Defective survival of naive CD8+ T lymphocytes in the absence of the beta3 regulatory subunit of voltage-gated calcium channels.

The survival of T lymphocytes requires sustained, Ca(2+) influx-dependent gene expression. The molecular mechanism that governs sustained Ca(2+) influx in naive T lymphocytes is unknown. Here we report an essential role for the beta3 regulatory subunit of voltage-gated calcium (Ca(v)) channels in the maintenance of naive CD8(+) T cells. Deficiency in beta3 resulted in a profound survival defect of CD8(+) T cells. This defect correlated with depletion of the pore-forming subunit Ca(v)1.4 and attenuation of T cell antigen receptor (TCR)-mediated global Ca(2+) entry in CD8(+) T cells. Ca(v)1.4 and beta3 associated with T cell signaling machinery and Ca(v)1.4 localized in lipid rafts. Our data demonstrate a mechanism by which Ca(2+) entry is controlled by a Ca(v)1.4-beta3 channel complex in T cells.

ORL1 receptor-mediated internalization of N-type calcium channels.

The inhibition of N-type calcium channels by opioid receptor like receptor 1 (ORL1) is a key mechanism for controlling the transmission of nociceptive signals. We recently reported that signaling complexes consisting of ORL1 receptors and N-type channels mediate a tonic inhibition of calcium entry. Here we show that prolonged ( approximately 30 min) exposure of ORL1 receptors to their agonist nociceptin triggers an internalization of these signaling complexes into vesicular compartments. This effect is dependent on protein kinase C activation, occurs selectively for N-type channels and cannot be observed with mu-opioid or angiotensin receptors. In expression systems and in rat dorsal root ganglion neurons, the nociceptin-mediated internalization of the channels is accompanied by a significant downregulation of calcium entry, which parallels the selective removal of N-type calcium channels from the plasma membrane. This may provide a new means for long-term regulation of calcium entry in the pain pathway.

Depolarization-induced Ca2+ release in ischemic spinal cord white matter involves L-type Ca2+ channel activation of ryanodine receptors.

The mechanisms of Ca(2+) release from intracellular stores in CNS white matter remain undefined. In rat dorsal columns, electrophysiological recordings showed that in vitro ischemia caused severe injury, which persisted after removal of extracellular Ca(2+); Ca(2+) imaging confirmed that an axoplasmic Ca(2+) rise persisted in Ca(2+)-free perfusate. However, depletion of Ca(2+) stores or reduction of ischemic depolarization (low Na(+), TTX) were protective, but only in Ca(2+)-free bath. Ryanodine or blockers of L-type Ca(2+) channel voltage sensors (nimodipine, diltiazem, but not Cd(2+)) were also protective in zero Ca(2+), but their effects were not additive with ryanodine. Immunoprecipitation revealed an association between L-type Ca(2+) channels and RyRs, and immunohistochemistry confirmed colocalization of Ca(2+) channels and RyR clusters on axons. Similar to "excitation-contraction coupling" in skeletal muscle, these results indicate a functional coupling whereby depolarization sensed by L-type Ca(2+) channels activates RyRs, thus releasing damaging amounts of Ca(2+) under pathological conditions in white matter.

Syntaxin 1A is required for normal in utero development.

We have generated a syntaxin 1A knockout mouse by deletion of exons 3 through 6 and a concomitant insertion of a stop codon in exon 2. Heterozygous knockout animals were viable with no apparent phenotype. In contrast, the vast majority of homozygous animals died in utero, with embryos examined at day E15 showing a drastic reduction in body size and development when compared to WT and heterozygous littermates. Surprisingly, out of a total of 204 offspring from heterozygous breeding pairs only four homozygous animals were born alive and viable. These animals exhibited reduced body weight, but showed only mild behavioral deficiencies. Taken together, our data indicate that syntaxin 1A is an important regulator of normal in utero development, but may not be essential for normal brain function later in life.

Agonist-independent modulation of N-type calcium channels by ORL1 receptors.

