Voltage-Gated N-Type and T-Type Calcium Channels and Excitability Disorders

Home , Calcium channel blocker, Mibefradil

2 VOLTAGE-GATED N-TYPE AND T-TYPE CALCIUM CHANNELS AND EXCITABILITY DISORDERS

1 1,2 ELIZABETH TRINGHAM AND TERRANCE P. SNUTCH 1Neuromed Pharmaceuticals, Rm 301, 2389 Health Sciences Mall, Vancouver, BC, Canada 2Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

2.1 INTRODUCTION

Calcium (Ca2þ) is a ubiquitous signaling molecule involved in a diverse array of cellular processes ranging from control of membrane excitability to gene transcription, all of which require that intracellular levels be tightly regulated. In particular, 2þ voltage-gated Ca channels (Cav) regulate transmembrane fluxes of calcium in response to membrane depolarization. Ca2þ currents can generally be grouped into two major classes of Cav channels: high voltage activated (HVA) and low voltage activated (LVAor “T-type”). Based on their pharmacological and biophysical properties, the HVA class can be further subdivided into L-, N-, P/Q-, and R-type channels. The main pore-forming a subunits of the Cav channels are encoded by 10 different genes, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1I, and CACNA1S, with a greater variety of subtypes for each class arising from a substantial degree of alternative splicing. Associated with the pore-forming subunit of HVA channels are several accessory subunits, a2d (four genes in mammals), b (four genes in mammals), and g (up to eight genes in mammals), although the precise complement is not well determined biochemically for all of the HVA channel types. Further, HVA channel complexity occurs as a result of the

35 36 VOLTAGE-GATED N-TYPE AND T-TYPE CALCIUM CHANNELS

FIGURE 2.1 Biophysical properties of high-voltage-activated Cav channels and T-type channels (LVA). (a) Voltage dependence of activation and inactivation of T-type channels is hyperpolarized compared to HVAchannels. T-type channels inactivate faster than HVAchannels (b), but deactivate (close) more slowly upon repolarization of the membrane potential (c).

interaction between the main pore-forming HVA Cav subunits and various alternatively spliced forms of the accessory subunits. This complexity in the molecular makeup underlying the Cav channel composition creates a daunting assortment of functional diversity in the physiological roles of Cav channels. As progress has been made in assigning physiological roles to particular Cav channel complexes, it has also unveiled their contributions to numerous pathophysiological conditions beyond the classically defined role of L-type channels in hyper- tension, angina, and cardiac arrhythmias. More recent interest has focused on understanding the roles of N- and T-type channels, which despite having quite distinct biophysical and pharmacological properties (Fig. 2.1) appear to contribute to the pathophysiology of disorders that are characterized by changes in neuronal excitability. Biophysically, N-type channels activate at higher potentials than T-type channels, exhibit slower kinetics of activation and inactivation, and recover more slowly from inactivation but deactivate more quickly. Three different genes, CACNA1G, CAC- NA1H, and CACNA1I, encode the pore-forming subunits of Cav3.1, Cav3.2, and Cav3.3 T-type channels, respectively, whereas N-type (Cav2.2) channels are encoded by a single gene, CACNA1B. Pharmacologically, to date there are no reported subtype- selective T-type channel blockers, which has limited the characterization of these channels in vivo. In contrast, selective peptide blockers with nanomolar affinity to N-type channels are available, providing insight into their roles in neuronal physiology. Despite some pharmacological limitations, the combination of available agents together with molecular genetic animal model and limited clinical data also provide valuable insight into the pathophysiological roles in which both N- and T-type channels are involved.

2.2 PATHOPHYSIOLOGY OF PAIN

Major advances in the field of pain have expanded our current understanding of the mechanisms underlying different pain states as well as some of the complexities of the signaling pathways involved in the processing of painful stimuli. One concept that has emerged as a common theme among different pain states, whether arising from neuropathic or inflammatory pain, is the spontaneous and persistent repetitive activation of primary afferent neurons (Kajander and Bennett, 1992; Kajander et al., PATHOPHYSIOLOGY OF PAIN 37

1992; McLachlan et al., 1993; Devor et al., 1994; Petersen et al., 1996; Ramer et al., 1997; Song et al., 1999; Zhang et al., 1999). These changes in excitability result in exaggerated transmitter release and enhanced postsynaptic excitability of neurons in the dorsal horn, which in turn leads to central sensitization or “windup.” Whether pain arises from damage of tissue as a result of sensitization of sensory terminals by peripheral chemical mediators or nerve damage as in neuropathic pain, both lead to maladaptive changes in central neuronal excitability and hence to behavioral phenotypes such as hyperalgesia and allodynia. Although a number of transmitters are released by primary afferent neurons, at the core of central sensitization is the excitatory neurotransmitter glutamate, the release of which from C and Ad fibers is controlled by Cav channels (Fig. 2.2). During inflammation and neuropathic pain states, both somatic and presynaptic Ca2þ channel activities are elevated leading to an increase in ectopic discharges and glutamate release. It is this increase that results in

FIGURE 2.2 Primary afferent nociceptive pathway. Following stimulation of primary afferent Ad and C fibers, second-order neurons in the spinal dorsal horn are activated and sensitized by neurotransmitters released into the presynaptic cleft. The second-order neurons ascend as part of the spinothalamic tract where they synapse onto third-order neurons in the thalamus that radiate to the somatosensory cortex where pain is perceived. A subset of neurons transmits proprioceptive information via the dorsal column. N-type calcium channels at primary afferent terminals in the spinal dorsal horn trigger the release of excitatory (glutamate) and modulatory (substance P and CGRP) transmitters onto second-order neurons. In the primary afferent pathway, T-type calcium channels are concentrated at both free nerve endings and DRG cell bodies. Both N-type and T-type calcium channels are also found in second-order spinal neurons as well as in higher brain regions such as the thalamus and cortex. 38 VOLTAGE-GATED N-TYPE AND T-TYPE CALCIUM CHANNELS the activation of glutamate receptors, specifically NMDA receptors through voltage- dependent Mg2þ unblock, which then prolongs neurotransmission and leads to “windup” in spinal nociceptive neurons of the dorsal horn. Further, as Cav channels are also known to fine-tune neuronal Ca2þ dynamics and hence shape action potential waveforms and control firing frequency, they present an interesting opportunity concerning nociceptive therapies.

