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.
In tottering mice, a point mutation in the gene encoding P-type (Cav2.1) voltage-gated calcium channels results in ataxia, absence epilepsy, and motor dystonia that appear 3–4 weeks postnatal. The aberrant motor behaviors have been linked to cerebellar dysfunction, and adult Purkinje cells (PCs) of tottering mice exhibit calcium-dependent changes in gene transcription suggestive of altered calcium homeostasis. In an attempt to identify early postnatal events important for the development of the behavioral phenotype, we examined calcium channel expression in cerebellar PCs from postnatal days 6 to 15 (P6–15). Whole cell recording was combined with selective calcium channel antagonists to allow discrimination of the various voltage-activated calcium channels types; early age-dependent differences between tottering and wild-type PCs were found. Wild-type PCs experienced a steady increase in P current density over this period, resulting in a two-fold change by P15. In tottering, by contrast, P current density remained unchanged from postnatal days 6 to 8 and was only 25% of the wild-type level by P8. A developmental delay in functional expression was implicated in this early deficit, since ensuing gains over the subsequent week brought tottering P current density close to the wild-type level by P15. At this age, tottering PCs also exhibited a 2.2-fold higher L-type calcium current density than that expressed by wild-type PCs. Increases in N current were apparent at some ages, most strikingly within a subset of tottering PCs at P15. Functional Rand T-type calcium current densities were equivalent to wild-type levels at all ages. We conclude that the tottering mutation brings about selective changes in functional calcium channel expression one to two weeks prior to the appearance of the behavioral deficits, raising the possibility that they represent an early, primary event along the path to motor dysfunction in tottering.
voltage; gated Ca2+ channel; channelopathy; neural development
Alternative pre-mRNA splicing predominates in the nervous systems of complex organisms including humans dramatically expanding the potential size of the proteome. Cell-specific alternative pre-mRNA splicing is thought to optimize protein function for specialized cellular tasks, but direct evidence for this is limited. Transmission of noxious thermal stimuli relies on the activity of N-type CaV2.2 calcium channels in nociceptors. Using an exon replacement strategy in mice, we show that mutually exclusive splicing in the CaV2.2 gene modulates N-type channel function in nociceptors leading to a change in morphine analgesia. Exon 37a enhances μ-opioid receptor mediated inhibition of N-type calcium channels by promoting activity-independent inhibition. In the absence of e37a spinal morphine analgesia is weakened in vivo without influencing the basal response to noxious thermal stimuli. Our data suggest that highly specialized, discrete cellular responsiveness in vivo can be attributed to alternative splicing events regulated at the level of individual neurons.
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.
Calcium channel blockers (CCBs) are widely used to treat cardiovascular diseases such as hypertension, angina pectoris, hypertrophic cardiomyopathy, and supraventricular tachycardia. CCBs selectively inhibit the inward flow of calcium ions through voltage-gated calcium channels, particularly Cav1.2, that are expressed in the cardiovascular system. Changes to the molecular structure of Cav1.2 channels could affect sensitivity of the channels to blockade by CCBs. Recently, extensive alternative splicing was found in Cav1.2 channels that generated wide phenotypic variations. Cardiac and smooth muscles express slightly different, but functionally important Cav1.2 splice variants. Alternative splicing could also modulate the gating properties of the channels and giving rise to different responses to inhibition by CCBs. Importantly, alternative splicing of Cav1.2 channels may play an important role to influence the outcome of many cardiovascular disorders. Therefore, the understanding of how alternative splicing impacts Cav1.2 channels pharmacology in various diseases and different organs may provide the possibility for individualized therapy with minimal side effects.
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
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
Caveolin-1 (Cav-1) interacts with large conductance Ca2+-activated potassium channels (BKCa) and likely exerts a negative regulatory effect on the channel activity. We investigated the role of Cav-1 in modulating BK(Ca) channel-mediated, endothelium-derived hyperpolarizing factor (EDHF)-dependent arteriolar dilation in normal condition and in an experimental model of obesity.