We have investigated modulation of voltage-gated calcium channels by nociceptin (ORL1) receptors. In rat DRG neurons and in tsA-201 cells, nociceptin mediated a pronounced inhibition of N-type calcium channels, whereas other calcium channel subtypes were unaffected. In tsA-201 cells, expression of N-type channels with human ORL1 resulted in a voltage-dependent G-protein inhibition of the channel that occurred in the absence of nociceptin, the ORL1 receptor agonist. Consistent with this observation, native N-type channels of small nociceptive dorsal root ganglion (DRG) neurons also had tonic inhibition by G proteins. Biochemical characterization showed the existence of an N-type calcium channel-ORL1 receptor signaling complex, which efficiently exposes N-type channels to constitutive ORL1 receptor activity. Calcium channel activity is thus regulated by changes in ORL1 receptor expression, which provides a possible molecular mechanism for the development of tolerance to opioid receptor agonists.

Differential inhibition of T-type calcium channels by neuroleptics.

T-type calcium channels play critical roles in cellular excitability and have been implicated in the pathogenesis of a variety of neurological disorders including epilepsy. Although there have been reports that certain neuroleptics that primarily target D2 dopamine receptors and are used to treat psychoses may also interact with T-type Ca channels, there has been no systematic examination of this phenomenon. In the present paper we provide a detailed analysis of the effects of several widely used neuroleptic agents on a family of exogenously expressed neuronal T-type Ca channels (alpha1G, alpha1H, and alpha1I subtypes). Among the neuroleptics tested, the diphenylbutylpiperidines pimozide and penfluridol were the most potent T-type channel blockers with Kd values (approximately 30-50 nm and approximately 70-100 nm, respectively), in the range of their antagonism of the D2 dopamine receptor. In contrast, the butyrophenone haloperidol was approximately 12- to 20-fold less potent at blocking the various T-type Ca channels. The diphenyldiperazine flunarizine was also less potent compared with the diphenylbutylpiperadines and preferentially blocked alpha1G and alpha1I T-type channels compared with alpha1H. The various neuroleptics did not significantly affect T-type channel activation or kinetic properties, although they shifted steady-state inactivation profiles to more negative values, indicating that these agents preferentially bind to channel inactivated states. Overall, our findings indicate that T-type Ca channels are potently blocked by a subset of neuroleptic agents and suggest that the action of these drugs on T-type Ca channels may significantly contribute to their therapeutic efficacy.

The CACNA1F gene encodes an L-type calcium channel with unique biophysical properties and tissue distribution.

Glutamate release from rod photoreceptors is dependent on a sustained calcium influx through L-type calcium channels. Missense mutations in the CACNA1F gene in patients with incomplete X-linked congenital stationary night blindness implicate the Ca(v)1.4 calcium channel subtype. Here, we describe the functional and pharmacological properties of transiently expressed human Ca(v)1.4 calcium channels. Ca(v)1.4 is shown to encode a dihydropyridine-sensitive calcium channel with unusually slow inactivation kinetics that are not affected by either calcium ions or by coexpression of ancillary calcium channel beta subunits. Additionally, the channel supports a large window current and activates near -40 mV in 2 mM external calcium, making Ca(v)1.4 ideally suited for tonic calcium influx at typical photoreceptor resting potentials. Introduction of base pair changes associated with four incomplete X-linked congenital night blindness mutations showed that only the G369D alteration affected channel activation properties. Immunohistochemical analyses show that, in contrast with previous reports, Ca(v)1.4 is widely distributed outside the retina, including in the immune system, thus suggesting a broader role in human physiology.

Modified Ca(v)1.4 expression in the Cacna1f(nob2) mouse due to alternative splicing of an ETn inserted in exon 2.

The Cacna1f(nob2) mouse is reported to be a naturally occurring null mutation for the Ca(v)1.4 calcium channel gene and the phenotype of this mouse is not identical to that of the targeted gene knockout model. We found two mRNA species in the Cacna1f(nob2) mouse: approximately 90% of the mRNA represents a transcript with an in-frame stop codon within exon 2 of CACNA1F, while approximately 10% of the mRNA represents a transcript in which alternative splicing within the ETn element has removed the stop codon. This latter mRNA codes for full length Ca(v)1.4 protein, detectable by Western blot analysis that is predicted to differ from wild type Ca(v)1.4 protein in a region of approximately 22 amino acids in the N-terminal portion of the protein. Electrophysiological analysis with either mouse Ca(v)1.4(wt) or Ca(v)1.4(nob2) cDNA revealed that the alternatively spliced protein does not differ from wild type with respect to activation and inactivation characteristics; however, while the wild type N-terminus interacted with filamin proteins in a biochemical pull-down experiment, the alternatively spliced N-terminus did not. The Cacna1f(nob2) mouse electroretinogram displayed reduced b-wave and oscillatory potential amplitudes, and the retina was morphologically disorganized, with substantial reduction in thickness of the outer plexiform layer and sprouting of bipolar cell dendrites ectopically into the outer nuclear layer. Nevertheless, the spatial contrast sensitivity (optokinetic response) of Cacna1f(nob2) mice was generally similar to that of wild type mice. These results suggest the Cacna1f(nob2) mouse is not a CACNA1F knockout model. Rather, alternative splicing within the ETn element can lead to full-length Ca(v)1.4 protein, albeit at reduced levels, and the functional Ca(v)1.4 mutant may be incapable of interacting with cytoskeletal filamin proteins. These changes, do not alter the ability of the Cacna1f(nob2) mouse to detect and follow moving sine-wave gratings compared to their wild type counterparts.

