cardiovascular system also control key physiological processes such as neuronal plasticity, sensory cell function (e.g. in the inner ear and retina) and endocrine function (e.g. in pancreatic beta cells and adrenal chromaffin cells). Research into L-VGCCs was stimulated by the discovery that the known L-VGCC isoforms (Ca(V)1.1, Ca(V)1.2, Ca(V)1.3 and Ca(V)1.4) possess different biophysical properties. However, no L-VGCC-isoform-selective drugs have yet been identified. In this review, we examine Ca(V)1.2 and Ca(V)1.3 isoforms at the level of genetic structure, splice variants, post-translational modifications and functional protein coupling. We discuss candidate Ca(V)1.2- and Ca(V)1.3-specific characteristics as future therapeutic targets in individual organs.

Are Ca(v)1.3 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 Ca(2+) channels expressed in these cells (Ca(v)1.2 and Ca(v)1.3). Using Ca(v)1.3(-/-) KO MCCs we have shown that Ca(v)1.3 possess all the prerequisites for carrying subthreshold currents that sustain low frequency cell firing near resting (0.5 to 2 Hz at -50 mV): low-threshold and steep voltage dependence of activation, slow and incomplete inactivation during pulses of several hundreds of milliseconds. Ca(v)1.2 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), Ca(v)1.3 carries equal amounts of Ca(2+) current to Ca(v)1.2 but activates at 9 mV more negative potentials. MCCs express only TTX-sensitive Na(v)1 channels that activate at 24 mV more positive potentials than Ca(v)1.3 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: Ca(v)1.3, Ca(v)1.2 and Na(v)1. 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 Ca(v)1.3 and Ca(v)1.2 as pacemaker channels in MCCs.

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 Ca (2+) -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 Ca (2+) currents and activate BK channels confers to CaV1.3 the unique feature of driving Ca (2+) loading during long interspike intervals and, possibly, to control the Ca (2+) -dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.

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 Ca(2+) 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 Ca(2+) 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 Ca(2+) current and the corresponding Ca(2+)-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.