Janette Mezeyova

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 D2dopamine 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 (α1G, α1H, and α1Isubtypes). Among the neuroleptics tested, the diphenylbutylpiperidines pimozide and penfluridol were the most potent T-type channel blockers with Kd values (∼30–50 nm and ∼70–100 nm, respectively), in the range of their antagonism of the D2 dopamine receptor. In contrast, the butyrophenone haloperidol was ∼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 α1G and α1I T-type channels compared with α1H. 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.

T-Type Calcium Channel Blockers That Attenuate Thalamic Burst Firing and Suppress Absence Seizures

Two high-affinity T-type calcium channel blockers attenuate neural activity in the thalamus and suppress seizures in a genetic model of absence epilepsy. To Soothe a Seizure Some epileptic children and adolescents experience “absence” seizures hundreds of times a day. Although apparently mild, these seizures—so named because they involve a sudden, brief absence of consciousness—can be dangerous if they occur during swimming or driving, for example. Unfortunately, the drugs available for treating such seizures are not completely effective. Tringham et al. sought to address this problem by rational drug design. Although the root cause of such seizures is not known, they are associated with abnormal, highly synchronous neuronal activity in certain brain regions. Voltage-gated ion channels, which have crucial functions in generating and propagating neuronal signals, likely play a key role. Several lines of evidence link one type of ion channel, low voltage–activated T-type calcium channels, to absence seizures. Using the structure of an N-type calcium channel blocker as a starting point, the researchers designed and screened small, focused libraries of compounds in a high-throughput assay that monitored calcium influx via a recombinant T-type channel. Two high-affinity T-type calcium channel blockers, termed Z941 and Z944, were identified; Z944 was highly selective for T-type channels and exhibited a preference for inactivated channels (the likely configuration in hyperexcited neurons). In a rat model of absence epilepsy, both compounds markedly reduced the time spent in seizures and the number of seizures per hour. In contrast to current first-line drugs for treating absence seizures, Z941 and Z944 also reduced the average seizure duration and cycle frequency. Both compounds were well tolerated in rats. Given its in vitro and in vivo activities, Z944 will progress to phase 1 clinical studies to test its safety in humans. Further studies will be needed to determine whether its marked effects in the rat model of absence epilepsy translate to the more complicated human condition. Absence seizures are a common seizure type in children with genetic generalized epilepsy and are characterized by a temporary loss of awareness, arrest of physical activity, and accompanying spike-and-wave discharges on an electroencephalogram. They arise from abnormal, hypersynchronous neuronal firing in brain thalamocortical circuits. Currently available therapeutic agents are only partially effective and act on multiple molecular targets, including γ-aminobutyric acid (GABA) transaminase, sodium channels, and calcium (Ca2+) channels. We sought to develop high-affinity T-type specific Ca2+ channel antagonists and to assess their efficacy against absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. Using a rational drug design strategy that used knowledge from a previous N-type Ca2+ channel pharmacophore and a high-throughput fluorometric Ca2+ influx assay, we identified the T-type Ca2+ channel blockers Z941 and Z944 as candidate agents and showed in thalamic slices that they attenuated burst firing of thalamic reticular nucleus neurons in GAERS. Upon administration to GAERS animals, Z941 and Z944 potently suppressed absence seizures by 85 to 90% via a mechanism distinct from the effects of ethosuximide and valproate, two first-line clinical drugs for absence seizures. The ability of the T-type Ca2+ channel antagonists to inhibit absence seizures and to reduce the duration and cycle frequency of spike-and-wave discharges suggests that these agents have a unique mechanism of action on pathological thalamocortical oscillatory activity distinct from current drugs used in clinical practice.

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 analysis of Cav3.2 T-type calcium channel mutations linked to childhood absence epilepsy

Summary:  Purpose: Childhood absence epilepsy (CAE) is an idiopathic form of seizure disorder that is believed to have a genetic basis.

