The neuronal beta 4 subunit increases the unitary conductance of L-type voltage-gated calcium channels in PC12 cells

beta subunits of voltage-gated calcium channels influence channel behavior in numerous ways, including enhancing the targeting of alpha1 subunits to the plasma membrane and shifting the voltage dependence of activation and inactivation. Of the four beta subunits that have been identified, beta 4 is of particular interest because mutation of its alpha1 subunit interaction domain produces severe neurological defects. Its differential distribution in the hippocampus prompted us to examine whether this subunit was responsible for the heterogeneity of hippocampal L-type calcium channels. To study the functional effects of the beta 4 subunit on native L-type calcium channels, we transfected beta 4 cDNA subcloned out of embryonic hippocampal neurons into PC12 cells, a cell line that contains the beta 1, beta 2, and beta 3 subunits but not the beta 4 subunit. Cell-attached single-channel recordings of L-type channel activity from untransfected and transfected PC12 cells compared with recordings obtained from hippocampal neurons revealed an effect of the beta 4 subunit on single-channel conductance. L-type channels in untransfected PC12 cells had a significantly smaller conductance (19.8 picosiemens (pS)) than L-type channels in hippocampal neurons (22 pS). After transfection of beta 4, however, L-type single-channel conductance was indistinguishable between the two cell types. Our data suggest that calcium channel beta 4 subunits affect the conductance of L-type calcium channels and that native hippocampal L-type channels contain the beta 4 subunit.     

Parkinson's disease: return of an old prime suspect

Pacemaking activity in adult substantia nigra (SN) dopamine neurons relies on L-type Ca2+ channels, but a surprising study in Nature by Chan et al. demonstrates that blockade of these channels by dihydropyridines re-establishes the pacemaking driven by sodium and HCN channels found in juvenile SN. This shift protects SN neurons in chemical models of Parkinson's disease (PD), suggesting that elevated intracellular Ca2+ participates in SN cell loss and that dihydropyridines may provide therapy in PD.     

Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain

Low voltage activation of Ca(V)1.3 L-type Ca(2+) channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Ca(V)1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in Ca(V)1.4 L-type Ca(2+) channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Ca(V)1.3 alpha1 subunit C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), we investigated if a C-terminal modulatory mechanism also controls Ca(V)1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with beta3 and alpha2delta1 subunits in HEK-293 cells. Activation of calcium current through Ca(V)1.3(42A) channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Ca(V)1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Ca(V)1.3(DeltaC116) channels showed gating properties similar to Ca(V)1.3(42A) that were reverted by co-expression of the corresponding C-terminal peptide C(116). Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Ca(V)1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Ca(V)1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Ca(V)1.3 channel activation at lower voltages expected to favor Ca(V)1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.     

Subthreshold sodium current from rapidly inactivating sodium channels drives spontaneous firing of tuberomammillary neurons

A role for "persistent," subthreshold, TTX-sensitive sodium current in driving the pacemaking of many central neurons has been proposed, but this has been impossible to test pharmacologically. Using isolated tuberomammillary neurons, we assessed the role of subthreshold sodium current in pacemaking by performing voltage-clamp experiments using a cell's own pacemaking cycle as voltage command. TTX-sensitive sodium current flows throughout the pacemaking cycle, even at voltages as negative as -70 mV, and this current is sufficient to drive spontaneous firing. When sodium channels underlying transient current were driven into slow inactivation by rapid stimulation, persistent current decreased in parallel, suggesting that persistent sodium current originates from subthreshold gating of the same sodium channels that underlie the phasic sodium current. This behavior of sodium channels may endow all neurons with an intrinsic propensity for rhythmic, spontaneous firing.     

Neurochemical characterization of the striatum and the nucleus accumbens in L-type Ca(v)1.3 channels knockout mice

L-type Ca(v)1.3 channels control the autonomous pacemaking of the substantia nigra (SN) dopamine (DA) neurons, which maintains the sustained release of DA in the striatum, its target structure. The persistent engagement of L-type channels during pacemaking might lead to increased vulnerability to environmental stressors or degenerative processes, providing a mechanism for the development of Parkinson's disease (PD). Interestingly, L-type channels are not necessary for pacemaking, opening the possible use of calcium channel antagonists as neuroprotective agents for PD without disturbing normal DA function. In this study we aimed to evaluate the consequences of Ca(v)1.3 channels deletion at the neurochemical level. For this purpose, tissue concentrations of DA and their respective metabolites were measured using high performance liquid chromatography (HPLC) in the striatum and the nucleus accumbens (NAcc) of mice lacking the gene for the Ca(v)1.3 channel subunit (CACNA1D) and compared to those in wild-type mice. Striatal DA level did not differ between the two groups. In contrast, the level of serotonin, glutamate, GABA, and taurine were increased by more than 50% in the striatum of Ca(v)1.3 null mice. Neurotransmitters levels in the NAcc did not differ between the different groups. In conclusion, our results neurochemically corroborate the robustness of the nigrostriatal DA neurons in the absence of Ca(v)1.3 channels, but suggest that complete deletion of this channel affected a variety of other transmitter systems.     