The Ca(v)1.4 calcium channel: more than meets the eye.

Ca(v)1.4 channels are the latest calcium channels to be described in the literature. Originally identified in 1997 from the human genome project, several reports have since been published describing mutations in the CACNA1F gene encoding Ca(v)1.4 channels, and implicated these mutations in human disorders such as X-linked cone rod dystrophy (CORDX3) and incomplete X-linked congenital stationary night blindness type 2 (CSNB2). The gene was subsequently cloned and expressed in heterologous expression systems beginning in 2003, and many of the mutations linked to CSNB2 have been tested. Here, we review literature describing the discovery of the CACNA1F gene, its tissue expression profile, alternative splicing events, and biophysical and pharmacological characteristics of the channel in various expression systems. Channel biophysics are also compared to those obtained from recordings made from vertebrate photoreceptors, suggesting that these studies may have been describing Ca(v)1.4 channels in native cells.

Ca(V)3 T-type calcium channel isoforms differentially distribute to somatic and dendritic compartments in rat central neurons.

Spike output in many neuronal cell types is affected by low-voltage-activated T-type calcium currents arising from the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 channel subtypes and their splice isoforms. The contributions of T-type current to cell output is often proposed to reflect a differential distribution of channels to somatic and dendritic compartments, but the subcellular distribution of the various rat T-type channel isoforms has not been fully determined. We used subtype-specific Ca(v)3 polyclonal antibodies to determine their distribution in key regions of adult Sprague-Dawley rat brain thought to exhibit T-type channel expression, and in particular, dendritic low-voltage-activated responses. We found a selective subcellular distribution of Ca(v)3 channel proteins in cell types of the neocortex and hippocampus, thalamus, and cerebellar input and output neurons. In general, the Ca(v)3.1 T-type channel immunolabel is prominent in the soma/proximal dendritic region and Ca(v)3.2 immunolabel in the soma and proximal-mid dendrites. Ca(v)3.3 channels are distinct in distributing to the soma and over extended lengths of the dendritic arbor of particular cell types. Ca(v)3 distribution overlaps with cell types previously established to exhibit rebound burst discharge as well as those not recognized for this activity. Additional immunolabel in the region of the nucleus in particular cell types was verified as corresponding to Ca(v)3 antigen through analysis of isolated protein fractions. These results provide evidence that different Ca(v)3 channel isoforms may contribute to low-voltage-activated calcium-dependent responses at the somatic and dendritic level, and the potential for T-type calcium channels to contribute to multiple aspects of neuronal activity.

Specific T-type calcium channel isoforms are associated with distinct burst phenotypes in deep cerebellar nuclear neurons.

T-type calcium channels are thought to transform neuronal output to a burst mode by generating low voltage-activated (LVA) calcium currents and rebound burst discharge. In this study we assess the expression pattern of the three different T-type channel isoforms (Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3) in cerebellar neurons and focus on their potential role in generating LVA spikes and rebound discharge in deep cerebellar nuclear (DCN) neurons. We detected expression of one or more Ca(v)3 channel isoforms in a wide range of cerebellar neurons and selective expression of different isoforms in DCN cells. We further identify two classes of large-diameter DCN neurons that exhibit either a strong or weak capability for rebound discharge, despite the ability to generate LVA spikes when calcium currents are pharmacologically isolated. By correlating the Ca(v)3 channel expression pattern with the electrophysiological profile of identified DCN cells, we show that Ca(v)3.1 channels are expressed in isolation in DCN-burst cells, whereas Ca(v)3.3 is expressed in DCN-weak burst cells. Ca(v)3.1-expressing DCN cells correspond to excitatory or GABAergic neurons, whereas Ca(v)3.3-expressing cells are non-GABAergic. The Ca(v)3 class of LVA calcium channels is thus expressed in specific combinations in a wide range of cerebellar neurons but contributes to rebound burst discharge in only a select number of cell classes.

