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 CBD's 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 CBD's 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.

Voltage-gated calcium channels represent a heterogenous family of calcium-selective channels that can be distinguished by their molecular, electrophysiological, and pharmacological characteristics. We report here the molecular cloning and functional expression of three members of the low voltage-activated calcium channel family from rat brain (␣ 1G , ␣ 1H , and ␣ 1I ). Northern blot and reverse transcriptase-polymerase chain reaction analyses show ␣ 1G , ␣ 1H , and ␣ 1I to be expressed throughout the newborn and juvenile rat brain. In contrast, while ␣ 1G and ␣ 1H mRNA are expressed in all regions in adult rat brain, ␣ 1I mRNA expression is restricted to the striatum. Expression of ␣ 1G , ␣ 1H , and ␣ 1I subunits in HEK293 cells resulted in calcium currents with typical T-type channel characteristics: low voltage activation, negative steady-state inactivation, strongly voltage-dependent activation and inactivation, and slow deactivation. In addition, the direct electrophysiological comparison of ␣ 1G , ␣ 1H , and ␣ 1I under identical recording conditions also identified unique characteristics including activation and inactivation kinetics and permeability to divalent cations. Simulation of ␣ 1G , ␣ 1H , and ␣ 1I T-type channels in a thalamic neuron model cell produced unique firing patterns (burst versus tonic) typical of different brain nuclei and suggests that the three channel types make distinct contributions to neuronal physiology.

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

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 (Ca(2+)) channels. We sought to develop high-affinity T-type specific Ca(2+) 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 Ca(2+) channel pharmacophore and a high-throughput fluorometric Ca(2+) influx assay, we identified the T-type Ca(2+) 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 Ca(2+) 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.

Childhood absence epilepsy (CAE) is a type of gener-We have functionally characterized five of these mutations (F161L, E282K, C456S, V831M, and D1463N) using rat Ca v 3.2 and whole-cell patch clamp recordings in transfected HEK293 cells. Two of the mutations, F161L and E282K, mediated an ϳ10-mV hyperpolarizing shift in the half-activation potential. Mutation V831M caused a ϳ50% slowing of inactivation relative to control and shifted half-inactivation potential ϳ10 mV toward more depolarized potentials. Mean time to peak was significantly increased by mutation V831M but was unchanged for all others. No resolvable changes in the parameters of the IV relation or current kinetics were observed with the remaining mutations. The findings suggest that several of the Ca v 3.2 mutants allow for greater calcium influx during physiological activation and in the case of F161L and E282K can result in channel openings at more hyperpolarized (close to resting) potentials. This may underlie the propensity for seizures in patients with CAE.Generalized epileptic disorders involve both brain hemispheres and are characterized by abnormal synchronous electrical (electroencephalographic) activity, recorded bilaterally at seizure onset (1). Childhood absence epilepsy (CAE) 1 is a type of idiopathic generalized epilepsy and is typified by sudden brief impairment of consciousness followed by ϳ3-Hz spikeand-wave discharges (SWDs) over both brain hemispheres (2). A typical absence seizure is without convulsions and there are no reported neuropathological changes associated with this disorder (3). Spike-wave discharges in absence epilepsy involve interactions between cortical and thalamic structures (4). The classical view of SWD-based seizures, including absence epilepsy, implicates the thalamus as the site of seizure generation (5, 6). Recently, an increasing body of evidence suggests that spike-wave seizures are initiated in the neocortex and then rapidly progress to involve thalamic structures (7-9). The thalamus and cortex then engage in complex interplay that underlies SWD generation and is dependent on the activation of low voltage-activated (T-type) calcium channels (4). Indeed, reticular thalamic neurons are endowed with large T-type currents that mediate bursting behavior associated with SWDs. The critical role of T-type channels in SWD epilepsies is also supported by treatment of absence seizures using ethosuximide, an inhibitor of T-type Ca 2ϩ currents (10, 11), and by the observation that expression of these channels is increased in thalamic neurons in a genetic rat absence model (12).We now know of three genes (subtypes) encoding different types of T-type channels (Ca v 3.1, Ca v 3.2, and Ca v 3.3), all of which are subject to alternative splicing resulting in a range of different isoforms with distinct biophysical, modulatory, and pharmacological properties (13-23). It was recently shown that Ca v 3.1 knock-out mice display reduced burst mode firing activity, and that the Ca v 3.1-deficient thalamus is specifically resilie...