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.     

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Electrostatic basis of valence selectivity in cationic channels

We examine how a variety of cationic channels discriminate between ions of differing charge. We construct models of the KcsA potassium channel, voltage gated sodium channel and L-type calcium channel, and show that they all conduct monovalent cations, but that only the calcium channel conducts divalent cations. In the KcsA and sodium channels divalent ions block the channel and prevent any further conduction. We demonstrate that in each case, this discrimination and some of the more complex conductance properties of the channels is a consequence of the electrostatic interaction of the ions with the charges in the channel protein. The KcsA and sodium channels bind divalent ions strongly enough that they cannot be displaced by other ions and thereby block the channel. On the other hand, the calcium channel binds them less strongly such that they can be destabilized by the repulsion of another incoming divalent ion, but not by the lesser repulsion from monovalent ions.     

Survival promotion of mesencephalic dopaminergic neurons by depolarizing concentrations of K+ requires concurrent inactivation of NMDA or AMPA/kainate receptors

The death of dopaminergic neurons that occurs spontaneously in mesencephalic cultures was prevented by depolarizing concentrations of K+ (20-50 mM). However, unlike that observed previously in other neuronal populations of the PNS or CNS, promotion of survival required concurrent blockade of either NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors by the specific antagonists, MK-801 and GYKI-52466, respectively. Rescued neurons appeared to be healthy and functional because the same treatment also dramatically enhanced their capacity to accumulate dopamine. The effects on survival and uptake were rather specific to dopaminergic neurons, rapidly reversible and still observed when treatment was delayed after plating. Glutamate release increased substantially in the presence of elevated concentrations of K+, and chronic treatment with glutamate induced a loss of dopaminergic neurons that was prevented by MK-801 or GYKI-52466 suggesting that an excitotoxic process interfered with survival when only the depolarizing treatment was applied. The effects of the depolarizing stimulus in the presence of MK-801 were mimicked by BAY K-8644 and abolished by nifedipine, suggesting that neuroprotection resulted from Ca(2+) influx through L-type calcium channels. Measurement of intracellular calcium revealed that MK-801 or GYKI-52466 were required to maintain Ca(2+) levels within a trophic range, thus preventing K+-induced excitotoxic stress and Ca(2+) overload. Altogether, our results suggest that dopaminergic neurons may require a finely tuned interplay between glutamatergic receptors and calcium channels for their development and maturation.     

The effect of variations in sodium conductances on pacemaking in a dopaminergic retinal neuron model

Dopaminergic neurons in the retina show spontaneous tetrodotoxin-sensitive pacemaking, which has been explained by a reduced Hodgkin-Huxley-type computer model. The present study used this model to investigate the effect of variations in transient and persistent sodium conductance values on pacemaking, under variable leakage conductance levels. This study indicated that transient sodium conductance plays an indispensable role in pacemaking, which occurs under conditions in which only a persistent sodium conductance is considerably reduced, thus contributing to a detailed understanding of the relationship between sodium conductance and pacemaking.     

Model of oscillatory activity in thalamic neurons: Role of voltage- and calcium-dependent ionic conductances

This paper describes a computer modeling study of the generation of 10 Hz oscillations in the electrical activity of guinea pig thalamic neurons in vitro. The computer model was based on experimental evidence suggesting that single thalamic neurons in guinea pig have a set of voltage- and calcium-dependent ionic conductances that is capable of generating self-sustained rhythmic oscillations. Simulation results are consistent with this hypothesis, and indicate that a model that contains dendritic calcium and calcium-dependent potassium conductances, as well as a voltage-dependent, slow sodium conductance, can indeed generate self-sustained oscillations like those seen in thalamic neurons. Moreover, simulations indicate that the occurrence of such oscillatory activity is strongly dependent on the location of the slow sodium conductance. Results predict that this slow sodium conductance is located in the dendrites.The authors express their appreciation to R. J. MacGregor for providing equations and computer programs for simulating a two-point neuronal model with active calcium-related conductances     

Different localizations and functions of L-type and N-type calcium channels during development of hippocampal neurons