2.2.1 Role of N-Type Channels in the Pathophysiology of Pain Since native N-type channels were first identified more than 20 years ago in sensory chick dorsal root ganglion (DRG) using electrophysiology techniques (Nowycky et al., 1985), RNA expression studies have revealed that they are exclusively expressed in neurons of both the central and peripheral nervous systems and in neurally derived cells such as neuroendocrine cells (Dubel et al., 1992). Immuno- histochemical studies show that N-type channels are distributed at the subcellular level along dendrites and cell bodies of many neurons and also at a subset of nerve terminals (Westenbroek et al., 1992). Their pivotal presynaptic location suggests that they are likely to be directly involved in synaptic transmission by controlling transmitter release and hence processing of sensory information. Indeed, the release of neuropeptides from the spinal cord, such as calcitonin gene-related peptide and substance P, is coupled to N-type channels (Holz et al., 1988; Maggi et al., 1990; Santicioli et al., 1992; Evans et al., 1996; Smith et al., 2002). Further, utilizing the peptide N-type Cav blocker w-conotoxin GVIA (w-CTx-GVIA, see below), the direct involvement of N-type channels in regulation of glutamatergic synaptic transmission, a major transmitter in afferent A and C fibers, has been demonstrated between primary afferent neurons and the spinal cord (Gruner and Silva, 1994). Following nerve injury, some inconsistencies have been noted with regard to changes in the expression of N-type channels. For example, N-type immunoreactivity increases in both the small dorsal root ganglion and lamina II of the spinal cord following chronic nerve compression injury (Cizkova et al., 2002), while no change in Cav2.2 mRNA levels is apparent in L5/L6 in a nerve ligation model of neuropathic pain (Luo et al., 2001). In other studies, whole-cell electrophysiological recordings of DRGs following nerve ligation or fura-2 microfluorometry of DRGs from axotomized neurons both demonstrate a reduction in the w-CTx-GVIA-sensitive N-type current (Baccei and Kocsis, 2000; Fuchs et al., 2007). While biochemical evidence implicates N-type channels in the pathophysiology of pain, the precise sensory modalities that N-type channels regulate have been elucidated by peptidic subtype-selective N-type channel blockers. Toxins derived from the venoms of cone snails and hunting spiders have been shown to be potent pain relievers invarious animal models of neuropathic and inflammatory pain. One such toxin, w-CTx-GVIA, is derived from the venom of the cone snail, Conus geographus. At nanomolar concentrations, w-CTx-GVIA is a selective and irreversible blocker of N-type channels that acts by occluding the pore of the channel (Olivera et al., 1984, 1987; Wagner et al., 1988; Boland et al., 1994; Feng et al., 2003). Early evidence for the involvement of N-type channels in nociceptive PATHOPHYSIOLOGY OF PAIN 39

responses was demonstrated by autoradiographic studies showing that radiolabeled w-CTx-GVIA binds in a highly localized manner in both the soma and presynaptic terminals of both myelinated and unmyelinated nociceptive primary afferent neurons at the level of superficial laminae I and II of the dorsal spinal cord (Kerr et al., 1988; Gohil et al., 1994). Intrathecal application of w-CTx-GVIA attenuates tactile allodynia and thermal hyperalgesia in the chronic constriction injury, partial sciatic nerve injury, and vincristine models of sensory peripheral neuropathy (Xiao and Bennett, 1995; Yamamoto and Sakashita, 1998; Scott et al., 2002; Fukuizumi Q1 et al., 2003). In the models of inflammatory pain in rats, w-CTx-GVIA reduces hyperalgesia induced by intraplantar injection of carrageenan (Yokoyama et al., 2003) and attenuates the algogenic response of bradykinin and a,b-methylene ATP when injected intraplantarly into mice (Kato et al., 2002). Antinociceptive effects have also been reported with w-CTx-GVIA in the formalin (Murakami et al., 2001, 2004) and capsaicin inflammatory pain models (Sluka, 1997), both of which activate unmyelinated C fibers (Dickenson et al., 1987; Winter et al., 1995; McCall et al., 1996). In a postoperative rat pain model, intrathecal administration of w-CTx- GVIA also dose-dependently attenuates incision-induced allodynia (Cheng et al., 2006). In contrast, responses to intrathecal administration of w-CTx-GVIA have provided mixed results in the acute tail-flick antinociception test, with some groups reporting no change in the basal tail-flick latencies (Suh et al., 1997; Scott et al., 2002) while others finding dose-dependent increases in tail-flick latencies in rats and mice (Omote et al., 1996; Lia and Prado, 1999; Fukuizumi et al., 2003). Spinal nociceptive responses to acute peripheral mechanical stimulation have also implicated N-type channels in increasing threshold responses in the tail and paw pressure tests (Omote et al., 1996; Fukuizumi et al., 2003). Considering the electrophysiological properties of the neurons involved in sensory signaling, it is interesting to note the commonality among different pain states such as neuropathic and inflammatory pain, namely, that both lead to spontaneous activity in primary afferent fibers (Wall and Devor, 1983; Babbedge et al., 1996; Boucher et al., 2000; Liu et al., 2000; Wu et al., 2001; Djouhri et al., 2006; Rojas-Piloni et al., 2007). This spontaneous activity originating in the peripheral afferents is thought to mediate maladaptive changes in the central nervous system (CNS) and is implicated in sensory abnormalities, including hyperalgesia and allodynia. This peripheral afferent signal is transmitted with a high degree of fidelity to the CNS and studies have shown that Ca2þ influx through N-type channels is critical to both sensitization and plasticity. Extracellular electrophysiological recordings within dorsal horn neurons following spinal nerve ligation show that increased neuronal excitability elicited by application of mechanical, thermal, or electrical stimulation in rats is dose-dependently reduced Q2 by application of w-CTx-GVIA to the spinal cord (Matthews and Dickenson, 2001). In spinal cord neurons that have become excitable following intraarticular injection of either mustard oil or kaolin and carrageenan, w-CTx-GVIA reduces neuronal excitability in both models suggesting a prominent role of N-type channels in afferent C-fiber-mediated hyperexcitability of spinal neurons (Neugebauer et al., 1996; Nebe et al., 1998). Intradermal application of formalin induces a biphasic 40 VOLTAGE-GATED N-TYPE AND T-TYPE CALCIUM CHANNELS increase in activity in C fibers that corresponds to the biphasic behavioral response. In agreement with its ability to suppress nociceptive behavior, w-CTx-GVIA reduces the spontaneous action potential firing rate in both phases (Dickenson and Sullivan, 1987; McCall et al., 1996; Diaz and Dickenson, 1997). Antinociceptive responses have also been reported for two other N-type channels preferring toxins, w-conotoxin CVID (w-CTx-CVID) and w-conotoxin MVIIA (w-CTx-MVIIA), both of which act by physically occluding the pore of Cav2.2 channels at nanomolar concentrations. w-CTx-MVIIA, derived from Conus magus (Olivera et al., 1985, 1987), reduces the neuropathy-induced allodynia following spinal nerve ligation (Bowersox et al., 1996; Urban et al., 2005) as well as thermal hyperalgesia in both a chronic constriction injury and a partial sciatic nerve injury model (Yamamoto et al., 1998). Mechanical hyperalgesia, assessed following inflammation induced by intraplantar injection of Freund’s complete adjuvant, is also dose-dependently reduced following intrathecal administration of w-CTx-MVIIA (Bowersox et al., 1996; Wang et al., 1998). Interestingly, in the formalin test, both phase I (acute phase) and phase II (tonic phase) are suppressed by w-CTx-MVIIA at doses that are 1000-fold more potent than morphine (Malmberg and Yaksh, 1994, 1995; Bowersox et al., 1996). Similarly, w-CTx-CVID, derived from Conus catus (Lewis et al., 2000; Nielsen et al., 2000), reduces mechanical and tactile allodynia in a spinal nerve ligation model (Scott et al., 2002; Blake et al., 2005) as well as being antinociceptive in the rat Freund’s complete adjuvant model (Smith et al., 2002). Acute nociceptive responses in the rat tail-flick model appear unaffected by either w- CTx-CVID or w-CTx-MVIIA (Scott et al., 2002; Blake et al., 2005). Following continuous intrathecal infusion with w-CTx-MVIIA, however, a significant reduction in the response to the hot plate is observed (Malmberg and Yaksh, 1995). Of interest is the observation that although the three subtype-selective w-conotoxins have similar affinity for the N-type channel, they are different in their rank order of potency in attenuating neuropathic pain (Scott et al., 2002). w-CTx-GVIA is the most potent with a three- to fourfold higher potency than w-CTx-MVIIA and w-CTx-CVID, but conversely w-CTx-CVID provides the greatest therapeutic margin, as animals display fewer adverse effects such as serpentine tail movements and body shaking. While this may possibly be explained by actions at off-target sites in the CNS other than N-type channels, as these effects are not noted in the Cav2.2 knockout mice, they support the notion that conotoxins have preferential affinities for different variants of N-type channels. For example, Adams et al. (2003) found that while transmitter release at preganglionic nerve terminals is regulated by N-type channels, w-CTx-CVID but not w-CTx-MVIIA blocks transmitter release. To address chronic pain conditions in patients refractory to current therapies, including morphine, the administration of a synthetic version of w-CTx-MVIIA, currently marketed as Prialt, has recently become available. Prialt was approved by the Food and Drug Administration (FDA) in 2005 to treat severe chronic cancer or AIDS pain and in some cases has resulted in complete or near-complete pain relief in patients whose symptoms were previously unmanageable even by intrathecal morphine (Brose et al., 1997; Mathur, 2000; Staats et al., 2004; Wallace, 2006). These results indicate that the animal models of pain used to evaluate the role of N-type PATHOPHYSIOLOGY OF PAIN 41

