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Introduction: Cannabidiol (CBD) has been clinically approved for intractable epilepsies, offering hope that novel anticonvulsants in the phytocannabinoid class might be developed. Looking beyond CBD, we have recently reported that a series of biosynthetic precursor molecules found in cannabis display anticonvulsant properties. However, information on the pharmacological activities of these compounds on CNS drug targets is limited. The current study aimed to fill this knowledge gap by investigating whether anticonvulsant phytocannabinoids affect T-type calcium channels, which are known to modulate neuronal excitability, and may be relevant to the anti-seizure effects of this class of compounds. Materials and methods: A fluorescence-based assay was used to screen the ability of the phytocannabinoids to inhibit human T-type calcium channels overexpressed in HEK-293 cells. A subset of compounds was further examined using patch-clamp electrophysiology. Alphascreen technology was used to characterise selected compounds against G-protein coupled-receptor 55 (GPR55) overexpressed in HEK-293 cells, as GPR55 is another target of the phytocannabinoids. Results: A single 10 µM concentration screen in the fluorescence-based assay showed that phytocannabinoids inhibited T-type channels with substantial effects on Ca v 3.1 and Ca v 3.2 channels compared to the Ca v 3.3 channel. The anticonvulsant phytocannabinoids cannabigerovarinic acid (CBGVA) and cannabidivarinic acid (CBDVA) had the greatest magnitudes of effect (≥80% inhibition against Ca v 3.1 and Ca v 3.2), so were fully characterized in concentration-response studies. CBGVA and CBDVA had IC 50 values of 6 μM and 2 µM on Ca v 3.1 channels; 2 μM and 11 µM on Ca v 3.2 channels, respectively. Biophysical studies at Ca v 3.1 showed that CBGVA caused a hyperpolarisation shift of steady-state inhibition. Both CBGVA and CBDVA had a use-dependent effect and preferentially inhibited Ca v 3.1 current in a slow inactivated state. CBGVA and CBDVA were also shown to antagonise GPR55. Conclusion and implications: These findings show that CBGVA and CBDVA inhibit T-type calcium channels and GPR55. These compounds should be further investigated to develop novel therapeutics for treating diseases associated with dysfunctional T-type channel activity., Competing Interests: JA and IM have served as expert witnesses in various medicolegal cases involving cannabis and cannabinoids. JA, LA, and IM hold patents on cannabinoid therapies (PCT/AU2018/05,089 and PCT/AU2019/050,554). JA has received consulting fees from Creo Inc. And Medicinal Cannabis Industry Australia (MCIA). IM acts as a consultant to Kinoxis Therapeutics and has received honoraria from Janssen. He has also received consulting fees from MCIA. CB is a founder and shareholder of Zymedyne Therapeutics. Zymedyne is not a publicly traded entity. The remaining authors have no conflicts of interest. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Udoh, Bladen, Heblinski, Luo, Janve, Anderson, McGregor and Arnold.)

Objective: The Epi4K Consortium recently identified 4 de novo mutations in the γ-aminobutyric acid type A (GABA A ) receptor β3 subunit gene GABRB3 and 1 in the β1 subunit gene GABRB1 in children with one of the epileptic encephalopathies (EEs) Lennox-Gastaut syndrome (LGS) and infantile spasms (IS). Because the etiology of EEs is often unknown, we determined the impact of GABRB mutations on GABA A receptor function and biogenesis., Methods: GABA A receptor α1 and γ2L subunits were coexpressed with wild-type and/or mutant β3 or β1 subunits in HEK 293T cells. Currents were measured using whole cell and single channel patch clamp techniques. Surface and total expression levels were measured using flow cytometry. Potential structural perturbations in mutant GABA A receptors were explored using structural modeling., Results: LGS-associated GABRB3(D120N, E180G, Y302C) mutations located at β + subunit interfaces reduced whole cell currents by decreasing single channel open probability without loss of surface receptors. In contrast, IS-associated GABRB3(N110D) and GABRB1(F246S) mutations at β - subunit interfaces produced minor changes in whole cell current peak amplitude but altered current deactivation by decreasing or increasing single channel burst duration, respectively. GABRB3(E180G) and GABRB1(F246S) mutations also produced spontaneous channel openings., Interpretation: All 5 de novo GABRB mutations impaired GABA A receptor function by rearranging conserved structural domains, supporting their role in EEs. The primary effect of LGS-associated mutations was reduced GABA-evoked peak current amplitudes, whereas the major impact of IS-associated mutations was on current kinetic properties. Despite lack of association with epilepsy syndromes, our results suggest GABRB1 as a candidate human epilepsy gene. Ann Neurol 2016;79:806-825., (© 2016 American Neurological Association.)