Michael E. Hildebrand

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.

Calcium-permeable ion channels in pain signaling.

The detection and processing of painful stimuli in afferent sensory neurons is critically dependent on a wide range of different types of voltage- and ligand-gated ion channels, including sodium, calcium, and TRP channels, to name a few. The functions of these channels include the detection of mechanical and chemical insults, the generation of action potentials and regulation of neuronal firing patterns, the initiation of neurotransmitter release at dorsal horn synapses, and the ensuing activation of spinal cord neurons that project to pain centers in the brain. Long-term changes in ion channel expression and function are thought to contribute to chronic pain states. Many of the channels involved in the afferent pain pathway are permeable to calcium ions, suggesting a role in cell signaling beyond the mere generation of electrical activity. In this article, we provide a broad overview of different calcium-permeable ion channels in the afferent pain pathway and their role in pain pathophysiology.

Functional Coupling between mGluR1 and Cav3.1 T-Type Calcium Channels Contributes to Parallel Fiber-Induced Fast Calcium Signaling within Purkinje Cell Dendritic Spines

T-type voltage-gated calcium channels are expressed in the dendrites of many neurons, although their functional interactions with postsynaptic receptors and contributions to synaptic signaling are not well understood. We combine electrophysiological and ultrafast two-photon calcium imaging to demonstrate that mGluR1 activation potentiates cerebellar Purkinje cell Cav3.1 T-type currents via a G-protein- and tyrosine-phosphatase-dependent pathway. Immunohistochemical and electron microscopic investigations on wild-type and Cav3.1 gene knock-out animals show that Cav3.1 T-type channels are preferentially expressed in Purkinje cell dendritic spines and colocalize with mGluR1s. We further demonstrate that parallel fiber stimulation induces fast subthreshold calcium signaling in dendritic spines and that the synaptic Cav3.1-mediated calcium transients are potentiated by mGluR1 selectively during bursts of excitatory parallel fiber inputs. Our data identify a new fast calcium signaling pathway in Purkinje cell dendritic spines triggered by short burst of parallel fiber inputs and mediated by T-type calcium channels and mGluR1s.

A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain.

&NA; Voltage‐gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including NaV1.7 and NaV1.8 sodium channels and CaV3.2 T‐type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow‐inactivated state. Slow inactivation of voltage‐gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state‐dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot‐plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state‐dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics. A novel organic compound stabilizes slow‐inactivated sodium and calcium channels to reduce the excitability of nociceptors and dorsal horn neurons and attenuate neuropathic pain signaling.

Functional Implications of a Novel EA2 Mutation in the P/Q-Type Calcium Channel

Episodic ataxia type 2 (EA2) is an autosomal dominant condition characterized by paroxysmal attacks of ataxia, vertigo, and nausea, typically lasting minutes to days in duration. These symptoms can be prevented or significantly attenuated by the oral administration of acetazolamide; however, the mechanism by which acetazolamide ameliorates EA2 symptoms is unknown. EA2 typically results from nonsense mutations in the CACNA1A gene that encodes the α1A (Cav2.1) subunit of the P/Q‐type calcium (Ca2+) channel. We have identified a novel H1736L missense mutation in the CACNA1A gene associated with the EA2 phenotype. This mutation is localized near the pore‐forming region of the P/Q‐type Ca2+ channel. Functional analysis of P/Q‐type channels containing the mutation show that the H1736L alteration affects several channel properties, including reduced current density, increased rate of inactivation, and a shift in the voltage dependence of activation to more positive values. Although these findings are consistent with an overall loss of P/Q‐type channel function, the mutation also caused some biophysical changes consistent with a gain of function. We also tested the direct effect of acetazolamide on both wild‐type and H1736L mutated P/Q‐type channels and did not observe any direct action on channel properties of this pharmacological agent used to treat EA2 patients. Ann Neurol 2004

Activation of Corticotropin-Releasing Factor Receptor 1 Selectively Inhibits CaV3.2 T-Type Calcium Channels

The corticotropin-releasing factor (CRF) peptides CRF and uro-cortins 1 to 3 are crucial regulators of mammalian stress and inflammatory responses, and they are also implicated in disorders such as anxiety, depression, and drug addiction. There is considerable interest in the physiological mechanisms by which CRF receptors mediate their widespread effects, and here we report that the native CRF receptor 1 (CRFR1) endogenous to the human embryonic kidney 293 cells can functionally couple to mammalian CaV3.2 T-type calcium channels. Activation of CRFR1 by either CRF or urocortin (UCN) 1 reversibly inhibits CaV3.2 currents (IC50 of ∼30 nM), but it does not affect CaV3.1 or CaV3.3 channels. Blockade of CRFR1 by the antagonist astressin abolished the inhibition of CaV3.2 channels. The CRFR1-dependent inhibition of CaV3.2 channels was independent of the activities of phospholipase C, tyrosine kinases, Ca2+/calmodulin-dependent protein kinase II, protein kinase C, and other kinase pathways, but it was dependent upon a cholera toxin-sensitive G protein-mediated mechanism relying upon G protein βγ subunits (Gβγ). The inhibition of CaV3.2 channels via the activation of CRFR1 was due to a hyperpolarized shift in their steady-state inactivation, and it was reversible upon washout of the agonists. Given that UCN affect multiple aspects of cardiac and neuronal physiology and that CaV3.2 channels are widespread throughout the cardiovascular and nervous systems, the results point to a novel and functionally relevant CRFR1-CaV3.2 T-type calcium channel signaling pathway.