channels in somatosensory processing of pain were highly predictive of human clinical outcome. The lack of tolerance of Prialt in humans was also predicted in animal models of pain as continuous intrathecal infusion of w-CTx-MVIIA in rats minimally developed tolerance after 7 days (Malmberg and Yaksh, 1995). Side effects with Prialt clinical use have been reported and can be present in the form of hallucinations, cognitive impairment, and alterations in mood and consciousness, thereby providing the opportunity for improved N-type channel blockers that more specifically target those channels resident in sensory pathways (Penn and Paice, 2000; Q3 Snutch, 2005). In support of in vivo behavioral tests using pharmacological tools to assign sensory modalities regulated by N-type channels, mice strains in which the Cav2.2 N-type has been genetically removed have for the most part substantiated the results using w-conotoxins. The data from three knockout strains from different laboratories clearly show the involvement of the N-type channels in the development of allodynia and hyperalgesia associated with neuropathic and inflammatory pain (Kim et al., 2001; Saegusa et al., 2001, 2002). However, for acute pain, the role of N-type channels is somewhat ambiguous as results are inconsistent among the labs. Interestingly, the three independent Cav2.2-deficient mouse strains exhibit surprisingly few deleterious effects despite the wide CNS distribution and known role of N-type channels in synaptic transmission. The most marked behavioral alteration related to CNS function is decreased anxiety, whereas in the autonomic nervous system, sympathetic nerve dysfunction is observed as a reduction in the baroreflex and elevated heart rate and blood pressure (Ino et al., 2001). As the assortment of splice variants encoding Cav channels is being unraveled, the precise alternatively spliced isoform of Cav2.2 expressed in sensory neurons has recently unfolded (see Lipscombe et al., Chapter 3 in this volume). A Cav2.2 splice isoform containing exon 37a is preferentially expressed in capsaicin receptor- expressing nociceptive neurons whereas the exon 37b variant is ubiquitously expressed (Bell et al., 2004). Of particular interest are the recent findings by Altier and colleagues showing that when splice isoform-specific small interfering RNA (siRNA) against exon 37a Cav2.2 channels is injected intrathecally, this results in a reduction in both thermal and mechanical hyperalgesia in inflammatory and neuropathic pain models, as well as in basal thermal nociception. Tactile neuropathic allodynia is, however, equally mediated by exon 37a- and 37b-containing neurons (Altier et al., 2007). In addition to these in vivo findings is the identification of a distinct mode of G-protein-mediated voltage-independent inhibition unique to Cav2.2e[37a] (Raingo et al., 2007). This is in contrast to the well-defined voltage-dependent G protein inhibition of Cav2.2 channels promoted by the liberation of Gbg from Gi/o heteromers following agonist–receptor binding (i.e., morphine) (Herlitze et al., 1996; Ikeda, 1996). Although both exon 37a- and 37b-containing Cav2.2 splice isoforms undergo varying degrees of voltage-dependent inhibition, voltage-independent inhibition is only present in the Cav2.2 channels containing the 37a variant. The signaling pathway coupling G-protein-coupled receptors (GPCRs) to voltage-independent inhibition of Cav2.2e[37a] is mediated by the Ga subunit with downstream activation of a tyrosine kinase, which phosphorylates a single tyrosine residue (Y1747) in exon 37a. The 42 VOLTAGE-GATED N-TYPE AND T-TYPE CALCIUM CHANNELS physiological significance that has been speculated of this bifurcating pathway is that it maximizes the efficiency of inhibition by using both Ga and Gbg subunits from 2þ Gi/o heteromers, while simultaneously providing adaptive changes to Ca influx depending on the level of neuronal activity (Ikeda and Dunlap, 2007). This will no doubt stimulate interest in generating a Cav2.2e[37a]-selective blocker that might have an increased therapeutic index while narrowing the risk of unwanted side effects.