A Fluorescence-Based High-Throughput Screening Assay for the Identification of T-Type Calcium Channel Blockers

T-type voltage-gated Ca(2+) channels have been implicated in contributing to a broad variety of human disorders, including pain, epilepsy, sleep disturbances, cardiac arrhythmias, and certain types of cancer. However, potent and selective T-type Ca(2+) channel modulators are not yet available for clinical use. This may in part be due to their unique biophysical properties that have delayed the development of high-throughput screening (HTS) assays for identifying blockers. One notable challenge is that at the normal resting membrane potential (V(m)) of cell lines commonly utilized for drug screening purposes, T-type Ca(2+) channels are largely inactivated and thus cannot be supported by typical formats of functional HTS assays to both evoke and quantify the Ca(2+) channel signal. Here we describe a simple method that can successfully support a fluorescence-based functional assay for compounds that modulate T-type Ca(2+)channels. The assay functions by exploiting the pore-forming properties of gramicidin to control the cellular V(m) in advance of T-type Ca(2+) channel activation. Using selected ionic conditions in the presence of gramicidin, T-type Ca(2+) channels are converted from the unavailable, inactivated state to the available, resting state, where they can be subsequently activated by application of extracellular K(+). The fidelity of the assay has been pharmacologically characterized with sample T-type Ca(2+) channel blockers whose potency has been determined by conventional manual patch-clamp techniques. This method has the potential for applications in high-throughput fluorometric imaging plate reader (FLIPR(R), Molecular Devices, Sunnyvale, CA) formats with cell lines expressing either recombinant or endogenous T-type Ca(2+) channels.

Identification of sodium channel isoforms that mediate action potential firing in lamina I/II spinal cord neurons

BackgroundVoltage-gated sodium channels play key roles in acute and chronic pain processing. The molecular, biophysical, and pharmacological properties of sodium channel currents have been extensively studied for peripheral nociceptors while the properties of sodium channel currents in dorsal horn spinal cord neurons remain incompletely understood. Thus far, investigations into the roles of sodium channel function in nociceptive signaling have primarily focused on recombinant channels or peripheral nociceptors. Here, we utilize recordings from lamina I/II neurons withdrawn from the surface of spinal cord slices to systematically determine the functional properties of sodium channels expressed within the superficial dorsal horn.ResultsSodium channel currents within lamina I/II neurons exhibited relatively hyperpolarized voltage-dependent properties and fast kinetics of both inactivation and recovery from inactivation, enabling small changes in neuronal membrane potentials to have large effects on intrinsic excitability. By combining biophysical and pharmacological channel properties with quantitative real-time PCR results, we demonstrate that functional sodium channel currents within lamina I/II neurons are predominantly composed of the NaV1.2 and NaV1.3 isoforms.ConclusionsOverall, lamina I/II neurons express a unique combination of functional sodium channels that are highly divergent from the sodium channel isoforms found within peripheral nociceptors, creating potentially complementary or distinct ion channel targets for future pain therapeutics.

Inhibitory effects of cannabidiol on voltage-dependent sodium currents

Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and nonpsychoactive compounds are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated antiepileptic effects has been from case reports or smaller surveys. The mechanisms for CBDs anticonvulsant effects are unclear and likely involve noncannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating the therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1–1.7 currents, with an IC50 of 1.9–3.8 μm, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of ∼3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade, became more potent at depolarized potentials, and also slowed recovery from inactivation, supporting the idea that CBD binding preferentially stabilizes inactivated Nav channel states. We also found that CBD inhibits other voltage-dependent currents from diverse channels, including bacterial homomeric Nav channel (NaChBac) and voltage-gated potassium channel subunit Kv2.1. Lastly, the CBD block of Nav was temperature-dependent, with potency increasing at lower temperatures. We conclude that CBDs mode of action likely involves 1) compound partitioning in lipid membranes, which alters membrane fluidity affecting gating, and 2) undetermined direct interactions with sodium and potassium channels, whose combined effects are loss of channel excitability.