Mutations in the CACNA1F gene encoding the L-type calcium channel pore-forming Cav1.4 (α1F) subunit in humans result in an incomplete form of congenital stationary night blindness (CSNB2) with residual photoreceptor function. It has been postulated that this residual function, at least in part, may be mediated by another L-type calcium channel subunit, Cav1.3 (α1D), expressed within cone photoreceptors. However, the expression of the calcium channel Cav1.3 (α1D) subunit within photoreceptors remains debatable due to discrepancies among the immunohistochemical studies reported in the literature. In order to get around the innate complications of utilizing unproven antibodies and to shed light on this discussion, we investigated the mRNA expression profile for the Cav1.3 (α1D) subunit in the mouse retina.
In situ hybridization was performed on wild type mouse retinal sections with two independent sets of digoxigenin-11-UTP-labeled Cav1.3 (α1D)-specific sense and antisense cRNA probes. The two probe sets employed correspond to two distinct regions of the Cav1.3 (α1D) subunit mRNA, each encoding a different fragment of the Cav1.3 (α1D) polypeptide. In situ hybridization of wild type mouse brain sections with these same probes was performed as an additional control for specificity.
Abundant L-type calcium channel Cav1.3 (α1D) subunit mRNA expression was confirmed in most cells of the outer nuclear layer using two independent Cav1.3 (α1D)-specific antisense cRNA probes, confirming expression in rod photoreceptors. Cav1.3 (α1D) mRNA expression was also observed within most cells of the inner nuclear layer and ganglion cell layers using these same antisense cRNA probes. No labeling of tissue was observed using either sense cRNA probe. In situ detection of concentrated Cav1.3 (α1D) mRNA expression within the hippocampus and Purkinje and granule cells of the cerebellum of wild type mouse brain with these same probes confirmed specificity of the probes.
Our finding of expression of the L-type calcium channel Cav1.3 (α1D) subunit mRNA in rods substantiates the possibility that this pore-forming subunit may be a competent component of channels mediating the residual photoreceptor responses observed in mutant mice lacking functional Cav1.4 (α1F) subunits and in humans with CSNB2. Furthermore, the combined observations of abundant expression of Cav1.3 (α1D) mRNA in wild type rods and the large reduction in the transmission of photoreceptor responses in mice lacking Cav1.4 (α1F) raises the possibility that Cav1.3 (α1D) protein expression levels, localization, or functioning might be concomitantly altered by disruption of the Cav1.4 (α1F) subunit in rods. To date, no studies of Cav1.3 (α1D) mRNA nor protein expression levels or localization in cacna1f mutant mice or humans with CSNB2 have been published. Our findings warrant such studies to address the abovementioned possibilities. Finally, the observation of Cav1.3 (α1D) mRNA expression in multiple retinal cell types suggests the potential for a broader role for this L-type calcium channel subunit in overall functioning of the normal retina than previously appreciated. We therefore suggest that lesions in either the gene encoding the L-type calcium channel Cav1.3 (α1D) subunit or other molecules that interact with and regulate it may underlie one or more retinopathies with currently unidentified molecular etiologies.
See Borgkvist et al. (doi:10.1093/brain/awu150) for a scientific commentary on this article.
D2 autoreceptors and L-type calcium channels are both implicated in Parkinson’s disease, but how they interact is unclear. Dragicevic et al. reveal that L-type calcium channels can modulate D2-autoreceptor responses via the neuronal calcium sensor NCS-1. This dopamine-dependent signalling network is altered in Parkinson’s disease and could represent a therapeutic target.
Dopamine midbrain neurons within the substantia nigra are particularly prone to degeneration in Parkinson’s disease. Their selective loss causes the major motor symptoms of Parkinson’s disease, but the causes for the high vulnerability of SN DA neurons, compared to neighbouring, more resistant ventral tegmental area dopamine neurons, are still unclear. Consequently, there is still no cure available for Parkinson’s disease. Current therapies compensate the progressive loss of dopamine by administering its precursor l-DOPA and/or dopamine D2-receptor agonists. D2-autoreceptors and Cav1.3-containing L-type Ca2+ channels both contribute to Parkinson’s disease pathology. L-type Ca2+ channel blockers protect SN DA neurons from degeneration in Parkinson’s disease and its mouse models, and they are in clinical trials for neuroprotective Parkinson’s disease therapy. However, their physiological functions in SN DA neurons remain unclear. D2-autoreceptors tune firing rates and dopamine release of SN DA neurons in a negative feedback loop through activation of G-protein coupled potassium channels (GIRK2, or KCNJ6). Mature SN DA neurons display prominent, non-desensitizing somatodendritic D2-autoreceptor responses that show pronounced desensitization in PARK-gene Parkinson’s disease mouse models. We analysed surviving human SN DA neurons from patients with Parkinson’s disease and from controls, and detected elevated messenger RNA levels of D2-autoreceptors and GIRK2 in Parkinson’s disease. By electrophysiological analysis of postnatal juvenile and adult mouse SN DA neurons in in vitro brain-slices, we observed that D2-autoreceptor desensitization is reduced with postnatal maturation. Furthermore, a transient high-dopamine state in vivo, caused by one injection of either l-DOPA or cocaine, induced adult-like, non-desensitizing D2-autoreceptor responses, selectively in juvenile SN DA neurons, but not ventral tegmental area dopamine neurons. With pharmacological and genetic tools, we identified that the expression of this sensitized D2-autoreceptor phenotype required Cav1.3 L-type Ca2+ channel activity, internal Ca2+, and the interaction of the neuronal calcium sensor NCS-1 with D2-autoreceptors. Thus, we identified a first physiological function of Cav1.3 L-type Ca2+ channels in SN DA neurons for homeostatic modulation of their D2-autoreceptor responses. L-type Ca2+ channel activity however, was not important for pacemaker activity of mouse SN DA neurons. Furthermore, we detected elevated substantia nigra dopamine messenger RNA levels of NCS-1 (but not Cav1.2 or Cav1.3) after cocaine in mice, as well as in remaining human SN DA neurons in Parkinson’s disease. Thus, our findings provide a novel homeostatic functional link in SN DA neurons between Cav1.3- L-type-Ca2+ channels and D2-autoreceptor activity, controlled by NCS-1, and indicate that this adaptive signalling network (Cav1.3/NCS-1/D2/GIRK2) is also active in human SN DA neurons, and contributes to Parkinson’s disease pathology. As it is accessible to pharmacological modulation, it provides a novel promising target for tuning substantia nigra dopamine neuron activity, and their vulnerability to degeneration.
