Biology subjects

Bladen, C.

Publications and source records attributed to Bladen, C..

3 recordsLinked to original sources

bioRxiv2020

Modulation of Recombinant Human T-type calcium channels by Δ9-tetrahydrocannabinolic acid in vitro

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. {Delta}9-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 {Delta}9-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. 1M 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 M) 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 (10M) 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 M 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.

pharmacology and toxicology↗

bioRxiv2020

Modulation of Human T-type Calcium Channels by Synthetic Cannabinoid Receptor Agonists in vitro

BACKGROUND AND PURPOSEConsumption 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 APPROACHFluorescence-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 RESULTSMDMB-CHMICA and AMB-CHMINACA potently blocked Cav3.2 with IC50 values of 1.5 and 0.74 M respectively. Current inhibition increased from 47 to 80% and 45 to 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 {Delta}9-THC. CONCLUSIONS and IMPLICATIONSSCRAs 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.

pharmacology and toxicology↗

bioRxiv2019

Differential activation of G protein-mediated signalling by synthetic cannabinoid receptor agonists

Synthetic cannabinoid receptor agonists (SCRAs) are new psychoactive substances associated with acute intoxication and even death. However, the molecular mechanisms through which SCRAs may exert their toxic effects remain unclear - including the potential differential activation of G protein subtypes by CB1, a major target of SCRA. We measured CB1-mediated activation of Gs and Gi/o proteins by SCRAs by examining stimulation (PTX-treated) as well as inhibition (non-PTX treated) of forskolin-induced cAMP accumulation in HEK cells stably expressing CB1. Real-time measurements of stimulation and inhibition of cAMP levels were made using a BRET biosensor. We found that the maximum concentration of SCRAs tested (10 M), increased cAMP levels 12 to 45% above that produced by forskolin alone, while the phytocannabinoid THC did not significantly alter cAMP levels in PTX-treated HEK-CB1 cells. All SCRAs had greater potency to inhibit of forskolin-induced cAMP levels than to stimulate cAMP levels. The rank order of potencies for SCRA stimulation of cAMP (Gs) was PB-22 > 5F-MDMB-PICA > JWH-018 > AB-FUBINACA > XLR-11. By contrast, the potency of SCRAs for inhibition of cAMP (Gi/o) was 5F-MDMB-PICA > AB-FUBINACA > PB-22 > JWH-018 > XLR-11. The different rank order of potency of the SCRAs to stimulate Gs-like signalling compared to Gi/o signalling suggests differences in G protein preference between SCRAs. Understanding the apparent differences among these drugs may contribute to unravelling their complex effects in humans.

pharmacology and toxicology↗