Methods and results
In isolated, pressurized (80 mmHg) gracilis muscle arterioles (∼100 µm) of Cav-1 knockout mice, acetylcholine (ACh)-induced, EDHF-mediated dilations were enhanced and were significantly reduced by the BK(Ca) channel inhibitor, iberiotoxin (IBTX), whereas IBTX had no effect on EDHF-mediated dilations in the wild-type mice. Dilations to the selective BK(Ca) channel opener, NS-1619 were augmented in the Cav-1 knockout mice. In high-fat diet-treated, obese rats ACh-induced coronary arteriolar dilations were preserved, whereas IBTX-sensitive, ACh-induced and also NS-1619-evoked vasodilations were augmented when compared with lean animals. In coronary arterioles of obese rats a reduced protein expression of Cav-1 was detected by western immunoblotting and immunohistochemistry. Moreover, in coronary arterioles of lean rats, disruption of caveolae with methyl-β-cyclodextrin augmented IBTX-sensitive, ACh-induced, and also NS-1619-evoked dilations.
Thus, under normal conditions, Cav-1 limits the contribution of the BK(Ca) channel to EDHF-mediated arteriolar dilation. In obesity, a reduced expression of Cav-1 leads to greater contribution of the BK(Ca) channel to EDHF-mediated response, which seems essential for maintained coronary dilation.
Coronary; Microcirculation; EDHF; MaxiK channel; Caveolae
The Kelch-like 1 protein (KLHL1) is a neuronal actin-binding protein that modulates calcium channel function. It increases the current density of Cav3.2 (α1H) calcium channels via direct interaction with α1H and actin-F, resulting in biophysical changes in Cav3.2 currents and an increase in recycling endosomal activity with subsequent increased α1H channel number at the plasma membrane. Interestingly, removal of the actin-binding Kelch motif (ΔKelch) prevents the increase in Cav3.2 current density seen with wild-type KLHL1 when tested with normal square pulse protocols but does not preclude the effect when tested using action potential waveforms (AP).
Here, we dissected the kinetic properties of the AP waveform that confer the mutant Kelch the ability to interact with Cav3.2 and induce an increase in calcium influx. We modified the action potential waveform by altering the slopes of repolarization and/or recovery from hyperpolarization or by changing the duration of the depolarization plateau or the hyperpolarization phase and tested the modulation of Cav3.2 by the mutant ΔKelch. Our results show that the recovery phase from hyperpolarization phase determines the conformational changes that allow the α1H subunit to properly interact with mutant KLHL1 lacking its actin-binding Kelch domains, leading to increased Ca influx.
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.
Voltage gated calcium channels (VGCCs) are well established mediators of pain signals in primary afferent neurons. N-type calcium channels are localized to synaptic nerve terminals in laminae 1 and 2 of the dorsal horn where their opening results in the release of neurotransmitters such as glutamate and substance P. The contribution of N-type channels to the processing of pain signals is regulated by alternate splicing of the N-type channel gene, with unique N-type channel splice variants being expressed in small nociceptive neurons. In contrast, T-type VGCCs of the Cav3.2 subtype are likely localized to nerve endings where they regulate cellular excitability. Consequently, inhibition of N-type and Cav3.2 T-type VGCCs has the propensity to mediate analgesia. T-type channel activity is regulated by redox modulation, and can be inhibited by a novel class of small organic blockers. N-type VGCC activity can be potently inhibited by highly selective peptide toxins that are delivered intrathecally, and the search for small organic blockers with clinical efficacy is ongoing. Here, we provide a brief overview of recent advances in this area, as presented at the Spring Pain Research conference (Grand Cayman, 2008).
Dorsal root ganglion; T-type channel; N-type channel; Conotoxin; Burst firing
Neurotransmitter release from mammalian sensory neurons is controlled by CaV2.2 N-type calcium channels. N-type channels are a major target of neurotransmitters and drugs that inhibit calcium entry, transmitter release and nociception through their specific G protein–coupled receptors. G protein–coupled receptor inhibition of these channels is typically voltage-dependent and mediated by Gβγ, whereas N-type channels in sensory neurons are sensitive to a second G protein–coupled receptor pathway that inhibits the channel independent of voltage. Here we show that preferential inclusion in nociceptors of exon 37a in rat Cacna1b (encoding CaV2.2) creates, de novo, a C-terminal module that mediates voltage-independent inhibition. This inhibitory pathway requires tyrosine kinase activation but not Gβγ. A tyrosine encoded within exon 37a constitutes a critical part of a molecular switch controlling N-type current density and G protein–mediated voltage-independent inhibition. Our data define the molecular origins of voltage-independent inhibition of N-type channels in the pain pathway.