2.2.2 Role of T-Type Channels in the Pathophysiology of Pain Despite being identified in rat and chick dorsal root ganglion over 20 years ago (Carbone and Lux, 1984a, 1984b; Nowycky et al., 1985), a prominent role of T-type channels in the pathophysiology of pain has only recently emerged. This has been attributed in part to the discovery of three distinct subunit genes, CACNA1G (Cav3.1), CACNA1H (Cav3.2), and CACNA1I (Cav3.3), that encode T-type channels, as well as the existence of several classes of nonselective T-type channel blockers that have been used to tease out physiological and pathophysiological roles. In comparison to N-type channels, T-type calcium channels are much more widely expressed. T-type calcium channel currents are found in many tissues and cell types, including both central and peripheral neurons, heart, adrenal glands, kidney, smooth muscle, embryonic skeletal muscle, pituitary, pancreas, retina, and testes (Perez- Reyes, 2003). In the somatosensory pathway, the expression of T-type channels is localized to the dorsal root ganglion, dorsal horn spinal neurons, and the thalamus. In the dorsal root ganglion neurons, characterization of these primary afferents using electrophysiological techniques determined that T-type channels are exclusively expressed in small- and medium-sized DRG neurons but are essentially devoid of expression in the large DRG neurons (Schroeder et al., 1990; Scroggs and Fox, 1992; Shin et al., 2003). More recently, a novel subset of small capsaicin-sensitive DRG neurons called “T-rich” neurons has been identified that express only T-type channels with little contribution ofthe calcium conductance attributed toHVAchannels (Nelson et al., 2005). Generally, the nociceptive C and Ad fibers are considerably smaller than Aa and Ab fibers that conduct primarily proprioceptive and tactile information (Harper and Lawson, 1985a, 1985b), which supports the contribution of T-type channels in the pathophysiology of pain. Of the three genes known to encode T-type channels, in situ hybridization and reverse-transcription PCR studies have identified the Cav3.2 subtype as the most prominent subtype in DRG neurons, whereas Cav3.3 expression is lower and Cav3.1 is virtually undetected in small to medium DRGs (Talley et al., 1999; Bourinet et al., 2005). In another subset of medium-sized DRG neurons, the Cav3.2 T-type channel is found to be present in D-hair cell mechan- oreceptors (Shin et al., 2003). At the level of the spinal cord, in situ hybridization studies have demonstrated that the dorsal horn neurons in the superficial lamina also express Cav3.2 (Talley et al., 1999). In contrast to the predominant role of the Cav3.2 subtype of T-type channel in the primary and secondary order neurons of the somatosensory pathway, the thalamus expresses all three subtypes, albeit differentially expressed in subsets of thalamic neurons. Cav3.1 is predominantly expressed in PATHOPHYSIOLOGY OF PAIN 43 the thalamocortical (TC) neurons, which relay information from the periphery to the cortex, and Cav3.2 and Cav3.3 are found in the reticular thalamic neurons that provide inhibitory input to TC neurons (Talley et al., 1999). In the absence of subtype-selective blockers for Cav3.1-, Cav3.2-, or Cav3.3- containing T-type channels, knockdown and knockout studies have provided the greatest insight into the sensory modalities conveyed by the individual isoforms. Intrathecal injection of antisense oligonucleotides against each subtype identified Cav3.2 T-type channel as having a major pronociceptive role, whereas antisense directed at Cav3.1 and Cav3.3 did not significantly affect the nociceptive behavioral responses in rats (Bourinet et al., 2005). Cav3.2 T-type channels have effects on both acute and chronic pain as knockdown of Cav3.2 results in antiallodynic and antihyperalgesic effects in rats with a chronic constriction nerve injury and is antinociceptive in na€ıve rats when a noxious pressure is applied to the paw or a noxious thermal stimulus is applied to the tail. Concomitantly, whole-cell recordings reveal that T-type currents are reduced by 70–90% in small- to medium-sized DRGs, following a 50% reduction in Cav3.2 mRNA levels. In contrast, in mice 2þ lacking the Cav3.2 subtype of T-type Ca channel, thermal hyperalgesia and mechanical allodynia are not reduced following spinal nerve ligation (Choi et al., 2007). Responses to acute pain, such as noxious mechanical (tail clip) and thermal € (tail flick and hot plate) stimuli in naıve mice, are, however, reduced in Cav3.2- deficient mice as are responses to intradermal capsaicin and formalin and visceral injections of acetic acid and MgSO4, both of which induce abdominal writhing. Although the role for Cav3.1 channels in processing of sensory inputs is predicted to be minimal as expression levels in the DRGs are extremely low or undetected, Cav3.1 knockout mice are unexpectedly found to have increased visceral pain response when induced by intraperitoneal injections of acetic acid or MgSO4 (Kim et al., 2003). In addition to increased visceral pain responses in the mutant mice, there are also changes in the firing patterns in the ventroposterolateral (VPL) thalamocortical neurons. Administration of acetic acid in wild-type mice increases both single spikes and bursts of spike activity in VPL neurons, but in the Cav3.1 knockout mice, only the frequency of the single spikes increases. However, the burst activity is not observed either prior to or following intraperitoneal administration of acetic acid in Cav3.1 knockout mice. This change in pattern of firing is likely to decouple the TC neurons from efficiently activating neurons in the nRT, which in turn provide the inhibitory feedback to the VPL neurons to induce burst firing. By preventing burst firing, the “sensory gate” is removed and sensory signals are able to reach the somatosensory cortex. This antinociceptive role of the thalamus is not thought to be effective at controlling acute pain responses, which are mediated by local reflexes in the periphery. Supporting this notion that Cav3.1 does not contribute to acute pain pathways is a lack of any difference in pain responses to thermal (tail flick and paw withdrawal) or mechanical stimuli (von Frey) in both Cav3.1 mutant and wild-type littermates. However, in the complete Freund’s adjuvant model of chronic inflammation, a model that involves central sensitization, hyperalgesia scores also do not differ in wild-type and mutant mice following intraplantar injection. 44 VOLTAGE-GATED N-TYPE AND T-TYPE CALCIUM CHANNELS

