Voltage-gated Ca(2+) channels couple membrane depolarization to Ca(2+)-dependent intracellular signaling events. This is achieved by mediating Ca(2+) ion influx or by direct conformational coupling to intracellular Ca(2+) release channels. The family of Ca(v)1 channels, also termed L-type Ca(2+) channels (LTCCs), is uniquely sensitive to organic Ca(2+) channel blockers and expressed in many electrically excitable tissues. In this review, we summarize the role of LTCCs for human diseases caused by genetic Ca(2+) channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within their pore-forming alpha1 subunits causing hypokalemic periodic paralysis and malignant hyperthermia sensitivity (Ca(v)1.1 alpha1), incomplete congenital stationary night blindness (CSNB2; Ca(v)1.4 alpha1), and Timothy syndrome (Ca(v)1.2 alpha1; reviewed separately in this issue). Ca(v)1.3 alpha1 mutations have not been reported yet in humans, but channel loss of function would likely affect sinoatrial node function and hearing. Studies in mice revealed that LTCCs indirectly also contribute to neurological symptoms in Ca(2+) channelopathies affecting non-LTCCs, such as Ca(v)2.1 alpha1 in tottering mice. Ca(2+) channelopathies provide exciting disease-related molecular detail that led to important novel insight not only into disease pathophysiology but also to mechanisms of channel function.

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.

Auxiliary beta subunits are critical determinants of membrane expression and gating properties of voltage-gated calcium channels. Mutations in the beta(4) subunit gene cause ataxia and epilepsy. However, the specific function of beta(4) in neurons and its causal relation to neurological diseases are unknown. Here we report the localization of the beta(4) subunit in the nuclei of cerebellar granule and Purkinje cells. beta(4b) was the only beta isoform showing nuclear targeting when expressed in neurons and skeletal myotubes. Its specific nuclear targeting property was mapped to an N-terminal double-arginine motif, which was necessary and sufficient for targeting beta subunits into the nucleus. Spontaneous electrical activity and calcium influx negatively regulated beta(4b) nuclear localization by a CRM-1-dependent nuclear export mechanism. The activity-dependent shuttling of beta(4b) into and out of the nucleus indicates a specific role of this beta subunit in neurons, in communicating the activity of calcium channels to the nucleus.

Mounting evidence suggests that voltage-gated L-type Ca2+ channels can modulate affective behaviour. We therefore explored the role of CaV1.3 L-type Ca2+ channels in depression- and anxiety-like behaviours using CaV1.3-deficient mice (CaV1.3-/-). We showed that CaV1.3-/- mice displayed less immobility in the forced swim test as well as in the tail suspension test, indicating an antidepressant-like phenotype. Locomotor activity in the home cage or a novel open-field test was not influenced. In the elevated plus maze (EPM), CaV1.3-/- mice entered the open arms more frequently and spent more time there indicating an anxiolytic-like phenotype which was, however, not supported in the stress-induced hyperthermia test. By performing parallel experiments in Claudin 14 knockout mice (Cldn14-/-), which like CaV1.3-/- mice are congenitally deaf, an influence of deafness on the antidepressant-like phenotype could be ruled out. On the other hand, a similar EPM behaviour indicative of an anxiolytic phenotype was also found in the Cldn14-/- animals. Using electroretinography and visual behavioural tasks we demonstrated that at least in mice, CaV1.3 channels do not significantly contribute to visual function. However, marked morphological changes were revealed in synaptic ribbons in the outer plexiform layer of CaV1.3-/- retinas by immunohistochemistry suggesting a possible role of this channel type in structural plasticity at the ribbon synapse. Taken together, our findings indicate that CaV1.3 L-type Ca2+ channels modulate depression-like behaviour but are not essential for visual function. The findings raise the possibility that selective modulation of CaV1.3 channels could be a promising new therapeutic concept for the treatment of mood disorders.

Migraine is a frequent and often disabling disease. Treatment is unsatisfactory in many patients. A disturbed dynamic balance between excitatory and inhibitory signal processing with enhanced cortical activity probably underlies common forms of migraine. Presynaptic voltage-gated Ca(2+) channels are critical determinants of neurotransmitter release and also contribute to trigeminovascular signal transduction. Because clinical evidence exists for migraine-prophylactic actions of Petasites hybridus extracts, we investigated whether petasins comprising the main constituents of the extract inhibit currents through presynaptic Ca(v)2.1 channels expressed in Xenopus laevis oocytes. P. hybridus extract (0.02 mg/ml), petasin, neopetasin, isopetasin, S-petasin, and iso-S-petasin (50 microM) were weak tonic blockers of Ca(v)2.1-mediated barium currents (I(Ba)) during infrequent depolarizations (0.1 Hz), but their inhibitory potency increased at higher stimulation rates (1 Hz), indicating preferential block of open and/or inactivated channels. Sulfur-containing compounds (S-petasin, Iso-S-petasin) were the most potent significantly promoting the accumulation of Ca(v)2.1 channel in inactivated states during pulse trains (I(Ba) decrease during 1-Hz pulse trains: control, 45%, S-petasin, 79%; iso-S-petasin, 80%). For the Eucalyptus williamsiania sesquiterpenes alpha- and gamma-eudesmol, a comparable use-dependent inhibition was found in addition to a tonic block component. Alpha-eudesmol and petasins accelerated the voltage-dependent inactivation of Ca(v)2.1 channels during depolarizations. We demonstrate that S-petasin, iso-S-petasin, and eudesmol are Ca(v)2.1 channel inhibitors preferentially acting as use-dependent channel blockers and with the sulfur-containing substituent in position 3 of the petasins serving as important functional feature. The Ca(v)2.1-inhibitory properties of these petasins may contribute to migraine-prophylactic properties described for P. hybridus extracts.