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
In T lymphocytes, calcium ion controls a variety of biological processes including development, survival, proliferation, and effector functions. These distinct and specific roles are regulated by different calcium signals, which are generated by various plasma membrane calcium channels. The repertoire of calcium-conducting proteins in T lymphocytes includes store-operated CRAC channels, transient receptor potential channels, P2X channels, and L-type voltage-gated calcium (Cav1) channels. In this paper, we will focus mainly on the role of the Cav1 channels found expressed by T lymphocytes, where these channels appear to operate in a T cell receptor stimulation-dependent and voltage sensor independent manner. We will review their expression profile at various differentiation stages of CD4 and CD8 T lymphocytes. Then, we will present crucial genetic evidence in favor of a role of these Cav1 channels and related regulatory proteins in both CD4 and CD8 T cell functions such as proliferation, survival, cytokine production, and cytolysis. Finally, we will provide evidence and speculate on how these voltage-gated channels might function in the T lymphocyte, a non-excitable cell.
Cav1 channels; calcium channels; CD4 T cells; CD8 T cells; CRAC channel
A loss of function of the L-type calcium channel, Cav1.2, results in a cardiac specific disease known as Brugada syndrome. Although many Brugada syndrome channelopathies reduce channel function, one point mutation in the N-terminus of Cav1.2 (A39V) has been shown to elicit disease a phenotype because of a loss of surface trafficking of the channel. This lack of cell membrane expression could not be rescued by the trafficking chaperone Cavβ.
We report that despite the striking loss of trafficking described previously in the cardiac Cav1.2 channel, the A39V mutation while in the background of the brain isoform traffics and functions normally. We detected no differences in biophysical properties between wild type Cav1.2 and A39V-Cav1.2 in the presence of either a cardiac (Cavβ2b), or a neuronal beta subunit (Cavβ1b). In addition, the A39V-Cav1.2 mutant showed a normal Cavβ2b mediated increase in surface expression in tsA-201 cells.
The Brugada syndrome mutation A39V when introduced into rat brain Cav1.2 does not trigger the loss-of-trafficking phenotype seen in a previous study on the human heart isoform of the channel.
L-type calcium channel; Beta subunit; Brugada; Channelopathy; Traffic; Cav1.2
CaV1.3 L-type channels control inner hair cell (IHC) sensory and sinoatrial node (SAN) function, and excitability in central neurons by means of their low-voltage activation and inactivation properties. In SAN cells CaV1.3 inward calcium current (ICa) inactivates rapidly whereas in IHCs inactivation is slow. A candidate suggested in slowing CaV1.3 channel inactivation is the presynaptically located ribbon-synapse protein RIM that is expressed in immature IHCs in presynaptic compartments also expressing CaV1.3 channels. CaV1.3 channel gating is also modulated by an intramolecular C-terminal mechanism. This mechanism was elicited during analysis of human C-terminal splice variants that differ in the length of their C-terminus and that modulates the channel's negative activation range and slows calcium-dependent inactivation.