D2-autoreceptor; isradipine; Parkinsons disease; l-DOPA; cocaine
Cav1.2 and Cav1.3 L-type Ca2+ channels (LTCCs) are believed to underlie Ca2+ currents in brain, pancreatic β cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause card iovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Cav1.2 α1 subunits (Cav1.2DHP–/–) without affecting function and expression. This allowed separation of the DHP effects of Cav1.2 from those of Cav1.3 and other LTCCs. DHP effects on pancreatic β cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Cav1.2DHP–/– mice, which rules out a direct role of Cav1.3 for these physiological processes. Using Cav1.2DHP–/– mice, we established DHPs as mood-modifying agents: LTCC activator–induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Cav1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.
Cardiac tubular-like membrane invaginations contain the membrane scaffolding protein BIN1, which tethers dynamic microtubules that deliver calcium channels directly to T-tubule membrane.
The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate T-tubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2) to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC) coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubule-based Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient, indicating that Cav1.2 targeting to BIN1 is functionally important to cardiac calcium signaling. We have identified that membrane-associated BIN1 not only induces membrane curvature but can direct specific antegrade delivery of microtubule-transported membrane proteins. Furthermore, this paradigm provides a microtubule and BIN1-dependent mechanism of Cav1.2 delivery to T-tubules. This novel Cav1.2 trafficking pathway should serve as an important regulatory aspect of EC coupling, affecting cardiac contractility in mammalian hearts.
Calcium plays a primary role in regulating heart function. During each heartbeat, calcium ions cross the membrane of individual cardiac muscle cells and trigger a rapid increase of calcium within the cell (called the calcium transient). Calcium causes the muscle cells to contract and determines the strength of the overall heartbeat. Each cardiac muscle cell has many small tubular-like membrane invaginations known as T-tubules where calcium channels localize, allowing calcium ions to enter and immediately encounter intracellular calcium release organelles. While this organization is well described, it is not known how calcium channels localize to T-tubule membrane. Here we show that in human and mouse heart cells, a membrane scaffolding protein known as BIN1 is localized together with calcium channels at T-tubules. Using high-resolution live cell microscopy, we found that microtubules, which are necessary for calcium channel delivery to the membrane, are also tethered by BIN1. Loss of BIN1 in cardiac cells impairs delivery of calcium channels to the membrane and diminishes the intracellular calcium transient. According to this model, microtubules function as highways that carry newly synthesized calcium channels to BIN1-containing membrane. Once tethered to T-tubules by BIN1, the microtubules can deliver their calcium channel cargo. We postulate that this calcium channel delivery pathway is important to the regulation of cardiac calcium signaling and beat-to-beat cardiac function.
The analgesic α-conotoxin Vc1.1 inhibits Cav2.3 channels through a GABAB receptor–dependent pathway involving c-Src.
Neuronal Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated through G protein–coupled receptor pathways. The analgesic α-conotoxin Vc1.1 acts through γ-aminobutyric acid type B (GABAB) receptors (GABABRs) to inhibit Cav2.2 channels. We investigated GABABR-mediated modulation by Vc1.1, a cyclized form of Vc1.1 (c-Vc1.1), and the GABABR agonist baclofen of human Cav2.1 or Cav2.3 channels heterologously expressed in human embryonic kidney cells. 50 µM baclofen inhibited Cav2.1 and Cav2.3 channel Ba2+ currents by ∼40%, whereas c-Vc1.1 did not affect Cav2.1 but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of ∼300 pM. Depolarizing paired pulses revealed that ∼75% of the baclofen inhibition of Cav2.1 was voltage dependent and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated through voltage-independent pathways that could be disrupted by pertussis toxin, guanosine 5′-[β-thio]diphosphate trilithium salt, or the GABABR antagonist CGP55845. Overexpression of the kinase c-Src significantly increased inhibition of Cav2.3 by c-Vc1.1. Conversely, coexpression of a catalytically inactive double mutant form of c-Src or pretreatment with a phosphorylated pp60c-Src peptide abolished the effect of c-Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for inhibition of Cav2.3 by c-Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conferred c-Vc1.1 sensitivity. Our findings show that Vc1.1 inhibition of Cav2.3, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins, is caused by specific c-Src phosphorylation sites in the C terminus.
Low voltage activation of CaV1.3 L-type Ca2+ channels
controls excitability in sensory cells and central neurons as well as
sinoatrial node pacemaking. CaV1.3-mediated pacemaking determines
neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson
disease. We have previously found that in CaV1.4 L-type
Ca2+ channels, activation, voltage, and calcium-dependent
inactivation are controlled by an intrinsic distal C-terminal modulator.
