John E. McRory

NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia

Central nervous system myelin is a specialized structure produced by oligodendrocytes that ensheaths axons, allowing rapid and efficient saltatory conduction of action potentials. Many disorders promote damage to and eventual loss of the myelin sheath, which often results in significant neurological morbidity. However, little is known about the fundamental mechanisms that initiate myelin damage, with the assumption being that its fate follows that of the parent oligodendrocyte. Here we show that NMDA (N-methyl-d-aspartate) glutamate receptors mediate Ca2+ accumulation in central myelin in response to chemical ischaemia in vitro. Using two-photon microscopy, we imaged fluorescence of the Ca2+ indicator X-rhod-1 loaded into oligodendrocytes and the cytoplasmic compartment of the myelin sheath in adult rat optic nerves. The AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor antagonist NBQX completely blocked the ischaemic Ca2+ increase in oligodendroglial cell bodies, but only modestly reduced the Ca2+ increase in myelin. In contrast, the Ca2+ increase in myelin was abolished by broad-spectrum NMDA receptor antagonists (MK-801, 7-chlorokynurenic acid, d-AP5), but not by more selective blockers of NR2A and NR2B subunit-containing receptors (NVP-AAM077 and ifenprodil). In vitro ischaemia causes ultrastructural damage to both axon cylinders and myelin. NMDA receptor antagonism greatly reduced the damage to myelin. NR1, NR2 and NR3 subunits were detected in myelin by immunohistochemistry and immunoprecipitation, indicating that all necessary subunits are present for the formation of functional NMDA receptors. Our data show that the mature myelin sheath can respond independently to injurious stimuli. Given that axons are known to release glutamate, our finding that the Ca2+ increase was mediated in large part by activation of myelinic NMDA receptors suggests a new mechanism of axo–myelinic signalling. Such a mechanism may represent a potentially important therapeutic target in disorders in which demyelination is a prominent feature, such as multiple sclerosis, neurotrauma, infections (for example, HIV encephalomyelopathy) and aspects of ischaemic brain injury.

Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception

Analgesic therapies are still limited and sometimes poorly effective, therefore finding new targets for the development of innovative drugs is urgently needed. In order to validate the potential utility of blocking T‐type calcium channels to reduce nociception, we explored the effects of intrathecally administered oligodeoxynucleotide antisenses, specific to the recently identified T‐type calcium channel family (CaV3.1, CaV3.2, and CaV3.3), on reactions to noxious stimuli in healthy and mononeuropathic rats. Our results demonstrate that the antisense targeting CaV3.2 induced a knockdown of the CaV3.2 mRNA and protein expression as well as a large reduction of ‘CaV3.2‐like’ T‐type currents in nociceptive dorsal root ganglion neurons. Concomitantly, the antisense treatment resulted in major antinociceptive, anti‐hyperalgesic, and anti‐allodynic effects, suggesting that CaV3.2 plays a major pronociceptive role in acute and chronic pain states. Taken together, the results provide direct evidence linking CaV3.2 T‐type channels to pain perception and suggest that CaV3.2 may offer a specific molecular target for the treatment of pain.

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 Cav1.4 calcium channel subtype. Here, we describe the functional and pharmacological properties of transiently expressed human Cav1.4 calcium channels. Cav1.4 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 β subunits. Additionally, the channel supports a large window current and activates near -40 mV in 2 mM external calcium, making Cav1.4 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, Cav1.4 is widely distributed outside the retina, including in the immune system, thus suggesting a broader role in human physiology.

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 Cav3.1, Cav3.2 and Cav3.3 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 Cav3 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 Cav3 channel proteins in cell types of the neocortex and hippocampus, thalamus, and cerebellar input and output neurons. In general, the Cav3.1 T‐type channel immunolabel is prominent in the soma/proximal dendritic region and Cav3.2 immunolabel in the soma and proximal‐mid dendrites. Cav3.3 channels are distinct in distributing to the soma and over extended lengths of the dendritic arbor of particular cell types. Cav3 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 Cav3 antigen through analysis of isolated protein fractions. These results provide evidence that different Cav3 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.

P/Q-type calcium channels mediate the activity-dependent feedback of syntaxin-1A

Spatial and temporal changes in intracellular calcium concentrations are critical for controlling gene expression in neurons. In many neurons, activity-dependent calcium influx through L-type channels stimulates transcription that depends on the transcription factor CREB by activating a calmodulin-dependent pathway. Here we show that selective influx of calcium through P/Q-type channels is responsible for activating expression of syntaxin-1A, a presynaptic protein that mediates vesicle docking, fusion and neurotransmitter release. The initial P/Q-type calcium signal is amplified by release of calcium from intracellular stores and acts through phosphorylation that is dependent on the calmodulin-dependent kinase CaM K II/IV, protein kinase A and mitogen-activated protein kinase kinase. Initiation of syntaxin-1A expression is rapid and short-lived, with syntaxin-1A ultimately interacting with the P/Q-type calcium channel to decrease channel availability. Our results define an activity-dependent feedback pathway that may regulate synaptic efficacy and function in the nervous system.

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 (∼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 μ-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.

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.

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 β and α2–δ subunits on expression levels and biophysical properties of three different types (Cav1.2, Cav2.1 and Cav2.3) of HVA calcium channels expressed in tsA‐201 cells. Our data show that Cav1.2 and Cav2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel β subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel α1 subunit, and β3 subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the α2–δ subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Cav2 channel family, appears to act synergistically with β subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by β and by α2–δ 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 α2–δ1 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.

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. Ann Neurol 2005

Defective survival of naive CD8+ T lymphocytes in the absence of the |[beta]|3 regulatory subunit of voltage-gated calcium channels

The survival of T lymphocytes requires sustained, Ca2+ influx–dependent gene expression. The molecular mechanism that governs sustained Ca2+ influx in naive T lymphocytes is unknown. Here we report an essential role for the β3 regulatory subunit of voltage-gated calcium (Cav) channels in the maintenance of naive CD8+ T cells. Deficiency in β3 resulted in a profound survival defect of CD8+ T cells. This defect correlated with depletion of the pore-forming subunit Cav1.4 and attenuation of T cell antigen receptor (TCR)-mediated global Ca2+ entry in CD8+ T cells. Cav1.4 and β3 associated with T cell signaling machinery and Cav1.4 localized in lipid rafts. Our data demonstrate a mechanism by which Ca2+ entry is controlled by a Cav1.4-β3 channel complex in T cells.