CaV1.3 L-type calcium channel; channel gating; C-terminal modulation; protein-protein interaction
The N-type voltage-gated calcium channel (Cav2.2) has gained immense prominence in the treatment of chronic pain. While decreased channel function is ultimately anti-nociceptive, directly targeting the channel can lead to multiple adverse side effects. Targeting modulators of channel activity may facilitate improved analgesic properties associated with channel block and a broader therapeutic window. A novel interaction between Cav2.2 and collapsin response mediator protein 2 (CRMP-2) positively regulates channel function by increasing surface trafficking. We recently identified a CRMP-2 peptide (TAT-CBD3), which effectively blocks this interaction, reduces or completely reverses pain behavior in a number of inflammatory and neuropathic models. Importantly, TAT-CBD3 did not produce many of the typical side effects often observed with Cav2.2 inhibitors. Notably chronic pain mechanisms offer unique challenges as they often encompass a mix of both neuropathic and inflammatory elements, whereby inflammation likely causes damage to the neuron leading to neuropathic pain, and neuronal injury may produce inflammatory reactions. To this end, we sought to further disseminate the ability of TAT-CBD3 to alter behavioral outcomes in two additional rodent pain models. While we observed that TAT-CBD3 reversed mechanical hypersensitivity associated with a model of chronic inflammatory pain due to lysophosphatidylcholine-induced sciatic nerve focal demyelination (LPC), injury to the tibial nerve (TNI) failed to respond to drug treatment. Moreover, a single amino acid mutation within the CBD3 sequence demonstrated amplified Cav2.2 binding and dramatically increased efficacy in an animal model of migraine. Taken together, TAT-CBD3 potentially represents a novel class of therapeutics targeting channel regulation as opposed to the channel itself.
N-type voltage-gated calcium channel/Cav2.2; CRMP-2; peptide; chronic pain; migraine; pain therapeutics
The C-terminus of the voltage-gated calcium channel Cav1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT), that regulates neurite extension and inhibits Cav1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of CACNA1C, the gene that encodes CaV1.2. Expression of CCAT is independent of Cav1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs), providing strong evidence that CCAT is not generated by cleavage of CaV1.2. Analysis of the transcriptional start sites in CACNA1C and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3′ end of the CACNA1C gene. This study provides new insights into the regulation of CACNA1C, and provides an example of how exonic promoters contribute to the complexity of mammalian genomes.
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
T-type calcium channels of the Cav3 family are unique among voltage-gated calcium channels due to their low activation voltage, rapid inactivation, and small single channel conductance. These special properties allow Cav3 calcium channels to regulate neuronal processing in the subthreshold voltage range. Here, we review two different subthreshold ion channel interactions involving Cav3 channels and explore the ability of these interactions to expand the functional roles of Cav3 channels. In cerebellar Purkinje cells, Cav3 and intermediate conductance calcium-activated potassium (IKCa) channels form a novel complex which creates a low voltage-activated, transient outward current capable of suppressing temporal summation of excitatory postsynaptic potentials (EPSPs). In large diameter neurons of the deep cerebellar nuclei, Cav3-mediated calcium current (IT) and hyperpolarization-activated cation current (IH) are activated during trains of inhibitory postsynaptic potentials. These currents have distinct, and yet synergistic, roles in the subthreshold domain with IT generating a rebound burst and IH controlling first spike latency and rebound spike precision. However, by shortening the membrane time constant the membrane returns towards resting value at a faster rate, allowing IH to increase the efficacy of IT and increase the range of burst frequencies that can be generated. The net effect of Cav3 channels thus depends on the channels with which they are paired. When expressed in a complex with a KCa channel, Cav3 channels reduce excitability when processing excitatory inputs. If functionally coupled with an HCN channel, the depolarizing effect of Cav3 channels is accentuated, allowing for efficient inversion of inhibitory inputs to generate a rebound burst output. Therefore, signal processing relies not only on the activity of individual subtypes of channels but also on complex interactions between ion channels whether based on a physical complex or by indirect effects on membrane properties.