Because alternative splicing in the CaV1.3 α1 subunit C
terminus gives rise to a long (CaV1.342) and a short
form (CaV1.342A), we investigated if a C-terminal
modulatory mechanism also controls CaV1.3 gating. The biophysical
properties of both splice variants were compared after heterologous expression
together with β3 and α2δ1 subunits in HEK-293 cells.
Activation of calcium current through CaV1.342A channels
was more pronounced at negative voltages, and inactivation was faster because
of enhanced calcium-dependent inactivation. By investigating several
CaV1.3 channel truncations, we restricted the modulator activity to
the last 116 amino acids of the C terminus. The resulting
CaV1.3ΔC116 channels showed gating properties
similar to CaV1.342A that were reverted by co-expression
of the corresponding C-terminal peptide C116. Fluorescence
resonance energy transfer experiments confirmed an intramolecular protein
interaction in the C terminus of CaV1.3 channels that also
modulates calmodulin binding. These experiments revealed a novel mechanism of
channel modulation enabling cells to tightly control CaV1.3 channel
activity by alternative splicing. The absence of the C-terminal modulator in
short splice forms facilitates CaV1.3 channel activation at lower
voltages expected to favor CaV1.3 activity at threshold voltages as
required for modulation of neuronal firing behavior and sinoatrial node
Among all voltage-gated calcium channels, the T-type Ca2+ channels encoded by the Cav3.2 genes are highly expressed in the hippocampus, which is associated with contextual, temporal and spatial learning and memory. However, the specific involvement of the Cav3.2 T-type Ca2+ channel in these hippocampus-dependent types of learning and memory remains unclear. To investigate the functional role of this channel in learning and memory, we subjected Cav3.2 homozygous and heterozygous knockout mice and their wild-type littermates to hippocampus-dependent behavioral tasks, including trace fear conditioning, the Morris water-maze and passive avoidance. The Cav3.2 −/− mice performed normally in the Morris water-maze and auditory trace fear conditioning tasks but were impaired in the context-cued trace fear conditioning, step-down and step-through passive avoidance tasks. Furthermore, long-term potentiation (LTP) could be induced for 180 minutes in hippocampal slices of WTs and Cav3.2 +/− mice, whereas LTP persisted for only 120 minutes in Cav3.2 −/− mice. To determine whether the hippocampal formation is responsible for the impaired behavioral phenotypes, we next performed experiments to knock down local function of the Cav3.2 T-type Ca2+ channel in the hippocampus. Wild-type mice infused with mibefradil, a T-type channel blocker, exhibited similar behaviors as homozygous knockouts. Taken together, our results demonstrate that retrieval of context-associated memory is dependent on the Cav3.2 T-type Ca2+ channel.
The fine-tuning of neuronal excitability relies on a tight control of Ca2+ homeostasis. The low voltage-activated (LVA) T-type calcium channels (Cav3.1, Cav3.2 and Cav3.3 isoforms) play a critical role in regulating these processes. Despite their wide expression throughout the central nervous system, the implication of T-type Cav3.2 isoform in brain functions is still poorly characterized. Here, we investigate the effect of genetic ablation of this isoform in affective disorders, including anxiety, cognitive functions as well as sensitivity to drugs of abuse. Using a wide range of behavioral assays we show that genetic ablation of the cacna1h gene results in an anxiety-like phenotype, whereas novelty-induced locomotor activity is unaffected. Deletion of the T-type channel Cav3.2 also triggers impairment of hippocampus-dependent recognition memories. Acute and sensitized hyperlocomotion induced by d-amphetamine and cocaine are dramatically reduced in T-type Cav3.2 deficient mice. In addition, the administration of the T-type blocker TTA-A2 prevented the expression of locomotor sensitization observed in wildtype mice. In conclusion, our data reveal that physiological activity of this specific Ca2+ channel is required for affective and cognitive behaviors. Moreover, our work highlights the interest of T-type channel blockers as therapeutic strategies to reverse drug-associated alterations.
T-type Ca2+ channels; anxiety; memory and learning; psychostimulants; drugs of abuse; spontaneous behavior
Previous data have indicated that T-type calcium channels (low-voltage activated, LVA, T-channels) are potently inhibited by volatile anesthetics. Although the interactions of T-channels with a number of anesthetics have been described, the mechanisms by which these agents modulate channel activity, and the functional consequences of such interactions, are not well studied. Here, we used patch-clamp recordings to explore the actions of a prototypical volatile anesthetic, isoflurane (Iso), on recombinant human CaV3.1 and CaV3.2 isoforms. We also performed behavioral testing of anesthetic end-points in mice lacking CaV3.2. Iso applied at resting channel states blocked current through both isoforms in a similar manner at clinically relevant concentrations (1 minimum alveolar concentration, MAC). Inhibition was more prominent at depolarized membrane potentials (−65 mV versus −100 mV) as evidenced by hyperpolarizing shifts in channel availability curves and a 2.5-fold decrease in IC50 values. Iso slowed recovery from inactivation and enhanced deactivation in both CaV3.1 and CaV3.2 in a comparable manner, but caused a depolarizing shift in activation curves and greater use-dependent block of CaV3.2 channels. In behavioral tests, CaV3.2 knockout (KO) mice showed significantly decreased MAC in comparison to wild type (WT) littermates. KO and WT mice did not differ in loss of righting reflex (LORR), but mutant mice displayed a delayed onset of anesthetic induction. We conclude that state-dependent inhibition of T-channel isoforms in the central and peripheral nervous system may contribute to isoflurane's important clinical effects.