Although there are no high-affinity subtype-selective T-type channel blockers available to support invivo knockout and knockdown studies, there are nonselective compounds known to inhibit these channels, which have provided insight into the role of T-type channels in processing nociceptive signals. Ethosuximide, a known antiepileptic drug, inhibits cloned Cav3.1, Cav3.2, and Cav3.3 (0.3–1 mM) at therapeutic plasma levels (0.3–0.7 mM; Gomora et al., 2001) and also dose- dependently suppresses both thermal hyperalgesia and mechanical allodynia following L5/L6 spinal nerve ligation when administered i.p. (Dogrul et al., 2003). This is in contrast with the lack of effect seen when ethosuximide is injected intrathecally, suggesting a peripheral mechanism of action. Similarly, no effect is seen when injected intraplantarly into the injured paw, suggesting that ethosuximide likely acts in the periphery at the level of the DRGs and not at sensory nerve endings. In other neuropathy models, paclitaxel- and vincristine-induced models of neuropathy, ethosuximide injected i.p. produces significant reversal of mechanical allodynia and hyperalgesia (Flatters and Bennett, 2004). Barton et al. 2005 also report antinociceptive effects of ethosuximide in both the early and late stages of the formalin test and in the acute tail-flick test as well as reversal of capsaicin-induced mechanical allodynia following i.p. injection. In contrast, intrathecal administration of ethosuximide fails to affect the nociceptive responses in the rat formalin test (Cheng, et al., 2007). In addition to the behavioral studies, during in vivo recordings from single neurons in the dorsal horn in which ethosuximide is directly applied to the spinal cord, the responses of the dorsal horn neurons to mechanical and thermal stimuli were suppressed in sham-operated and spinal nerve-ligated animals (Matthews and Dickenson, 2001). Two other antiepileptic drugs that are structurally diverse from ethosuximide and for which T-type inhibition has been reported are zonisamide and phenytoin. In cultured neurons, 50 mM zonisamide inhibits 40% of the T-type current in neuroblastoma cells (Kito et al., 1996) and 60% at 500 mM in cerebral cortex neurons (Suzuki et al., 1992). Interestingly, this compound suppresses neuropathy-induced thermal hyperalgesia, but not mechanical allodynia, in the Bennett chronic constriction rat model (Bennett and Xie, 1988; Hord et al., 2003) and is reported to provide Q4 relief in humans with refractory neuropathic pain (Guay, 2003; Takahashi et al., 2004) as well as prophylaxis for migraine patients (Drake et al., 2004). Phenytoin, which is used clinically in humans to treat neuropathic pain (McCleane, 1999; Finnerup et al., 2005), blocks Cav3.1 and Cav3.2 (140 and 8.3 mM, respectively; Todorovic et al., 2000) expressed in human embryonic kidney (HEK) cells, as well as native T-type channels in DRG neurons (IC50 ¼8.3 mM) and N1E-115 neuroblastoma (3–100 mM; Matsuki et al., 1984; Twombly et al., 1988). Though not studied in wide variety of animal models, phenytoin dose-dependently reduces bradykinin-induced pain when applied subcutaneously (Foong and Satoh, 1983); however, in acute models, i.p. administration of phenytoin is superior at reducing thermal pain in paw- withdrawal test than mechanical pain as measured in the tail-pressure test (Sakaue et al., 2004). Some of the most potent T-type channel blockers are comprised of neuroleptics used to treat a variety of psychiatric disorders, including schizophrenia, Tourette’s PATHOPHYSIOLOGY OF PAIN 45 disorder, and obsessive-compulsive disorder. In particular, pimozide inhibits in a nonpreferential manner Cav3.1, Cav3.2, and Cav3.3 with IC50 values of 50 nM (Santi et al., 2002). Although not extensively tested in a variety of pain models, pimozide when administered at low doses in the mouse formalin test is not highly efficacious (Saddi and Abbott, 2000), yet in humans it suppresses the pain associated with trigeminal neuralgia (Lechin et al., 1989; Green and Selman, 1991). Another compound well known for its inhibition of the T-type channels as well as HVA channels (P/Q-, R-, and L-types) is mibefradil, which inhibits cloned and native T-type channels with IC50 values ranging from 0.1to4.7mM depending on the assay conditions and cell type (Jimenez et al., 2000; Martin et al., 2000). In contrast to ethosuximide, when mibefradil is administered both intraperitoneally and injected directly into the injured limb of rats, it effectively reduces the tactile allodynia induced by spinal nerve ligation of L5/L6 (Dogrul et al., 2003). A reduction in thermal hyperalgesia is also reported following i.p. administration. Of relevance is the fact that mibefradil does not cross the blood–brain barrier when administered systemically and also does not suppress neuropathic behaviors upon direct i.t. administration, again suggesting a peripheral mechanism of action (Ertel et al., 1997). In the capsaicin assay, the results were mixed in that systemic (i.p) administration has no effect on allodynia, yet when injected intracisternally (i.c.), capsaicin-induced mechanical allodynia is dose-dependently reduced (Barton et al., 2005). In a postoperative pain model involving an incision into the paw, no antiallodynic action is observed following i.t. administration of mibefradil (Cheng et al., 2006). However, mibefradil inhibits both phases of the formalin response when injected i.p. or i.t., but is ineffective in suppressing acute nociceptive responses in the tail-flick reflex when administered i.p. (Barton et al., 2005; Cheng et al., 2007). Also noted is a lack of effect on an acute nociceptive stimulus (tail-flick test) when mibefradil is administered following i.t. administration (Dogrul et al., 2001; Barton et al., 2005). Another class of compounds that inhibit native T-type channels in DRG neurons is 5a-reduced neuroactive steroids, a class of compounds also known to potentiate GABAA ligand-gated channels. Of the various 5a-reduced neuroactive steroids examined, (3b,5a,17b)-17-hydroxyestrane-3-carbonitrile (ECN) was identified as inhibiting T-type channels in DRGs (IC50 300 nM) but being devoid of effects on GABAA receptors (Todorovic et al., 1998). To assess the peripheral role of T-type channels in nociceptive processing of acute noxious thermal stimulus to the paw, ECN injected intradermally dose-dependently produces analgesia as seen by the increase in latency to paw withdrawal (Pathirathna et al., 2005a). In rats with neuropathic pain (Bennett and Xie, 1988), intradermal injection of ECN alleviates both thermal and mechanical hyperalgesia in ligated rats and thermal and mechanical nociception in sham-operated rats (Pathirathna et al., 2005b). This antinociceptive effect on the thermal stimulus in ligated and sham-operated rats is not reversed by bicuculline, a GABAA antagonist, suggesting that the effect is most likely due to T-type channel inhibition. A novel role of peripheral T-type channels in amplifying nociceptive signals was unveiled when the effects of redox agents were examined on cloned and native T-type 46 VOLTAGE-GATED N-TYPE AND T-TYPE CALCIUM CHANNELS channels. Todorovic et al. (2001) found that the endogenous reducing agent, L-cysteine, augments T-type currents by 130% in small DRG neurons and in recombinant Cav3.2 channels by a similar amount and is reversed by the oxidizing agent 5,50-dithio-bis-(2-nitrobenzoic acid) (DTNB). The effects are mimicked by the reducing agent dithiothreitol (DTT). Conversely, the oxidizing agent DTNB, when applied alone, inhibits the peak T-type calcium channel current in small DRG neurons and Cav3.2 channels expressed in HEK cells, both by about 50%. When applied intradermally into the ventral side of the paw, the reducing agents decrease paw- withdrawallatencies (PWLs), while the oxidizing agent prolongs PWLs. Cysteine also produces a hyperalgesic response to noxious mechanical stimulus, while DTNB is analgesic and reverses the effect of L-cysteine. In providing further evidence that T-type channels boost nociceptive signals, mibefradil reverses the thermal hyperalgesic response of L-cysteine and DTT and enhances the analgesic effect of DTNB. Similarly, reducing cysteine analogues, L-cysteine, D-cysteine, and D,L-homocysteine, induce potent dose- and time-dependent hyperalgesia and conversely endogenous oxidizing cysteine analogues, L-cysteine, D-cysteine, and D,L-homocysteine, induce potent dose- and time-dependent analgesia in an acute model of thermal peripheral nociception in intact rats (Pathirathna et al., 2006). In a neuropathic pain model induced by chronic constriction of the sciatic nerve, both L-cysteine and DTT increase the thermal hyperalgesic response in both nerve-injured and sham-operated rats, while DTNB reduces the neuropathy-induced thermal hyperalgesia and produces analgesia in sham-operated rats (Todorovic et al., 2004). Both DTNB and mibefradil are able to abolish the L-cysteine-induced increase in thermal hyperalgesia rats with neuropathic pain and in sham-operated rats. At the cellular the level, the mechanisms underlying changes in neuronal excitability that lead to the development of allodynia and hyperalgesia are not well understood (Kajander and Bennett, 1992; Kajander et al., 1992; McLachlan et al., 1993; Devor et al., 1994; Petersen et al., 1996; Ramer et al., 1997; Song et al., 1999; Zhang et al., 1999.). Recent molecular and behavioral pharmacology data have provided a strong linkage for the contribution of T-type channels to the pathological perceptions of pain, but the biophysical properties of T-type channels themselves provide even further evidence that they contribute to hyperexcitability observed in DRGs after peripheral trauma or inflammation. Under normal physiological conditions, T-type channels are capable of regulating the pacemaker activity of neurons, contributing to rebound burst firing and oscillatory behavior and hence making them likely contributors to the hyperexcitability of primary afferent neurons in neuropathic and inflammatory pain states. One of the first studies investigating the involvement of T-type channels in processing sensory information was by White et al. (1989) who reported that Ca2þ influx by LVA channels produces an after- hyperpolarizing potential in dorsal root ganglion that triggers a burst of action þ potentials, which is abolished by 100 mMNi2 . Supporting evidence using the “T- rich” subset of DRGs that express a high density of T-type channels showed that the endogenous redox agent, L-cysteine, which promotes an increase in the current amplitude and shifts the gating properties, also lowers the nociceptor excitability threshold and induces burst firing (Nelson et al., 2005). Also in the D-hair CONTRIBUTIONS OF T-TYPE CHANNELS TO THE PATHOPHYSIOLOGY OF EPILEPSY 47 mechanoreceptor neurons, a role of Cav3.2 T-type channels in inducing a slow depolarization has been identified, which leads to a lowering of the voltage threshold for the generation of action potentials and hence an increase in neuronal excitability (Dubreuil et al., 2004). In a streptozotocin model of diabetic neuropathy, the T-type currents were shown to have increased by twofold in medium-sized DRGs with a concomitant depolarizing shift in the steady-state inactivation, which results in a greater “window current,” suggesting that T-type channels can activate more readily at physiological membrane potentials (Jagodic et al., 2007). Interest- ingly, Jagodic et al. also identified a newly expressed T-type channel that only partially inactivates at a membrane potential of 40 mV, a voltage that completely inactivates the T-type channels in DRGs from control rats, and likely contributes to the reduced threshold for burst firing in DRGs neurons from diabetic rats. In contrast, T-type channel currents in DRGs were reduced after neuropathic injury was induced by chronic constriction of the sciatic nerve or axotomy of L5 neurons in rats (McCallum et al., 2003; Hogan, 2007). Also observed in these DRGs following the induction of neuropathic pain is an increase in the rate of deactivation, which also supports the loss of T-type channels that deactivate slowly in a voltage- dependent manner. As the ability of neurons to fire repetitively requires that there is sufficient influx of Ca2þ to activate Ca2þ-activated Kþ channels that in turn hyperpolarize the cell, a reduction in Ca2þ at the entry during the slow deactivation mode at the end of an action potential may contribute to increased excitability of the DRGs from injured rats. Ultimately it will be interesting to determine whether the ability of T-type channel blockers to provide pain relief is dependent solely on their affinity of inhibiting Cav3.2 versus Cav3.1 and Cav3.3 channels or some combination thereof. Although the available evidence is suggestive of a peripheral mechanism of action, whether the efficacy is enhanced by the CNS-penetrant compounds also needs to be determined. Given the critical ability of Cav3.1 to alter the sensory gate in the thalamus, the effects of modulators on these central channels, which remain to be fully elucidated, may be quite important.