Functional analysis of Ca3.2 T-type calcium channel mutations linked to childhood absence epilepsy.

PURPOSE: Childhood absence epilepsy (CAE) is an idiopathic form of seizure disorder that is believed to have a genetic basis. METHODS: We examined the biophysical consequences of seven mutations in the Ca(v)3.2 T-type calcium channel gene linked to CAE. RESULTS: Of the channel variants examined, one of the mutants, a replacement of glycine 848 in the domain II-S2 region with serine, resulted in significant slowing of the time courses of both activation and inactivation across a wide range of membrane potentials. These changes are consistent with increased channel activity in response to prolonged membrane depolarizations. CONCLUSIONS: Taken together, these findings suggest that such little changes in channel gating may contribute to the etiology of CAE.

Cav1.4 encodes a calcium channel with low open probability and unitary conductance.

When transiently expressed in tsA-201 cells, Ca(v)1.4 calcium channels support only modest whole-cell currents with unusually slow voltage-dependent inactivation kinetics. To examine the basis for this unique behavior we used cell-attached patch single-channel recordings using 100 mM external barium as the charge carrier to determine the single-channel properties of Ca(v)1.4 and to compare them to those of the Ca(v)1.2. Ca(v)1.4 channel openings occurred infrequently and were of brief duration. Moreover, openings occurred throughout the duration of the test depolarization, indicating that the slow inactivation kinetics observed at the whole-cell level are caused by sustained channel activity. Ca(v)1.4 and Ca(v)1.2 channels displayed similar latencies to first opening. Because of the rare occurrence of events, the probability of opening could not be precisely determined but was estimated to be <0.015 over a voltage range of -20 to +20 mV. The single-channel conductance of Ca(v)1.4 channels was approximately 4 pS compared with approximately 20 pS for Ca(v)1.2 under the same experimental conditions. Additionally, in the absence of divalent cations, Ca(v)1.4 channels pass cesium ions with a single-channel conductance of approximately 21 pS. Although Ca(v)1.2 opening events were best described kinetically with two open time constants, Ca(v)1.4 open times were best described by a single time constant. BayK8644 slightly enhanced the single-channel conductance in addition to increasing the open time constant for Ca(v)1.4 channels by approximately 45% without, however, causing the appearance of an additional slower gating mode. Overall, our data indicate that single Ca(v)1.4 channels support only minute amounts of calcium entry, suggesting that large numbers of these channels are needed to allow for significant whole-cell current activity, and providing a mechanism to reduce noise in the visual system.

Effects of Cav3.2 channel mutations linked to idiopathic generalized epilepsy.

Heron and colleagues (Ann Neurol 2004;55:595-596) identified three missense mutations in the Cav3.2 T-type calcium channel gene (CACNA1H) in patients with idiopathic generalized epilepsy. None of the variants were associated with a specific epilepsy phenotype and were not found in patients with juvenile absence epilepsy or childhood absence epilepsy. Here, we introduced and functionally characterized these three mutations using transiently expressed human Cav3.2 channels. Two of the mutations exhibited functional changes that are consistent with increased channel function. Taken together, these findings along with previous reports, strongly implicate CACNA1H as a susceptibility gene in complex idiopathic generalized epilepsy.

Mammalian voltage-gated calcium channels are potently blocked by the pyrethroid insecticide allethrin.