The L-type calcium channel Cav1.2 is important for brain and heart function. The ubiquitous calcium sensing protein calmodulin (CaM) regulates calcium dependent gating of Cav1.2 channels by reducing calcium influx, a process known as calcium-dependent inactivation (CDI). Dissecting the calcium-dependence of CaM in this process has benefited greatly from the use of mutant CaM molecules which are unable to bind calcium to their low affinity (N-lobe) and high affinity (C-lobe) binding sites. Unlike CDI, it is unknown whether CaM can modulate the activation gating of Cav1.2 channels.
We examined a Cav1.2 point mutant in the N-terminus region of the channel (A39V) that has been previously linked to Brugada syndrome. Using mutant CaM constructs in which the N- and/or C-lobe calcium binding sites were ablated, we were able to show that this Brugada syndrome mutation disrupts N-lobe CDI of the channel. In the course of these experiments, we discovered that all mutant CaM molecules were able to alter the kinetics of channel activation even in the absence of calcium for WT-Cav1.2, but not A39V-Cav1.2 channels. Moreover, CaM mutants differentially shifted the voltage-dependence of activation for WT and A39V-Cav1.2 channels to hyperpolarized potentials. Our data therefore suggest that structural changes in CaM that arise directly from site directed mutagenesis of calcium binding domains alter activation gating of Cav1.2 channels independently of their effects on calcium binding, and that the N-terminus of the channel contributes to this CaM dependent process.
Our data indicate that caution must be exercised when interpreting the effects of CaM mutants on ion channel gating.
Calcium channel; Calmodulin mutant; CDI; N-terminus; Brugada; Activation; Cav1.2; L-type; IQ; Channelopathy; Voltage; Gating; CACNA1C
Familial hemiplegic migraine type 1 (FHM1), a monogenic subtype of migraine with aura, is caused by gain-of-function mutations in CaV2.1 (P/Q-type) calcium channels. In FHM1 knockin mice, excitatory neurotransmission at cortical pyramidal cell synapses is enhanced, but inhibitory neurotransmission at connected pairs of fast-spiking (FS) interneurons and pyramidal cells is unaltered, despite being initiated by CaV2.1 channels. The mechanism underlying the unaltered GABA release at cortical FS interneuron synapses remains unknown. Here, we show that the FHM1 R192Q mutation does not affect inhibitory transmission at autapses of cortical FS and other types of multipolar interneurons in microculture from R192Q knockin mice, and investigate the underlying mechanism. Lowering the extracellular [Ca2+] did not reveal gain-of-function of evoked transmission neither in control nor after prolongation of the action potential (AP) with tetraethylammonium, indicating unaltered AP-evoked presynaptic calcium influx at inhibitory autapses in FHM1 KI mice. Neither saturation of the presynaptic calcium sensor nor short duration of the AP can explain the unaltered inhibitory transmission in the mutant mice. Recordings of the P/Q-type calcium current in multipolar interneurons in microculture revealed that the current density and the gating properties of the CaV2.1 channels expressed in these interneurons are barely affected by the FHM1 mutation, in contrast with the enhanced current density and left-shifted activation gating of mutant CaV2.1 channels in cortical pyramidal cells. Our findings suggest that expression of specific CaV2.1 channels differentially sensitive to modulation by FHM1 mutations in inhibitory and excitatory cortical neurons underlies the gain-of-function of excitatory but unaltered inhibitory synaptic transmission and the likely consequent dysregulation of the cortical excitatory–inhibitory balance in FHM1.
•Multipolar interneuron autaptic transmission is unaltered in FHM1 knockin mice.•This is due to unaltered action potential (AP)-evoked presynaptic Ca influx.•Unaltered AP-evoked Ca influx is not due to short duration of interneuron APs.•CaV2.1 channels of multipolar interneurons are barely affected by the FHM1 mutation.•This may explain unaltered inhibitory neurotransmission in FHM1.
AHP, after-hyperpolarization; AP, action potential; APhw, action potential half width; BME, basal Eagle's medium; BSA, bovine serum albumin; CGRP, calcitonin gene-related peptide; CSD, cortical spreading depression; DIV, days in vitro; FHM1, familial hemiplegic migraine type 1; FS, fast-spiking; GABA, gamma-aminobutyric acid; GAD, glutamic acid decarboxylase; 5HT3aR, 5-hydroxytryptamine 3a receptor; KI, knockin; LJP, liquid junction potential; PBS, phosphate buffered saline; PV, parvalbumin; siRNA, small interfering ribonucleic acid; SNAP25, synaptosomal-associated protein of 25 kDa; SOM, somatostatin; VIP, vasoactive intestinal peptide; Migraine, calcium channel, inhibitory synaptic transmission; Fast-spiking interneuron; Knockin mouse model; Channelopathy; Excitatory–inhibitory balance
Voltage-gated L-type Ca2+ channels (L-VGCCs) like CaV1.2 are assumed to play a crucial role for controlling release of trophic peptides including brain-derived neurotrophic factor (BDNF). In the inner ear of the adult mouse, besides the well-described L-VGCC CaV1.3, CaV1.2 is also expressed. Due to lethality of constitutive CaV1.2 knock-out mice, the function of this ion channel as well as its putative relationship to BDNF in the auditory system is entirely elusive. We recently described that BDNF plays a differential role for inner hair cell (IHC) vesicles release in normal and traumatized condition. To elucidate a presumptive role of CaV1.2 during this process, two tissue-specific conditional mouse lines were generated. To distinguish the impact of CaV1.2 on the cochlea from that on feedback loops from higher auditory centers CaV1.2 was deleted, in one mouse line, under the Pax2 promoter (CaV1.2Pax2) leading to a deletion in the spiral ganglion neurons, dorsal cochlear nucleus, and inferior colliculus. In the second mouse line, the Egr2 promoter was used for deleting CaV1.2 (CaV1.2Egr2) in auditory brainstem nuclei. In both mouse lines, normal hearing threshold and equal number of IHC release sites were observed. We found a slight reduction of auditory brainstem response wave I amplitudes in the CaV1.2Pax2 mice, but not in the CaV1.2Egr2 mice. After noise exposure, CaV1.2Pax2 mice had less-pronounced hearing loss that correlated with maintenance of ribbons in IHCs and less reduced activity in auditory nerve fibers, as well as in higher brain centers at supra-threshold sound stimulation. As reduced cochlear BDNF mRNA levels were found in CaV1.2Pax2 mice, we suggest that a CaV1.2-dependent step may participate in triggering part of the beneficial and deteriorating effects of cochlear BDNF in intact systems and during noise exposure through a pathway that is independent of CaV1.2 function in efferent circuits.