2.3 CONTRIBUTIONS OF T-TYPE CHANNELS TO THE PATHOPHYSIOLOGY OF EPILEPSY

Epilepsy is a complex disorder of spontaneous recurrent seizures characterized by neuronal hyperexcitability leading to hypersynchronization of neural networks. Phenotypically, seizures present themselves in different forms depending on the site of origin and subsequent recruitment of additional CNS structures. Although the cellular substrate(s) underlying the genesis of seizure activity are largely unknown, genetic, pharmacological, and physiological evidence all implicate an involvement of T-type calcium channels. The involvement of T-type channels in seizure disorders is primarily implicated in idiopathic generalized epilepsies (IGEs) with evidence arising from studies using transgenic and mutant animals as well as population analysis studying linkages to genetic mutations in humans. 48 VOLTAGE-GATED N-TYPE AND T-TYPE CALCIUM CHANNELS

Of the many IGEs, T-type channels have been shown to be critical for seizure activity in absence epilepsy. Absence epilepsy is characterized by a brief and sudden loss of consciousness, which is temporally correlated with 3 Hz bilateral synchronous spike-wave discharges (SWDs) involving the thalamocortical circuitry (Williams, 1950; Niedermeyer and Primary, 1996). Specifically, rebound burst firing in neocortical cells, thalamic reticular neurons, and thalamocortical relay neurons is known to be evoked by low-threshold Ca2þ potentials, which are thought to give rise to SWDs (Llinas and Jahnsen, 1982; Deschênes et al., 1984; Jahnsen and Llinas, 1984; Coulter et al., 1989a; Huguenard and Prince, 1992; De la Pena and Geijo-Barrientos, 1996; Destexhe and Sejnowski, 2002). In rats, the mRNAs of all three T-type isoforms are expressed in the thalamocortical pathway, though differentially in thalamus and neocortex (Talley et al., 1999). Cav3.2 and Cav3.3 are expressed in the thalamic reticular nucleus, whereas Cav3.1 is dominantly expressed in the thalamocortical neurons. More recently, splice variants of Cav3.1 have been differentially localized in the thalamic circuitry, which may account for the divergent burst firing in TC and GABAergic interneurons (Broicher et al., 2007a). In the neocortex, Cav3.1 and Cav3.3 mRNA levels are diffusely distributed through most layers of the cortex. Conversely, Cav3.2 mRNA is regionally restricted to layer 5 cortical pyramidal neurons, with little expression in other cortical areas. Of interest is that although the neural networks involved in SWDs all express one or more T-type channel isoform, the initiation and generalization has only recently been demonstrated as arising from the cortex. Meeren et al. (2002) have demonstrated through nonlinear association analysis that SWDs originate from the perioral region of the somatosensory cortex of Wistar Albino Glaxo rats from Rijswijk (WAG/Rij), a genetic model of absence epilepsy. Multisite field potential recordings from other cortical areas consistently lag behind the perioral region, with the cortical focus leading the thalamus during the first 500 ms. More in-depth electrophysiological studies using in vivo intracellular recordings from genetic absence epilepsy rats from Strasbourg (GAERS) demonstrate that the neural substrate involved in the initiation and intracortical propagation of ictal activity leading to generalization of related thalamic nuclei is found in layer 5/6 of the perioral somatosensory cortex (Polack et al., 2007). These neurons display pronounced hyperactivity relative to more superficial cortical neurons in that their membrane potentials are more depolarized and show distinctive interictal and preictal 9–11 Hz oscillations as well as enhanced bursting activity. It is postulated that these changes in layer 5/6 neurons create local oscillations (Silva et al., 1991) that lead to the generation of SWDs that propagate secondarily and intra- and interhemispherically throughout the somatosensory cortex and other cortical regions and the thalamic nuclei. Syn- chronization of SWDs in the thalamocortical and corticothalamic neurons sets into motion a unified oscillatory network that is driven by the ictogenic properties of the cortical neurons while the thalamocortical neurons provide resonant circuitry to sustain the activity. In support of the “cortical focus theory” of absence epilepsy, infusion of ethosuximide (a first choice antiabsence drug) into the perioral region of GAERS animals but not into the thalamus immediately reduces SWDs (Sherwin, CONTRIBUTIONS OF T-TYPE CHANNELS TO THE PATHOPHYSIOLOGY OF EPILEPSY 49