Pyrethroids are commonly used insecticides for both household and agricultural applications. It is generally reported that voltage-gated sodium channels are the primary target for toxicity of these chemicals to humans. The phylogenetic and structural relatedness between sodium channels and voltage-gated calcium (Ca) channels prompted us to examine the effects of the type 1 pyrethroid allethrin on the three major classes of mammalian calcium channels exogenously expressed in human embryonic kidney 293 cells. We report that all classes of mammalian calcium channels are targets for allethrin at concentrations very similar to those reported for interaction with sodium channels. Allethrin caused blockade with IC(50) values of 7.0 microM for T-type alpha(1G) (Ca(v)3.1), 6.8 microM for L-type alpha(1C) (Ca(v)1.2), and 6.7 microM for P/Q-type alpha(1A) (Ca(v)2.1) channels. Mechanistically, the blockade of calcium channels was found to be significantly different than the prolonged opening of mammalian sodium channels caused by pyrethroids. In all calcium channel subtypes tested, allethrin caused a significant acceleration of the inactivation kinetics and a hyperpolarizing shift in the voltage dependence of inactivation. The high-voltage-activated P/Q- and L-type channels showed a frequency of stimulation-dependent increase in block by allethrin, whereas the low-voltage-activated alpha(1G) subtype did not. Allethrin did not significantly modify the deactivation kinetics or current-voltage relationships of any of the calcium channel types. Our study indicates that calcium channels are another primary target for allethrin and suggests that blockade of different types of calcium channels may underlie some of the chronic effects of low-level pyrethroid poisoning.

Several structural domains contribute to the regulation of N-type calcium channel inactivation by the beta 3 subunit.

Calcium channel beta subunits are essential regulatory elements of the gating properties of high voltage-activated calcium channels. Co-expression with beta(3) subunits typically accelerates inactivation, whereas co-expression with beta(4) subunits results in a slowly inactivating phenotype. Here, we have examined the molecular basis of the differential effect of these two subunits on the inactivation characteristics of Ca(v)2.2 + alpha(2)-delta(1) N-type calcium channels by creating a series of 22 chimeric beta subunits that are based on various combinations of variable and conserved regions of the parent beta subunit isoforms. Our data show that replacement of the N terminus region of beta(4) with a corresponding 14-amino acid stretch of beta(3) sequence accelerates the inactivation kinetics to levels seen with wild type beta(3). A similar kinetic speeding is observed by a concomitant substitution of the second conserved and variable regions, but not when these regions are substituted individually, suggesting that 1) the second variable and conserved regions cooperatively regulate N-type calcium channel inactivation and 2) that there are two redundant mechanisms that allow the beta(3) subunit to accelerate N-type channel inactivation. In contrast with previous reports in Ca(v)2.1 calcium channels, deletion of the C-terminal region of Ca(v)2.2 did not alter the regulation of the channel by wild type and chimeric beta subunits. Hence, the molecular underpinnings of beta subunit regulation of voltage-gated calcium channels appear to vary with calcium channel subtype.

Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cells.

The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel beta and alpha(2)-delta subunits on expression levels and biophysical properties of three different types (Ca(v)1.2, Ca(v)2.1 and Ca(v)2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Ca(v)1.2 and Ca(v)2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel beta subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel alpha(1) subunit, and beta(3) subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the alpha(2)-delta subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Ca(v)2 channel family, appears to act synergistically with beta subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by beta and by alpha(2)-delta subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the alpha(2)-delta(1) subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes.

Gating effects of mutations in the Cav3.2 T-type calcium channel associated with childhood absence epilepsy.

Childhood absence epilepsy (CAE) is a type of generalized epilepsy observed in 2-10% of epileptic children. In a recent study by Chen et al. (Chen, Y., Lu, J., Pan, H., Zhang, Y., Wu, H., Xu, K., Liu, X., Jiang, Y., Bao, X., Yao, Z., Ding, K., Lo, W. H., Qiang, B., Chan, P., Shen, Y., and Wu, X. (2003) Ann. Neurol. 54, 239-243) 12 missense mutations were identified in the CACNA1H (Ca(v)3.2) gene in 14 of 118 patients with CAE but not in 230 control individuals. We have functionally characterized five of these mutations (F161L, E282K, C456S, V831M, and D1463N) using rat Ca(v)3.2 and whole-cell patch clamp recordings in transfected HEK293 cells. Two of the mutations, F161L and E282K, mediated an approximately 10-mV hyperpolarizing shift in the half-activation potential. Mutation V831M caused a approximately 50% slowing of inactivation relative to control and shifted half-inactivation potential approximately 10 mV toward more depolarized potentials. Mean time to peak was significantly increased by mutation V831M but was unchanged for all others. No resolvable changes in the parameters of the IV relation or current kinetics were observed with the remaining mutations. The findings suggest that several of the Ca(v)3.2 mutants allow for greater calcium influx during physiological activation and in the case of F161L and E282K can result in channel openings at more hyperpolarized (close to resting) potentials. This may underlie the propensity for seizures in patients with CAE.