L-VGCCs; CaV1.2; inner ear; SOC; ABR; BDNF
Neurons express multiple types of voltage-gated calcium (Ca2+) channels. Two subtypes of neuronal L-type Ca2+ channels are encoded by CaV1.2 and CaV1.3 pore-forming subunits. To compare targeting of CaV1.2 and CaV1.3 L-type Ca2+ channels, we transfected rat hippocampal neuronal cultures with surface-epitope-tagged sHA-CaV1.2 or sHA-CaV1.3a constructs and found that: (i) both sHA-CaV1.2 and sHA-CaV1.3a form clusters on the neuronal plasma membrane surface; (ii) when compared with sHA-CaV1.2 surface clusters, the sHA-CaV1.3a surface clusters were 10% larger and 25% brighter, but 35% less abundant; (iii) 81% of sHA-CaV1.2 surface clusters, but only 48% of sHA-CaV1.3a surface clusters, co-localized with synapsin clusters; (iv) co-expression with GFP-Shank-1B had no significant effect on sHA-CaV1.2 surface clusters, but promoted formation and synaptic localization of sHA-CaV1.3a surface clusters. In experiments with dihydropyridine-resistant CaV1.2 and CaV1.3a mutants we demonstrated that CaV1.3a L-type Ca2+ channels preferentially mediate nuclear pCREB signaling in hippocampal neurons at low, but not at high, levels of stimulation. In experiments with primary neuronal cultures from CaV1.3 knockout mice we discovered that CaV1.3 channels play a more important role in pCREB signaling in striatal medium spiny neurons than in hippocampal neurons. Our results provide novel insights into the function of CaV1.2 and CaV1.3 L-type Ca2+ channels in the brain.
calcium channels; CREB; postsynaptic density; synapse; synaptic plasticity
Voltage-dependent calcium channels are widely distributed in animal cells, including spermatozoa. Calcium is fundamental in many sperm functions such as: motility, capacitation and the acrosome reaction, all essential for fertilization. Pharmacological evidence has suggested T-type calcium channels participate in the acrosome reaction. Niflumic acid (NA), a non-steroidal anti-inflammatory drug commonly used as chloride channel blocker, blocks T-currents in mouse spermatogenic cells and Cl− channels in testicular sperm. Here we examine the mechanism of NA blockade and explore if it can be used to separate the contribution of different CaV3 members previously detected in these cells. Electrophysiological patch-clamp recordings were performed in isolated mouse spermatogenic cells and in HEK cells heterologously expressing CaV3 channels. NA blocks mouse spermatogenic cell T-type currents with an IC50 of 73.5 µM, without major voltage-dependent effects. The NA blockade is more potent in the open and in the inactivated state than in the closed state of the T-type channels. Interestingly, we found that heterologously expressed CaV3.1 and CaV3.3 channels were more sensitive to NA than CaV3.2 channels, and this drug substantially slowed the recovery from inactivation of the three isoforms. Molecular docking modeling of drug-channel binding predicts that NA binds preferentially to the extracellular face of CaV3.1 channels. The biophysical characteristics of mouse spermatogenic cell T-type currents more closely resemble those from heterologously expressed CaV3.1 channels, including their sensitivity to NA. As CaV3.1 null mice maintain their spermatogenic cell T-currents, it is likely that a novel CaV3.2 isoform is responsible for them.
Niflumic acid; T-type channels; Blockade Mechanism
Kelch-like 1 (KLHL1) is a neuronal actin-binding protein that modulates voltage-gated CaV2.1 (P/Q-type) and CaV3.2 (α1H T-type) calcium channels; KLHL1 knockdown experiments (KD) cause down-regulation of both channel types and altered synaptic properties in cultured rat hippocampal neurons (Perissinotti et al., 2014). Here, we studied the effect of ablation of KLHL1 on calcium channel function and synaptic properties in cultured hippocampal neurons from KLHL1 knockout (KO) mice. Western blot data showed the P/Q-type channel α1A subunit was less abundant in KO hippocampus compared to wildtype (WT); and P/Q-type calcium currents were smaller in KO neurons than WT during early days in vitro, although this decrease was compensated for at late stages by increases in L-type calcium current. In contrast, T-type currents did not change in culture. However, biophysical properties and western blot analysis revealed a differential contribution of T-type channel isoforms in the KO, with CaV3.2 α1H subunit being down-regulated and CaV3.1 α1G up-regulated. Synapsin I levels were also reduced in the KO hippocampus and cultured neurons displayed a concomitant reduction in synapsin I puncta and decreased miniature excitatory postsynaptic current (mEPSC) frequency. In summary, genetic ablation of the calcium channel modulator resulted in compensatory mechanisms to maintain calcium current homeostasis in hippocampal KO neurons; however, synaptic alterations resulted in a reduction of excitatory synapse number, causing an imbalance of the excitatory-inhibitory synaptic input ratio favoring inhibition.
actin-binding proteins; Kelch-like 1 protein; T-type voltage-gated calcium channels; P/Q-type voltage-gated calcium channels; LVA; HVA; synapsin I; SCA8
Adhesion and migration are relevant physiological functions that must be regulated by the cell under both normal and pathological conditions. The dioxin receptor (AhR) has emerged as a transcription factor regulating both processes in mesenchymal, epithelial and endothelial cells. Indirect results suggest that AhR could cooperate not only with additional transcription factors but also with membrane-associated proteins to drive such processes.