Using immunocytochemical assays and patch-clamp and calcium-imaging recordings, we demonstrate that L-type and N-type calcium channels have distinct patterns of expression and distribution and play different functional roles during hippocampal neuron differentiation. L-type channels, which support the depolarization-induced calcium influx in neurons from the very early developmental stages, are functionally restricted to the somatodendritic compartment throughout neuronal development and play a crucial role in supporting neurite outgrowth at early developmental stages. N-type channels, which start contributing at later neuronal differentiation stages (3-4 DIV), are also functionally expressed in the axons of immature neurons. At this developmental stage preceding synaptogenesis, N-type (but not L-type) channels are involved in controlling synaptic vesicle recycling. It is only at later developmental stages (10-12 DIV), when the neurons have established a clear axodendritic polarity and form synaptic contacts, that N-type channels are progressively excluded from the axon. Electrophysiological recordings of single neurons growing in microislands revealed that synaptic maturation coincides with a progressive increase in N-type channels in the somatodendritic region and a progressive decrease in the N-type channels supporting glutamate release from the presynaptic terminal. These results indicate that L-type and N-type calcium channels undergo dynamic, developmentally regulated rearrangements in regional distribution and function and also suggest that different mechanisms may be involved in the sorting and/or stabilization of these two types of channels in different plasma membrane domains during neuronal differentiation.     

Calcium current subtypes in GnRH neurons

Calcium plays roles in excitability, rhythm generation, and neurosecretion. Identifying channel subtypes that regulate calcium influx is thus important to understanding rhythmic GnRH secretion, which is a prerequisite for reproduction. Whole-cell voltage-clamp recordings were made from short-term dissociated GnRH adult ovariectomized (OVX) mice (n = 21) to identify channel subtypes that carry calcium current using selective channel blockers and voltage characteristics. Low-voltage activated (LVA) currents were not observed in 42 GnRH neurons tested, although most non-GnRH neurons (4/6) displayed LVA current. The L-type component of the high-voltage activated (HVA) calcium current was 25% +/- 2%. The remaining HVA calcium current passed through N-type (27% +/- 3%), P-type (15% +/- 1%), Q-type (18% +/- 3%), and R-type (15% +/- 1%) channels. Because these data differ substantially from reports on cultured GnRH neurons, which may represent reproductively immature models, we also examined GnRH neurons from gonadal-intact young (Postnatal Days 4-10, n = 8 mice) mice. LVA currents were still rare (2/28) in young mice. Although the same HVA components were observed, the proportions were shifted toward significantly more L-type and less N-type current, suggesting a possible developmental shift in calcium currents in GnRH neurons. These data suggest that calcium channel subtypes in GnRH neurons prepared in the short term from brain slices differ substantially from those in long-term cultured GnRH models. These findings provide a vital foundation to examine the role of calcium channels in the secretory and rhythmic machinery of GnRH neurons.     

An Alpha-Catulin Homologue Controls Neuromuscular Function through Localization of the Dystrophin Complex and BK Channels in Caenorhabditis elegans

The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.     

Interactions among toxins that inhibit N-type and P-type calcium channels

A number of peptide toxins from venoms of spiders and cone snails are high affinity ligands for voltage-gated calcium channels and are useful tools for studying calcium channel function and structure. Using whole-cell recordings from rat sympathetic ganglion and cerebellar Purkinje neurons, we studied toxins that target neuronal N-type (Ca(V)2.2) and P-type (Ca(V)2.1) calcium channels. We asked whether different toxins targeting the same channels bind to the same or different sites on the channel. Five toxins (omega-conotoxin-GVIA, omega-conotoxin MVIIC, omega-agatoxin-IIIA, omega-grammotoxin-SIA, and omega-agatoxin-IVA) were applied in pairwise combinations to either N- or P-type channels. Differences in the characteristics of inhibition, including voltage dependence, reversal kinetics, and fractional inhibition of current, were used to detect additive or mutually occlusive effects of toxins. Results suggest at least two distinct toxin binding sites on the N-type channel and three on the P-type channel. On N-type channels, results are consistent with blockade of the channel pore by omega-CgTx-GVIA, omega-Aga-IIIA, and omega-CTx-MVIIC, whereas grammotoxin likely binds to a separate region coupled to channel gating. omega-Aga-IIIA produces partial channel block by decreasing single-channel conductance. On P-type channels, omega-CTx-MVIIC and omega-Aga-IIIA both likely bind near the mouth of the pore. omega-Aga-IVA and grammotoxin each bind to distinct regions associated with channel gating that do not overlap with the binding region of pore blockers. For both N- and P-type channels, omega-CTx-MVIIC binding produces complete channel block, but is prevented by previous partial channel block by omega-Aga-IIIA, suggesting that omega-CTx-MVIIC binds closer to the external mouth of the pore than does omega-Aga-IIIA.     

Effect of firing rate on the calcium permeability in adult neurons during spontaneous action potentials.

Calcium channels in neurons mediate a wide variety of essential functions. In addition to contributing to action potential shape, they furnish a substrate that acts as an intracellular second messenger. This study shows that the shape of the neuronal action potential has characteristics that promote long openings of L-type (high threshold) calcium channels. We also present evidence that a change in the firing rate of isolated neurons modulates gating of single calcium channels. This mechanism could be important in modulating neuron excitability and providing a rise in intracellular Ca, when needed.     

Morphological and electrophysiological aspects of dorsal median paired neurons generating plateau action potentials in the terminal abdominal ganglion of the insect central nervous system

Among the three clusters of dorsal unpaired median neurons of the Periplaneta americana terminal abdominal ganglion, another type of neuron has been characterized by anterograde cobalt stainings and microelectrode technique. These neurons are bilaterally distributed in the ganglion. Their axons ipsilaterally exit the ganglion via the anterior proctodeal nerves, to innervate the proctodeum. They are characterized by a long-duration overshooting action potentials and a low firing frequency. Most often the depolarizing phase is composed of two peaks: a fast spike followed by a slow phase. Tetrodotoxin suppressed the fast peak and blocked the spontaneous activity suggesting that sodium channels are involved in the depolarizing phase as well as in the initiation of the action potential. Calcium channel blockers induced a disappearing of the slow depolarizing phase indicating the participation of calcium ions and a reduction of the afterhyperpolarization reflecting the participation of calcium-activated potassium channels. Furthermore, cadmium, as lanthanum or barium, induced a long-lasting plateau potential, which would be due to a persistent sodium conductance. Tetraethylammonium increased the duration of the action potential indicating that potassium channels are implicated in the falling phase. The results demonstrate that these neurons are different from other cells, especially dorsal unpaired median neurons, of the central nervous system of the cockroach.Abbreviations DUM dorsal unpaired median - SDP slow depolarizing phase - AP action potential - PAP plateau action potential - TAG terminal abdominal ganglion - CNS central nervous system     

Role of Calcium Electrogenesis in Apical Dendrites: Generation of Intrinsic Oscillations by an Axial Current

Dendrites are covered with conductances whose function is still mysterious. Using intracellular recording and calcium imaging, we describe an electrogenic band of calcium channels in distal apical dendrites of layer 5 pyramidal neurons (Yuste et al., 1994). We now explore the functional consequences of this distal electrogenic area with multicompartmental numerical simulations. A calcium imaging and electrophysiological database from a single neuron, recorded under blocked sodium and potassium conductances, is replicated by simulations having increased dendritic calcium current. In these models a significant axial current flows from the apical dendrite into the somatic region, activating low-threshold calcium channels and generating oscillations similar to those seen in the electrophysiological data. We propose that the distal electrogenic area in apical dendrites serves to inject current into the soma and produce intrinsic oscillatory dynamics.     

Co-variation of ionic conductances supports phase maintenance in stomatogastric neurons

Neuronal networks produce reliable functional output throughout the lifespan of an animal despite ceaseless molecular turnover and a constantly changing environment. Central pattern generators, such as those of the crustacean stomatogastric ganglion (STG), are able to robustly maintain their functionality over a wide range of burst periods. Previous experimental work involving extracellular recordings of the pyloric pattern of the STG has demonstrated that as the burst period varies, the inter-neuronal delays are altered proportionally, resulting in burst phases that are roughly invariant. The question whether spike delays within bursts are also proportional to pyloric period has not been explored in detail. The mechanism by which the pyloric neurons accomplish phase maintenance is currently not obvious. Previous studies suggest that the co-regulation of certain ion channel properties may play a role in governing neuronal activity. Here, we observed in long-term recordings of the pyloric rhythm that spike delays can vary proportionally with burst period, so that spike phase is maintained. We then used a conductance-based model neuron to determine whether co-varying ionic membrane conductances results in neural output that emulates the experimentally observed phenomenon of spike phase maintenance. Next, we utilized a model neuron database to determine whether conductance correlations exist in model neuron populations with highly maintained spike phases. We found that co-varying certain conductances, including the sodium and transient calcium conductance pair, causes the model neuron to maintain a specific spike phase pattern. Results indicate a possible relationship between conductance co-regulation and phase maintenance in STG neurons.     

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 (Ca_V1.2 and Ca_V1.3), Ca_V1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using Ca_V1 .3^-/- KO mice and the AP-clamp it has been possible to resolve the time course of Ca_V1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells Ca_V1.3 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 Ca_V1.3 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.