Selective Inhibition of Cav3.3 T-type Calcium Channels by Gαq/11-coupled Muscarinic Acetylcholine Receptors

T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca2+ channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α) T-type Ca2+1Ichannels were co-expressed with the M1 Gαq/11-coupled mAChR. Perforated patch recordings demonstrate that activation of M1 receptors has a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a moderate stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. This differential modulation was observed for both rat and human T-type Ca2+ channel variants. The inhibition of Cav3.3 channels by M1 receptors is reversible, use-independent, and associated with a concomitant increase in inactivation kinetics. Loss-of-function experiments with genetically encoded antagonists of Gα and Gβγ proteins and gain-of-function experiments with genetically encoded Gα subtypes indicate that M1 receptor-mediated inhibition of Cav3.3 occurs through Gαq/11. This is supported by experiments showing that activation of the M3 and M5 Gαq/11-coupled mAChRs also causes inhibition of Cav3.3 currents, although Gαi-coupled mAChRs (M2 and M4) have no effect. Examining Cav3.1-Cav3.3 chimeric channels demonstrates that two distinct regions of the Cav3.3 channel are necessary and sufficient for complete M1 receptor-mediated channel inhibition and represent novel sites not previously implicated in T-type channel modulation.

Contributions of T-Type Voltage-Gated Calcium Channels to Postsynaptic Calcium Signaling within Purkinje Neurons

Low threshold voltage-gated T-type calcium channels have long been implicated in the electrical excitability and calcium signaling of cerebellar Purkinje neurons although the molecular composition, localization, and modulation of T-type channels within Purkinje cells have only recently been addressed. The specific functional roles that T-type channels play in local synaptic integration within Purkinje spines are also currently being unraveled. Overall, Purkinje neurons represent a powerful model system to explore the potential roles of postsynaptic T-type channels throughout the nervous system. In this review, we present an overview of T-type calcium channel biophysical, pharmacological, and physiological characteristics that provides a foundation for understanding T-type channels within Purkinje neurons. We also describe the biophysical properties of T-type channels in context of other voltage-gated calcium channel currents found within Purkinje cells. The data thus far suggest that one specific T-type isoform, Cav3.1, is highly expressed within Purkinje spines and both physically and functionally couples to mGluR1 and other effectors within putative signaling microdomains. Finally, we discuss how the selective potentiation of Cav3.1 channels via activation of mGluR1 by parallel fiber inputs affects local synaptic integration and how this interaction may relate to the overall excitability of Purkinje neuron dendrites.

Potentiation of Synaptic GluN2B NMDAR Currents by Fyn Kinase Is Gated through BDNF-Mediated Disinhibition in Spinal Pain Processing

In chronic pain states, the neurotrophin brain-derived neurotrophic factor (BDNF) transforms the output of lamina I spinal neurons by decreasing synaptic inhibition. Pain hypersensitivity also depends on N-methyl-D-aspartate receptors (NMDARs) and Src-family kinases, but the locus of NMDAR dysregulation remains unknown. Here, we show that NMDAR-mediated currents at lamina I synapses are potentiated in a peripheral nerve injury model of neuropathic pain. We find that BDNF mediates NMDAR potentiation through activation of TrkB and phosphorylation of the GluN2B subunit by the Src-family kinase Fyn. Surprisingly, we find that Cl--dependent disinhibition is necessary and sufficient to prime potentiation of synaptic NMDARs by BDNF. Thus, we propose that spinal pain amplification is mediated by a feedforward mechanism whereby loss of inhibition gates the increase in synaptic excitation within individual lamina I neurons. Given that neither disinhibition alone nor BDNF-TrkB signaling is sufficient to potentiate NMDARs, we have discovered a form of molecular coincidence detection in lamina I neurons.

GluN2B and GluN2D NMDARs dominate synaptic responses in the adult spinal cord

The composition of the postsynaptic ionotropic receptors that receive presynaptically released transmitter is critical not only for transducing and integrating electrical signals but also for coordinating downstream biochemical signaling pathways. At glutamatergic synapses in the adult CNS an overwhelming body of evidence indicates that the NMDA receptor (NMDAR) component of synaptic responses is dominated by NMDARs containing the GluN2A subunit, while NMDARs containing GluN2B, GluN2C, or GluN2D play minor roles in synaptic transmission. Here, we discovered NMDAR-mediated synaptic responses with characteristics not described elsewhere in the adult CNS. We found that GluN2A-containing receptors contribute little to synaptic NMDAR responses while GluN2B dominates at synapses of lamina I neurons in the adult spinal cord. In addition, we provide evidence for a GluN2D-mediated synaptic NMDAR component in adult lamina I neurons. Strikingly, the charge transfer mediated by GluN2D far exceeds that of GluN2A and is comparable to that of GluN2B. Lamina I forms a distinct output pathway from the spinal pain processing network to the pain networks in the brain. The GluN2D-mediated synaptic responses we have discovered in lamina I neurons provide the molecular underpinning for slow, prolonged and feedforward amplification that is a fundamental characteristic of pain.