In this study, we have used immortalized and primary dermal fibroblasts from wild type (AhR+/+) and AhR-null (AhR−/−) mice to show that AhR modulates membrane distribution and mobilization of caveolin-1 (Cav-1) during directional cell migration. AhR co-immunoprecipitated with Cav-1 and a fraction of both proteins co-localized to detergent-resistant membrane microdomains (DRM). Consistent with a role of AhR in the process, AhR−/− cells had a significant reduction in Cav-1 in DRMs. Moreover, high cell density reduced AhR nuclear levels and moved Cav-1 from DRMs to the soluble membrane in AhR+/+ but not in AhR−/− cells. Tyrosine-14 phosphorylation had a complex role in the mechanism since its upregulation reduced Cav-1 in DRMs in both AhR+/+ and AhR−/−cells, despite the lower basal levels of Y14-Cav-1 in the null cells. Fluorescence recovery after photobleaching revealed that AhR knock-down blocked Cav-1 transport to the plasma membrane, a deficit possibly influencing its depleted levels in DRMs. Membrane distribution of Cav-1 in AhR-null fibroblasts correlated with higher levels of cholesterol and with disrupted membrane microdomains, whereas addition of exogenous cholesterol changed the Cav-1 distribution of AhR+/+ cells to the null phenotype. Consistently, higher cholesterol levels enhanced caveolae-dependent endocytosis in AhR-null cells.
These results suggest that AhR modulates Cav-1 distribution in migrating cells through the control of cholesterol-enriched membrane microdomains. Our study also supports the likely possibility of membrane-related, transcription factor independent, functions of AhR.
Electronic supplementary material
The online version of this article (doi:10.1186/s12964-014-0057-7) contains supplementary material, which is available to authorized users.
Dioxin receptor; Caveolin-1; Membrane microdomains; Endocytosis; Cholesterol
L-type Ca2+ channel (LTCC)-activated signaling cascades contribute significantly to psychostimulant-induced locomotor sensitization; however, the precise contribution of the two brain-specific subunits Cav1.2 and Cav1.3 remains largely unknown. In this study by utilizing amphetamine and cocaine locomotor sensitization in mutant mice expressing dihydropyridine (DHP)-insensitive Cav1.2 LTCCs (Cav1.2DHP−/−) we find that as opposed to a previously identified role of the Cav1.3 subunit of LTCCs in development of sensitization, the Cav1.2 subunit mediates expression of amphetamine and cocaine sensitization when examined following a fourteen day drug-free period. Molecular studies to further elucidate the role of Cav1.2 versus Cav1.3 LTCCs in activating signaling pathways in the nucleus accumbens (NAc) of drug naïve versus drug pre-exposed mice examined fourteen days later revealed that an acute amphetamine and cocaine challenge in drug naïve mice increases Ser 133 CREB phosphorylation in the NAc via Cav1.3 channels and via a dopamine D1-dependent mechanism, independent of the ERK pathway, an important mediator of psychostimulant-induced plasticity. In contrast, in amphetamine and cocaine pre-exposed mice, an amphetamine or cocaine challenge no longer activates CREB unless Cav1.2 LTCCs are blocked. This Cav1.2-dependent blunting of CREB activation that underlies expression of locomotor sensitization occurs only following extended drug-free periods and involves recruitment of D1 receptors and the ERK pathway. Thus, our results demonstrate that specific LTCC subunits are required for the development (Cav1.3) versus expression (Cav1.2) of psychostimulant sensitization and that subunit-specific signaling pathways recruited by psychostimulants, underlies long-term drug-induced behavioral responses.
Cav1.2; Cav1.3; amphetamine; cocaine; ERK; CREB
The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human CaV2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, P.F. Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J. Gen. Physiol. 124:445–461). Here, we report the characterization of the so-called b gating mode. A CaV2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability, and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human CaV2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human CaV2.1 channels. In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly. Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes. Regulation of the complex modal gating of human CaV2.1 channels could be a potent and versatile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes.
Ca2+ channel; gating mode; synaptic transmission; familial hemiplegic migraine; channelopathy
It is well established that idiopathic generalized epilepsies (IGEs) show a polygenic origin and may arise from dysfunction of various types of voltage- and ligand-gated ion channels. There is an increasing body of literature implicating both high and low voltage-activated (HVA and LVA) calcium channels and their ancillary subunits in IGEs. Cav2.1 (P/Q-type) calcium channels control synaptic transmission at presynaptic nerve terminals, and mutations in the gene encoding the Cav2.1 α1 subunit (CACNA1A) have been linked to absence seizures in both humans and rodents. Similarly, mutations and loss of function mutations in ancillary HVA calcium channel subunits known to coassemble with Cav2.1 result in IGE phenotypes in mice. It is important to note that in all these mouse models with mutations in HVA subunits there is a compensatory increase in thalamic LVA currents, which likely leads to the seizure phenotype. In fact, gain of function mutations have been identified in Cav3.2 (an LVA or T-type calcium channel encoded by the CACNA1H gene) in patients with congenital forms of IGEs, consistent with increased excitability of neurons as a result of enhanced T-type channel function. Here we provide a broad overview of the roles of voltage-gated calcium channels, their mutations, and how they might contribute to the river that terminates in epilepsy.
