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T-type Ca channels (IA fluorometric (fluorescence imaging plate reader [FLIPR]) assay was used to investigate modulation of human T-type IIn the FLIPR assay, all 11 phytocannabinoids tested modulated T-type IModulation of T-type calcium channels is a common property of phytocannabinoids, which all increase steady-state inactivation at physiological membrane potentials, with some also affecting channel activation. Thus, T-type I

IntroductionLow voltage-activated T-type calcium channels (T-type ICa), CaV3.1, CaV3.2, and CaV3.3 are opened by small depolarizations from the resting membrane potential in many cells and have been associated with neurological disorders including absence epilepsy and pain. Δ9-tetrahydrocannabinol (THC) is the principal psychoactive compound in Cannabis and also directly modulates T-type ICa, however, there is no information about functional activity of most phytocannabinoids on T-type calcium channels, including Δ9-tetrahydrocannabinol acid (THCA), the natural non-psychoactive precursor of THC. The aim of this work was to characterize THCA effects on T-type calcium channels.Materials and MethodsWe used HEK293 Flp-In-TREx cells stably expressing CaV3.1, 3.2 or 3.3. Whole-cell patch clamp recordings were made to investigate cannabinoid modulation of ICa.ResultsTHCA and THC inhibited the peak current amplitude CaV3.1 with a pEC50s of 6.0 ± 0.7 and 5.6 ± 0.4, respectively. 1μM THCA or THC produced a significant negative shift in half activation and inactivation of CaV3.1 and both drugs prolonged CaV3.1 deactivation kinetics. THCA (10 μM) inhibited CaV3.2 by 53% ± 4 and both THCA and THC produced a substantial negative shift in the voltage for half inactivation and modest negative shift in half activation of CaV3.2. THC prolonged the deactivation time of CaV3.2 while THCA did not. THCA inhibited the peak current of CaV3.3 by 43% ± 2 (10μM) but did not notably affect CaV3.3 channel activation or inactivation, however, THC caused significant hyperpolarizing shift in CaV3.3 steady state inactivation.DiscussionTHCA modulated T-type ICa currents in vitro, with significant modulation of kinetics and voltage dependence at low μM concentrations. This study suggests that THCA may have potential for therapeutic use in pain and epilepsy via T-type channel modulation without the unwanted psychoactive effects associated with THC.

Introduction: Low voltage-activated T-type calcium channels (T-type I(Ca)), Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3, are opened by small depolarizations from the resting membrane potential in many cells and have been associated with neurological disorders, including absence epilepsy and pain. Δ(9)-tetrahydrocannabinol (THC) is the principal psychoactive compound in Cannabis and also directly modulates T-type I(Ca); however, there is no information about functional activity of most phytocannabinoids on T-type calcium channels, including Δ(9)-tetrahydrocannabinolic acid (THCA), the natural nonpsychoactive precursor of THC. The aim of this work was to characterize THCA effects on T-type calcium channels. Materials and Methods: We used HEK293 Flp-In-TREx cells stably expressing Ca(V)3.1, 3.2, or 3.3. Whole-cell patch clamp recordings were made to investigate cannabinoid modulation of I(Ca). Results: THCA and THC inhibited the peak current amplitude Ca(V)3.1 with pEC(50)s of 6.0±0.7 and 5.6±0.4, respectively. THC (1 μM) or THC produced a significant negative shift in half activation and inactivation of Ca(V)3.1, and both drugs prolonged Ca(V)3.1 deactivation kinetics. THCA (10 μM) inhibited Ca(V)3.2 by 53%±4%, and both THCA and THC produced a substantial negative shift in the voltage for half inactivation and modest negative shift in half activation of Ca(V)3.2. THC prolonged the deactivation time of Ca(V)3.2, while THCA did not. THCA inhibited the peak current of Ca(V)3.3 by 43%±2% (10 μM) but did not notably affect Ca(V)3.3 channel activation or inactivation; however, THC caused significant hyperpolarizing shift in Ca(V)3.3 steady-state inactivation. Discussion: THCA modulated T-type I(Ca) currents in vitro, with significant modulation of kinetics and voltage dependence at low μM concentrations. This study suggests that THCA may have potential for therapeutic use in pain and epilepsy through T-type calcium channel modulation without the unwanted psychoactive effects associated with THC.

Synthetic cannabinoid receptor agonists (SCRAs) are a large and growing class of new psychoactive substances (NPSs). Two recently identified compounds, MEPIRAPIM and 5F-BEPIRAPIM (NNL-2), have not been confirmed as agonists of either cannabinoid receptor subtype but share structural similarities with both SCRAs and a class of T-type calcium channel (Ca

Background and purpose Consumption of Synthetic Cannabinoid Receptor agonists (SCRAs) is associated with severe adverse reactions including seizures, arrhythmias and death, but the molecular mechanisms surrounding SCRA toxicity are not yet established. These disease-like symptoms are also synonymous with altered T-type calcium channel activity which controls rhythmicity in the heart and brain. This study examined whether SCRAs alter T-type activity and whether this represents a possible mechanism of toxicity. Experimental approach Fluorescence-based and electrophysiology assays were used to screen 16 structurally related synthetic cannabinoids for their ability to inhibit human T-type calcium channels expressed in HEK293 cells. The most potent compounds were then further examined using patch clamp electrophysiology. Key results MDMB-CHMICA and AMB-CHMINACA potently blocked Cav3.2 with IC50 values of 1.5 and 0.74 μM respectively. Current inhibition increased from 47 to 80% and 45–87% respectively when the channel was in slow-inactivated state. Both SCRAs had little effect on steady state inactivation, however MDMB-CHMICA significantly shifted the half activation potential by -7mV. Neither drug produced frequency dependent block, in contrast to the phytocannabinoid Δ9-THC. Conclusions and implications SCRAs are potent agonists of CB1 receptors and can be extremely toxic, but observed toxicity also resembles symptoms associated with altered Cav3.2 activity. Many SCRAs tested were potent modulators of Cav3.2, raising the possibility that SC toxicity may be due in part to Cav3.2 modulation. This potent T-type channel modulation suggests the possibility of SCRAs as a new drug class with potential to treat diseases associated with altered T-type channel activity. This article is part of the special issue on ‘Cannabinoids’.