1989; Richards et al., 2003; Manning et al., 2004). Actions of ethosuximide on native T-type currents from isolated thalamocortical neurons and neurons from the thalamic reticular nucleus have, however, been mixed, with some groups demon- strating up to a 40% reduction in current amplitude at therapeutic concentrations (Coulter et al., 1989; Huguenard and Prince, 1994; Huguenard, 2002) while other studies observing no inhibition (Pfrieger et al., 1992; Leresche et al., 1998). More recently, a study on cloned channels confirms that ethosuximide inhibits cloned Cav3.1, Cav3.2, and Cav3.3 channels (IC50s 0.3–1 mM) at therapeutic plasma levels (0.3–0.7 mM; Gomora et al., 2001). Further evidence providing insight into the role of T-type channels in the generation of SWDs and absence-like seizures is from Cav3.1 knockout mice (Kim et al., 2001). Not only are Cav3.1 KO mice resistant to baclofen-induced 3–5 Hz spike-wave discharges as measured by EEG, intracellular recordings from thalamocortical neurons show an absence of rebound burst firing action potentials in thalamic slices when injected with negative current. In freely moving mice, field potential recordings from the ventroposteromedial and ventroposterolateral nuclei also show that responses to baclofen-mediated intrathalamic oscillations, which are required for SWD discharges, are also diminished. On the other hand, low doses of bicuculline, which evokes SWDs originating from the cortex (Steriade and Contreras, 1998), when injected into Cav3.1 KO mice are capable of generating SWDs in both the thalamus and cortex. Similarly, Cav3.1 KO mice are not resistant to 4-aminopyridine (4-AP)- induced tonic–clonic seizures. While these experiments highlight a critical role of Cav3.1 in generating absence-like seizures, the lack of protection in other models may be indicative of the dominant involvement of either Cav3.2 or Cav3.3 in the cortex for bicuculline-induced SWDs and mechanistically different pathways for 4-AP-induced seizures. Cross-breeding Cav3.1 KO mice with other models of absence seizures, such as lethargic (b4lh/lh), tottering (a1Atg/tg), or stargazer (g2stg/stg) mutant mice models or mice harboring a null mutation for the pore-forming Cav2.1 subunit of P/Q-type channels, results in strongly or completely suppressed cortical SWD paroxysmal activities (Song et al., 2004). Interestingly, in all these murine models, T-type channel currents are elevated in thalamocortical neurons by up to 50%, although no alteration in mRNA levels are apparent (Zhang et al., 2002; Nahm et al., 2005). In GAERS animals, elevated increases in reticular thalamic T-typechannel currents (55%) have also been reported with a concomitant increase in Cav3.1 and Cav3.2 mRNA levels in the ventral posterior thalamic relay nuclei and thalamic reticular nucleus, respectively (Tsakiridou et al., 1995; Talley et al., 2000). More recently, a homozygous, missense, single nucleotide (G–C) mutation has been identified in the Cav3.2 gene from GAERS, / though the functional effects are unknown (Kyi et al., 2006). However, in Cav2.1 / /þ Cav3.1 mice, T-type channel currents are decreased by 25%, yet animals are still capable of generating SWDs indicating that baseline levels, but not necessarily augmented levels, of T-type channel currents are sufficient for the genesis of spontaneous SWD activity. As protection against absence seizures was afforded by deletion of Cav3.1, it will be interesting to determine whether complete and selective pharmacological inhibition of Cav3.1 channels provides complete 50 VOLTAGE-GATED N-TYPE AND T-TYPE CALCIUM CHANNELS suppression of SWDs in these genetic models and pharmacological models of absence seizures. In humans, the role of T-type channels as a susceptibility gene leading to the pathophysiology of absence epilepsy has been speculated due to the known function of T-type channels in the thalamic and cortical physiology. All three subtypes, Cav3.1, Cav3.2, and Cav3.3, have been examined in patients with childhood absence epilepsy (CAE) and other forms of IGEs. Population analyses studying linkages to genetic mutations in humans have identified numerous missense mutations in the Cav3.2 gene. Chen et al. (2003a) reported 12 mutations in 14 patients of Chinese Han ethnicity that were not observed in 230 control patients. In some patients, two or more mutations were identified and numerous polymorphisms were reported in both patients and seizure-free individuals. In an expanded study, which included patients with CAE, juvenile myoclonic epilepsy, and febrile convulsions, three new missense mutations have been identified, though they were also found in the control group (Heron et al., 2004). More recently, 28 new variants have been identified in the Cav3.2 gene from the Chinese Han population of which only some were found in CAE patients (Liang et al., 2006). In a study attempting to investigate whether common polymorphisms in the Cav3.2 gene are associated with CAE in the Chinese Han population, carriers with three different polymorphisms were identified as at a higher risk of developing CAE than noncarriers (Liang et al., 2007). In a predominantly Caucasian population, over 100 variants of the Cav3.2 gene have been found in patients with IGEs as well as temporal lobe epilepsy (Heron et al., 2007). In contrast, in an evaluation of Caucasian European patients with CAE, linkage analysis was unable to detect any of the Chinese variants in 220 patients (Chioza et al., 2006). The human Cav3.2 mutations are located mainly in exons 6–12 encoding the domain I–II linker region. Exogenous expression of the mutations introduced into rat and human Cav3.2 channels reveals that in some instances channel biophysical properties are altered while other changes are “biophysically silent” (Khosravani et al., 2004, 2005; Vitko et al., 2005; Peloquin et al., 2006). Alterations in the biophysical properties mainly result in gain-of-function phenotypes as seen by a more depolarized steady-state inactivation and hyperpolarized voltage dependence of activation. Further insight into the possible roles of the “biophysically silent” mutations and SNPs in loop I–II was revealed when deletion of this region was found to increase Cav3.2 plasma membrane expression with a predicted overactivity to occur in neurons (Vitko et al., 2007). Therefore, the effect of the gain offunction as a result of changes in biophysical properties on the Cav3.2 channels as well as biophysically silent mutations that may serve to promote increased plasma membrane expression both result in increased T-type channel activity. A study investigating whether Cav3.1 channels are involved in the etiology of IGEs has proposed a linkage to the CACNA1G gene (Singh et al., 2007). However, while 13 variants were identified in 123 Japanese and Hispanic patients with IGEs, many were found in both patients and control individuals. In the Han Chinese population, no Cav3.1 mutations were identified in patients with CAE, but six single nucleotide polymorphisms (SNPs) were found (Chen et al., 2003b). Overall, the distribution of SNPs was not significantly different in control and CAE patients in the Chinese Han CONTRIBUTIONS OF T-TYPE CHANNELS TO THE PATHOPHYSIOLOGY OF EPILEPSY 51 population and therefore the SNPs are not considered important for susceptibility to nonconvulsive seizures (Wang et al., 2006). Though the pathophysiological role of T-type channels has primarily focused on IGEs, they are also likely to be involved in other seizure types as RNA expression studies have shown that Cav3.1 and Cav3.3 are prominently expressed in the human CNS, including structures known to generate seizure activity, such as the thalamus, cortex, hippocampus, and amygdala. In situ hybridization studies in rat brain have also revealed diffuse mRNAexpression ofCav3.1, Cav3.2, and Cav3.3 in all brain structures (Craig et al., 1999; Talley et al., 1999). Interestingly, at the subcellular level subtype- specific polyclonal antibodies against Cav3 channel proteins indicate that the three T-channel subtypes are differentially localized in the soma and dendrites (McKay et al., 2006). Cav3.1 immunolabeling is prominent in the soma and proximal region of the dendrites, while somatic and proximal middendritic regions largely express Cav3.2. Distribution of Cav3.3 is further distinct in that expression is found in the soma as well as extended throughout the arborization of the dendrites in selective neurons. In concordance with this immunohistochemical study are previous electrophysiological studies that have identified T-type currents in both the somatic and dendritic compartments of central neurons (Karst et al., 1993; Markram and Sakmann, 1994; Magee and Johnston, 1995; Mouginot et al., 1997; Jung et al., 2001; Isope and Murphy, 2005). Two particular animal models of seizures have substantiated the potential role of T-type channels in seizure types other than IGEs. In the pilocarpine-induced status epilepticus rat model, a model of human complex partial seizures with secondary generalization, a threefold increase in T-type tail current density is observed in hippocampal neurons with a concomitant 54% increase in the number of neurons with intrinsic burst firing, which normally fire in a regular mode; this was inhibited by 2þ Ni with an IC50 of 27 mM (Su et al., 2002). In a focal model of epilepsy induced by kindling of the rat hippocampus, patch-clamp recordings from in situ slices show an 80% increase in T-type channel currents in hippocampal neurons compared to recordings from control rats (Faas et al., 1996). These elevated T-typechannel currents are still present 6 weeks after the last kindling stimulation and support a role of T-type channels in epileptogenesis. Definitive pharmacological proof-of-concept studies confirming that T-type calcium channels represent important drug targets for the treatment of different epilepsies have been hampered by the lack of high-affinity subtype-selective blockers. Besides ethosuximide, which is used to treat patients with absence epilepsy and inhibits both native (0.2–24 mM) and cloned T-type channels (Cav3.1, Cav3.2, and Cav3.3; 0.3– 1 mM) at therapeutically relevant concentrations of 0.3–0.7 mM (Coulter et al., 1989b; Huguenard and Prince, 1994; Gomora et al., 2001; Huguenard, 2002), only a handful of other AEDs with activity at T-type Ca2þ channels have been identified. Valproate, a broad spectrum antiepileptic drug used in the treatment of patients with IGEs, is thought to primarily provide efficacy by inhibiting GABA transaminase to promote GABAergic transmission and through inhibition of sodium channels; this compound is also known to block T-type channels (reviewed in Czapinski et al., 2005). Initial studies report the partial inhibition of T-type channel currents at 52 VOLTAGE-GATED N-TYPE AND T-TYPE CALCIUM CHANNELS high concentrations in rat nodose ganglion neurons relative to the therapeutic plasma concentration (Kelly et al., 1990; Todorovic and Lingle, 1998). However, even at 2þ cloned Cav3.1 channels, 10 mM valproate only modestly inhibited the Ca channel current by 10% (Lacinova et al., 2000). More recently, the antagonistic action of valproate and ethosuximide on LVA channels of thalamic relay neurons in WAG/Rij rats has been systematically compared to nonepileptic control rats (Broicher et al., 2007b). Both ethosuximide (0.25–0.75 mM) and valproate (1 mM) inhibit T-type Ca2þ channel currents with higher affinity in acutely isolated thalamocortical relay neurons from WAG/Rij rats than in comparable neurons from nonepileptic rats. In addition, ethosuximide delays the onset of the low-threshold spike and increases the tonic action potentials in thalamocortical neurons from WAG/Rij rats, an effect that is different to that observed in control rats. A role for T-type channels in temporal lobe seizures has been suggested as fluoxetine, a reuptake inhibitor of serotonin used to treat depression, not only inhibits LVA (IC50 ¼ 6.8 mM) and HVA (1–2 mM) calcium channels in cultured hippocampal pyramidal cells, but also reduces Kþ-induced seizure-like activity in hippocampal slices (Wong et al., 1995; Deak et al., 2000). In addition, in rodents, fluoxetine dose-dependently protects animals from limbic seizures evoked by bicuculline applied into the deep prepiriform cortex (Prendiville and Gale, 1993) as well as by increasing the after-discharge threshold of hippocampal seizures induced by electrical stimulation (Wada et al., 1995). In genetically epilepsy-prone rats (GEPR), fluoxetine in a dose-dependent manner reduces convulsions induced by sound stimulus (Dailey et al., 1996). Interestingly, in humans when fluoxetine is coadministered with valproate, carbamazepine, or phenobarbital or in mice with phenytoin, carbamazepine, and ameltolide, the effects of the AEDS are enhanced (Leander, 1992; Favale et al., 1995). Other studies examining roles of T-type channels in partial and generalized seizures have proposed that the therapeutic action of phenytoin and zonisamide may be in part due to T-type inhibition (Suzuki et al., 1992; Todorovic et al., 2000). Phenytoin is used clinically to treat partial and generalized seizures and is known to primarily inhibit sodium channels.Italso inhibitsrecombinantT-typechannels atconcentrationscloseto the maximal therapeutic concentration (Cav3.1 IC50 ¼ 124 mM; Cav3.2 IC50 ¼ 8.3 and 192 mM). Zonisamide, an adjunctive therapy in adults with partial onset seizures with multiple sites of action, shows T-type current blockade in cultured neurons isolated from ratcerebralcortex of38% at 50 mM (Suzuki et al., 1992). In addition, flunarizine,a potent blocker of recombinant Cav3.1 (IC50 ¼ 0.53 mM), Cav3.2 (IC50 ¼ 0.36 mM), and Cav3.3 (IC50 ¼ 0.84 mM) channels, also exhibits anticonvulsant properties in both animals and humans (Santi et al., 2002). In humans, flunarizine administered as an adjuvant therapy is efficacious in patients with complex partial seizures with or without secondarily generalized seizures as well as reflex seizures (Starreveld et al., 1989; Durrheim et al., 1992; Pledger et al., 1994). In various chemoconvulsant and electro- convulsant animal models, flunarizine monotherapy or coadministration results in protection (Desmedt et al., 1975; De Sarro et al., 1986, 1992; Drago et al., 1986; Pohl and Mares, 1987; Mack and Gilbert, 1992; Rodger and Pleuvry, 1993; Becker and Grecksch, 1995; Joseph et al., 1998a, 1998b), though in some models no protection is REFERENCES 53 observed for cocaine-induced seizures, cortical stimulation, amygdala kindling, or pentylenetetrazol seizures (Trommer and Pasternak, 1989; Gasior et al., 1996, 1999).

2.4 CONCLUSIONS

In summary, there is considerable pharmacological, genetic, and physiological evidence implicating N-type and T-type calcium channels in a number of pathophysiological conditions centered around neuronal hyperexcitability. Although there exist clinical agents that nonselectively block T-type calcium channels, there are no subtype-specific drugs yet available; thus, there remains both significant clinical challenge and opportunity. In the case of N-type channels and intractable pain, while Prialt provides excellent clinical proof of concept, its peptidic nature requiring intrathecal administration together with its narrow therapeutic window provides the opportunity for developing equally efficacious but safer and orally available N-type channel blockers.

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