calcium channel; P/Q-type channels; T-type channels; epilepsy; seizures
The atrioventricular node controls cardiac impulse conduction and generates pacemaker activity in case of failure of the sino-atrial node. Understanding the mechanisms of atrioventricular automaticity is important for managing human pathologies of heart rate and conduction. However, the physiology of atrioventricular automaticity is still poorly understood. We have investigated the role of three key ion channel-mediated pacemaker mechanisms namely, Cav1.3, Cav3.1 and HCN channels in automaticity of atrioventricular node cells (AVNCs). We studied atrioventricular conduction and pacemaking of AVNCs in wild-type mice and mice lacking Cav3.1 (Cav3.1−/−), Cav1.3 (Cav1.3−/−), channels or both (Cav1.3−/−/Cav3.1−/−). The role of HCN channels in the modulation of atrioventricular cells pacemaking was studied by conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity. Inactivation of Cav3.1 channels impaired AVNCs pacemaker activity by favoring sporadic block of automaticity leading to cellular arrhythmia. Furthermore, Cav3.1 channels were critical for AVNCs to reach high pacemaking rates under isoproterenol. Unexpectedly, Cav1.3 channels were required for spontaneous automaticity, because Cav1.3−/− and Cav1.3−/−/Cav3.1−/− AVNCs were completely silent under physiological conditions. Abolition of the cAMP sensitivity of HCN channels reduced automaticity under basal conditions, but maximal rates of AVNCs could be restored to that of control mice by isoproterenol. In conclusion, while Cav1.3 channels are required for automaticity, Cav3.1 channels are important for maximal pacing rates of mouse AVNCs. HCN channels are important for basal AVNCs automaticity but do not appear to be determinant for β-adrenergic regulation.
genetically-engineered mice; pacemaker activity; atrioventricular node; congenital heart block; sino-atrial node dysfunction; ion channels; Cav1.3 channels; Cav3.1 channels; HCN channels; electrophysiology; conduction; heart rate
The function of voltage-gated calcium (Cav) channels greatly depends on coupling to cytoplasmic accessory β subunits, which not only promote surface expression, but also modulate gating and kinetic properties of the α1 subunit. Schistosomes, parasitic platyhelminths that cause schistosomiasis, express two β subunit subtypes: a structurally conventional β subunit and a variant β subunit with unusual functional properties. We have previously characterized the functional properties of the variant Cavβ subunit. Here, we focus on the modulatory phenotype of the conventional Cavβ subunit (SmCavβ) using the human Cav2.3 channel as the substrate for SmCavβ and the whole-cell patch-clamp technique.
The conventional Schistosoma mansoni Cavβ subunit markedly increases Cav2.3 currents, slows macroscopic inactivation and shifts steady state inactivation in the hyperpolarizing direction. However, currents produced by Cav2.3 in the presence of SmCavβ run-down to approximately 75% of their initial amplitudes within two minutes of establishing the whole-cell configuration. This suppressive effect was independent of Ca2+, but dependent on intracellular Mg2+-ATP. Additional experiments revealed that SmCavβ lends the Cav2.3/SmCavβ complex sensitivity to Na+ ions. A mutant version of the Cavβ subunit lacking the first forty-six amino acids, including a string of twenty-two acidic residues, no longer conferred sensitivity to intracellular Mg2+-ATP and Na+ ions, while continuing to show wild type modulation of current amplitude and inactivation of Cav2.3.
The data presented in this article provide insights into novel mechanisms employed by platyhelminth Cavβ subunits to modulate voltage-gated Ca2+ currents that indicate interactions between the Ca2+ channel complex and chelated forms of ATP as well as Na+ ions. These results have potentially important implications for understanding previously unknown mechanisms by which platyhelminths and perhaps other organisms modulate Ca2+ currents in excitable cells.
Mice that have defects in their low-threshold T-type calcium channel (T-channel) genes show altered pain behaviors. The changes in the ratio of nociceptive neurons and the burst firing property of reticular thalamic (RT) and ventroposterior (VP) neurons in Cav3.2 knockout (KO) mice were studied to test the involvement of thalamic T-channel and burst firing activity in pain function.
Under pentobarbital or urethane anesthesia, the patterns of tonic and burst firings were recorded in functionally characterized RT and VPL neurons of Cav3.2 KO mice. Many RT neurons were nociceptive (64% under pentobarbital anesthesia and 50% under urethane anesthesia). Compared to their wild-type (WT) controls, fewer nociceptive RT neurons were found in Cav3.2 KO mice. Both nociceptive and tactile RT neurons showed fewer bursts in Cav3.2 KO mice. Within a burst, RT neurons of Cav3.2 KO mice had a lower spike frequency and less-prominent accelerando-decelerando change. In contrast, VP neurons of Cav3.2 KO mice showed a higher ratio of bursts and a higher discharge rate within a burst than those of the WT control. In addition, the long-lasting tonic firing episodes in RT neurons of the Cav3.2 KO had less stereotypic regularity than their counterparts in WT mice.
RT might be important in nociception of the mouse. In addition, we showed an important role of Cav3.2 subtype of T-channel in RT burst firing pattern. The decreased occurrence and slowing of the bursts in RT neurons might cause the increased VP bursts. These changes would be factors contributing to alternation of pain behavior in the Cav3.2 KO mice.