L-Type Ca(2+) channels (LTCCs) play a key role in the regulation of vascular smooth muscle contraction, and substances that interfere with their function (Ca(2+) channel blockers) are widely used in the therapy of hypertension. Here, we report anthracene-maleimide derivatives as new LTCC blockers. Among these, 3, lacking intracellular effects, was investigated in more detail. The results show that 3 binds preferentially to inactivated LTCCs, directly interacting with the pore-forming subunit of the channel.

The L-type calcium channel (LTCC) isoforms Cav1.2 and Cav1.3 display similar 1,4-Dihydropyridine binding properties and are both expressed in mammalian brain. Recent work implicates Cav1.3 channels as interesting drug targets, but no isoform selective modulators exist. It is also unknown to which extent Cav1.1 and Cav1.4 contribute to L-type specific dihydropyridine (DHP) binding activity in brain. To address this question and to determine if DHPs can discriminate between Cav1.2 and Cav1.3 binding pockets we combined radioreceptor assays and quantitative PCR (qPCR). We bred double mutants (Cav-DM) from mice expressing mutant Cav1.2 channels (Cav1.2DHP(-/-)) lacking high affinity for DHPs and from Cav1.3 knockouts (Cav1.3(-/-)).(+)-[(3)H]isradipine binding to Cav1.2DHP(-/-) and Cav-DM brains was reduced to 15.1 and 4.4% of wildtype, respectively, indicating that Cav1.3 accounts for 10.7% of brain LTCCs. qPCR revealed that Cav1.1 and Cav1.4 alpha1 subunits comprised 0.08% of the LTCC transcripts in mouse whole brain, suggesting that they cannot account for the residual binding. Instead, this could be explained by low affinity binding (127-fold Kd increase) to the mutated Cav1.2 channels. Inhibition of (+)-[(3)H]isradipine binding to Cav1.2DHP(-/-)(predominantly Cav1.3) and wildtype (predominantly Cav1.2) brain membranes by unlabeled DHPs revealed a 3-4-fold selectivity of nitrendipine and nifedipine for the Cav1.2 binding pocket, a finding further confirmed with heterologously expressed channels. This suggests that small differences in their binding pockets may allow development of isoform-selective modulators for LTCCs and that, due to their very low expression, Cav1.1 and Cav1.4 are unlikely to serve as drug targets to treat CNS diseases.

Depolarisation-induced Ca(2+) influx into electrically excitable cells is determined by the density of voltage-gated Ca(2+) channels at the cell surface. Surface expression is modulated by physiological stimuli as well as by drugs and can be altered under pathological conditions. Extracellular epitope-tagging of channel subunits allows to quantify their surface expression and to distinguish surface channels from those in intracellular compartments. Here we report the first systematic characterisation of extracellularly epitope-tagged Ca(V)2.1 channels. We identified a permissive region in the pore-loop of repeat IV within the Ca(V)2.1 alpha(1) subunit, which allowed integration of several different tags (hemagluttinine [HA], double HA; 6-histidine tag [His], 9-His, bungarotoxin-binding site) without compromising alpha1 subunit protein expression (in transfected tsA-201 cells) and function (after expression in X. laevis oocytes). Immunofluorescence studies revealed that the double-HA tagged construct (1722-HAGHA) was targeted to presynaptic sites in transfected cultured hippocampal neurons as expected for Ca(V)2.1 channels. We also demonstrate that introduction of tags into this permissive position creates artificial sites for channel modulation. This was demonstrated by partial inhibition of 1722-HA channel currents with anti-HA antibodies and the concentration-dependent stimulation or partial inhibition by Ni-nitrilo triacetic acid (NTA) and novel bulkier derivatives (Ni-trisNTA, Ni-tetrakisNTA, Ni-nitro-o-phenyl-bisNTA, Ni-nitro-p-phenyl-bisNTA). Therefore our data also provide evidence for the concept that artificial modulatory sites for small ligands can be introduced into voltage-gated Ca(2+) channel for their selective modulation.