Cav3; HCN; T-type; IKCa; KCa3.1; channel complex
Previously, we demonstrated that mice in which the gene for the L-type voltage-gated calcium channel CaV1.3 is deleted (CaV1.3 knockout mice) exhibit an impaired ability to consolidate contextually-conditioned fear. Given that this form of Pavlovian fear conditioning is critically dependent on the basolateral complex of the amygdala (BLA), we were interested in the mechanisms by which CaV1.3 contributes to BLA neurophysiology. In the present study, we used in vitro amygdala slices prepared from CaV1.3 knockout mice and wild-type littermates to explore the role of CaV1.3 in long-term potentiation (LTP) and intrinsic neuronal excitability in the BLA. We found that LTP in the lateral nucleus (LA) of the BLA, induced by high-frequency stimulation of the external capsule, was significantly reduced in CaV1.3 knockout mice. Additionally, we found that BLA principal neurons from CaV1.3 knockout mice were hyperexcitable, exhibiting significant increases in firing rates and decreased interspike intervals in response to prolonged somatic depolarization. This aberrant increase in neuronal excitability appears to be at least in part due to a concomitant reduction in the slow component of the post-burst afterhyperpolarization. Together, these results demonstrate altered neuronal function in the BLA of CaV1.3 knockout mice which may account for the impaired ability of these mice to consolidate contextually-conditioned fear.
L-type voltage-gated calcium channels; afterhyperpolarization; long-term potentiation; conditioned fear; memory mechanisms; spike accommodation
L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through β adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of CaV1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.1 Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Cav1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of CaV1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of CaV1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Cav1.2 intracellular regions.
calcium channels; L-type; Cav1.2; AKAP79; AKAP18; Proline rich domain; Leucine Zipper motif
The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels and their modulation by the auxiliary β1b, β2e, β3a, and β4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human CaV2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463–474). Here, we report the characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). CaV2.1 channels containing any of the four β subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, CaV2.1 channels display different rates of inactivation and different steady-state inactivation depending on the β subtype. The type of β subunit also modulates the relative occurrence of the slow and the fast gating mode of CaV2.1 channels; β3a promotes the fast mode, whereas β4a promotes the slow mode. The prevailing mode of gating of CaV2.1 channels lacking a β subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode.
Ca2+ channel; gating mode; synaptic transmission; familial hemiplegic migraine; auxiliary subunit
Influx of calcium through voltage-dependent channels regulates processes throughout the nervous system. Specifically, influx through L-type channels plays a variety of roles in early neuronal development and is commonly modulated by G-protein-coupled receptors such as GABAB receptors. Of the four isoforms of L-type channels, only CaV1.2 and CaV1.3 are predominately expressed in the nervous system. Both isoforms are inhibited by the same pharmacological agents, so it has been difficult to determine the role of specific isoforms in physiological processes. In the present study, Western blot analysis and confocal microscopy were utilized to study developmental expression levels and patterns of CaV1.2 and CaV1.3 in the CA1 region of rat hippocampus. Steady-state expression of CaV1.2 predominated during the early neonatal period decreasing by day 12. Steady-state expression of CaV1.3 was low at birth and gradually rose to adult levels by postnatal day 15. In immunohistochemical studies, antibodies against CaV1.2 and CaV1.3 demonstrated the highest intensity of labeling in the proximal dendrites at all ages studied (P1–72). Immunohistochemical studies on one-week-old hippocampi demonstrated significantly more colocalization of GABAB receptors with CaV1.2 than with CaV1.3, suggesting that modulation of L-type calcium current in early development is mediated through CaV1.2 channels.
Low-voltage-activated T-type calcium channels act as a major pathway for calcium entry near the resting membrane potential in a wide range of neuronal cell types. Several reports have uncovered an unrecognized feature of T-type channels in the control of vesicular neurotransmitter and hormone release, a process so far thought to be mediated exclusively by high-voltage-activated calcium channels. However, the underlying molecular mechanisms linking T-type calcium channels to vesicular exocytosis have remained enigmatic. In a recent study, we have reported that Cav3.2 T-type channel forms a signaling complex with the neuronal Q-SNARE syntaxin-1A and SNAP-25. This interaction that relies on specific Cav3.2 molecular determinants, not only modulates T-type channel activity, but was also found essential to support low-threshold exocytosis upon Cav3.2 channel expression in MPC 9/3L-AH chromaffin cells. Overall, we have indentified an unrecognized regulation pathway of T-type calcium channels by SNARE proteins, and proposed the first molecular mechanism by which T-type channels could mediate low-threshold exocytosis.
Cav3.2 channel; calcium channel; chromaffin cell; exocytosis; MPC cell; neuron; SNAP-25; SNARE; syntaxin-1A; T-type