Low voltage-activated (T-type) calcium channels are responsible for burst firing and transmitter release in neurons and are important for exocytosis and hormone secretion in pituitary cells. T-type channels contain an α1 subunit, of which there are three subtypes, Cav3.1, 3.2 and 3.3, and each subtype has distinct kinetic characteristics. Although 17β-estradiol modulates T-type calcium channel expression and function, little is known about the molecular mechanisms involved. Presently, we used real-time PCR quantification of RNA extracted from hypothalamic nuclei and pituitary in vehicle and E2-treated C57BL/6 mice to elucidate E2-mediated regulation of Cav3.1, 3.2 and 3.3 subunits. The three subunits were expressed in both the hypothalamus and the pituitary. E2 treatment increased the mRNA expression of Cav3.1 and 3.2, but not Cav3.3, in the medial preoptic area and the arcuate nucleus. In the pituitary, Cav3.1 was increased with E2-treatment and Cav3.2 and 3.3 were decreased. In order to examine whether the classical estrogen receptors (ERs) were involved in the regulation, we used ERα- and ERβ-deficient C57BL/6 mice and explored the effects of E2 on T-type channel subtypes. Indeed, we found that the E2-induced increase in Cav3.1 in the hypothalamus was dependent on ERα, whereas the E2 effect on Cav3.2 was dependent on both ERα and ERβ. However, the E2-induced effects in the pituitary were dependent on only the expression of ERα. The robust E2-regulation of the T-type calcium channels could be an important mechanism by which E2 increases the excitability of hypothalamic neurons and modulates pituitary secretion.
Cav3.1; Cav3.2; Cav3.3; α1 subunits; hypothalamus; pituitary
In heart, Ca2+ entering myocytes via CaV1.2 channels controls essential functions including, excitation-contraction (EC) coupling, action potential duration (APD), and gene expression. RGK GTPases potently inhibit CaV1.2 channels, an effect that may figure prominently in cardiac Ca2+ homeostasis under physiological and disease conditions.
To define the mechanisms and molecular determinants underlying Rem GTPase inhibition of CaV1.2 channels in heart, and determine whether such inhibited channels can be pharmacologically rescued.
Methods and Results
Over-expressing Rem in adult guinea pig heart cells dramatically depresses L-type calcium current (ICa,L; ~90% inhibition) and moderately reduces maximum gating charge (Qmax; 33%), without appreciably diminishing the physical number of channels in the membrane. Rem-inhibited CaV1.2 channels were supra-modulated by BAY K 8644 (10-fold increase) compared to control channels (3-fold increase). However, Rem prevented protein kinase A (PKA)-mediated up-regulation of ICa,L, an effect achieved without disrupting the sympathetic signaling cascade since PKA modulation of IKS remained intact. In accord with its functional impact on ICa,L, Rem selectively prevented PKA-, but not BAY K 8644-induced prolongation of the cardiac APD. A GTP-binding-deficient Rem[T94N] mutant was functionally inert with respect to ICa,L inhibition. A chimeric construct, Rem265-H, featuring a swap of the Rem C-terminus tail for the analogous domain from H-Ras, inhibited ICa,L and Qmax to the same extent as wild type Rem despite lacking the capacity to autonomously localize to the sarcolemma.
Rem predominantly inhibits ICa,L in heart by arresting surface CaV1.2 channels in a low open probability (Po) gating mode, rather than by interfering with channel trafficking. Moreover, Rem-inhibited CaV1.2 channels can be selectively rescued by BAY K 8644 but not PKA-dependent phosphorylation. Contrary to findings in reconstituted systems, Rem-induced ablation of cardiac ICa,L requires GTP-binding, but not membrane-targeting of the nucleotide binding domain (NBD). These findings provide a different perspective on the molecular mechanisms and structural determinants underlying RGK GTPase inhibition of CaV1.2 channels in heart, and suggest new (patho)physiological dimensions of this crosstalk.
L-type calcium channels; Rem; RGK GTPase; cardiac myocytes
Intravascular pressure-induced vasoconstriction (the “myogenic response”) is intrinsic to smooth muscle cells, but mechanisms that underlie this response are unresolved. Here we investigated the physiological function of arterial smooth muscle cell caveolae in mediating the myogenic response. Since caveolin-1 (cav-1) ablation abolishes caveolae formation in arterial smooth muscle cells, myogenic mechanisms were compared in cerebral arteries from control (cav-1+/+) and cav-1-deficient (cav-1−/−) mice. At low intravascular pressure (10 mmHg), wall membrane potential, intracellular calcium concentration ([Ca2+]i), and myogenic tone were similar in cav-1+/+ and cav-1−/− arteries. In contrast, pressure elevations to between 30 and 70 mmHg induced a smaller depolarization, [Ca2+]i elevation, and myogenic response in cav-1−/− arteries. Depolarization induced by 60 mM K+ also produced an attenuated [Ca2+]i elevation and constriction in cav-1−/− arteries, whereas extracellular Ca2+ removal and diltiazem, an L-type Ca2+ channel blocker, similarly dilated cav-1+/+ and cav-1−/− arteries. Nω-nitro-l-arginine, an nitric oxide synthase inhibitor, did not restore myogenic tone in cav-1−/− arteries. Iberiotoxin, a selective Ca2+-activated K+ (KCa) channel blocker, induced a similar depolarization and constriction in pressurized cav-1+/+ and cav-1−/− arteries. Since pressurized cav-1−/− arteries are more hyperpolarized and this effect would reduce KCa current, these data suggest that cav-1 ablation leads to functional KCa channel activation, an effect that should contribute to the attenuated myogenic constriction. In summary, data indicate that cav-1 ablation reduces pressure-induced depolarization and depolarization-induced Ca2+ influx, and these effects combine to produce a diminished arterial wall [Ca2+]i elevation and constriction.
calcium-activated potassium channels; membrane depolarization; arterial wall calcium