Modulation of Recombinant Human T-Type Calcium Channels by Δ⁹-Tetrahydrocannabinolic Acid In Vitro

Abstract

Introduction: Low voltage-activated T-type calcium channels (T-type I_Ca), Ca_V3.1, Ca_V3.2, and Ca_V3.3, 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 I_Ca; however, there is no information about functional activity of most phytocannabinoids on T-type calcium channels, including Δ⁹-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_V3.1, 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_V3.1 with pEC₅₀s 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_V3.1, and both drugs prolonged Ca_V3.1 deactivation kinetics. THCA (10 μM) inhibited Ca_V3.2 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_V3.2. THC prolonged the deactivation time of Ca_V3.2, while THCA did not. THCA inhibited the peak current of Ca_V3.3 by 43%±2% (10 μM) but did not notably affect Ca_V3.3 channel activation or inactivation; however, THC caused significant hyperpolarizing shift in Ca_V3.3 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.

Introduction

Cannabis sativa has been used for thousands of years as a medicinal plant for the relief of pain and seizures.^1–3 There is a growing body of evidence suggesting that cannabinoids are beneficial for a range of clinical conditions, including pain,⁴ inflammation,⁵ epilepsy,^6–8sleep disorders,⁹ muscle spasticity associated with multiple sclerosis,¹⁰ and other conditions.^11,12 Phytocannabinoids, derived from diterpenes in Cannabis, have a range of distinct pharmacological actions.¹³ The best characterized phytocannabinoid is Δ⁹-tetrahydrocannabinol (THC), well known for its psychoactive effects,¹⁴ mediated by its activation of the cannabinoid receptor CB₁.¹⁵ The next most abundant phytocannabinoid is cannabidiol (CBD), which is nonpsychotomimetic and proposed to have potential therapeutic effects in a broad range of neurological disorders.^16–18 CBD inhibits signaling at both CB₁ and CB₂ receptors^16,19,20 and may also act at a range of other G protein-coupled receptors (GPCR), including 5-HT1A, GPR55, and GPR18.²¹

Cannabinoids can also interact with a wide variety of ion channels, including transient receptor potential (TRP) channels TRPV1–4, TRPA1, and TRPM8; ligand-gated channels; and voltage dependent channels.²² THC was identified as a prototypic agonist of TRPA1, and subsequently, it and other phytocannabinoids have been reported to activate or inhibit many other TRP channels.²³ THC and CBD inhibit evoked currents through recombinant 5-HT3 receptors independent of cannabinoid receptors²⁴; and THC caused significant inhibition of native receptors in mammalian neurons.²⁵ THC and CBD also potentiate glycine receptor function through an allosteric mechanism.²⁶

Voltage-gated ion channels are also modulated by phytocannabinoids. CBD and cannabigerol are able to inhibit voltage-gated Na (Na_V) channels in vitro,^27,28 which have been suggested to contribute to antiepileptic effects. A wide range of cannabinoids have been shown to modulate T-type I_Ca channels, including endogenous cannabinoids anandamide and N-arachidonoyl dopamine,²⁹ endogenous lipoamino acids such as N-arachidonoyl 5-HT and N-arachidonoyl glycine, as well as the phytocannabinoids THC and CBD.^30–32 These effects are thought to be mediated by direct interaction of the ligands with channels, as the experiments were done in HEK293 cells, which do not express cannabinoid receptors.

Voltage-gated calcium channels are categorized into three families: L-type channels (Ca_V1), the neuronal N-, P/Q-, and R-type channels (Ca_V2), and the T-type channels (Ca_V3).³³ T-type calcium channels (Ca_V3) can activate upon small depolarizations of the plasma membrane and are present in many excitable cells³⁴ where they are critical for neuronal firing and neurotransmitter release and physiological processes such as slow-wave sleep.^35–37 Cells expressing T-type calcium channels are involved in epilepsy, pain, and other diseases, and there is substantial evidence supporting the idea that modulating T-type calcium channels is a potential therapeutic option in these conditions.^38–40 T-type calcium channels are composed of three Ca_V3 alpha subunits (Ca_V3.1, Ca_V3.2, and Ca_V3.3). Much smaller membrane depolarizations are required for opening, and at typical neuronal resting membrane potentials, a significant number of T-type calcium channels are inactivated. They markedly differ in some of their electrophysiological properties.^41,42 The most notable of these are that Ca_V3.1 and Ca_V3.2 have much faster activation and inactivation kinetics than Ca_V3.3.^43,44

Δ⁹-tetrahydrocannabinolic acid (THCA) is the precursor of THC in Cannabis. THCA is acutely decarboxylated to form THC by heating.⁴⁵ Importantly, THCA has low affinity at CB₁ receptor⁴⁶ but, interestingly, THCA has been reported to have neuroprotective, anti-inflammatory, and immunomodulatory effects,⁴⁵ raising the possibility of therapeutic activity without unwanted psychotropic effects.

Previous work from our laboratory have shown that THC and CBD modulate T-type calcium channels⁴⁷; however, there is no information about the effects of other phytocannabinoids, including THCA on these channels. This work aimed to characterize THCA modulatory effects on the T-type calcium channels and compare its effects with THC. If THCA could also modulate Ca_V3 channels, this may provide potential therapeutic activity in pain and other disorders involving the peripheral nervous system without having psychoactive properties.

Methods

Transfection and cell culture

Flp-In T-REx 293 HEK cells (Thermo Fisher) were stably transfected with pcDNA5/FRT/TO vector encoding human Ca_V3.1 (NM 018896.4), Ca_V3.2 (NM 021098.2), or Ca_V3.3 (NM 021096.3) (GenScript). The integration of this vector to the Flp-In site was mediated by Flp-recombinase expression vector pOG44, which was cotransfected as per manufacturer's recommendation (ratio 9:1). Transfections were done using FuGENE HD transfection agent (Promega) at ratio 1:4 (w/v) total DNA: FuGENE HD. Selection of stably expressing cells was performed using 150 μg/mL Hygromycin B Gold (InvivoGen) as per kill curve (data not shown). Flp-In T-Rex 293 HEK cells (expressing Ca_V3.1, Ca_V3.2, or Ca_V3.3) do not express CB₁ or CB₂ receptors.⁴⁸ Cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin–streptomycin. HEK-Ca_V3.1, Ca_V3.2, and Ca_V3.3 were passaged in media with 15 μg/mL Blasticidin (InvivoGen) and 100 μg/mL Hygromycin. Cells were maintained in 5% CO₂ at 37°C in a humidified atmosphere. Channel expression was induced by adding 2 μg/mL tetracycline.

Electrophysiology

Currents in Flp-In T-REx 293 HEK cells expressing Ca_V3.1, Ca_V3.2, or Ca_V3.3 channels were recorded in the whole-cell configuration of the patch clamp method at room temperature. Dishes were constantly perfused with external recording solution containing (in mM) (1 MgCl₂, HEPES, 10 Glucose, 114 CsCl, 5 BaCl₂) (pH to 7.4 with CsOH, osmolarity=330). 2–4 MΩ recording electrodes were filled with internal solutions containing (in mM):126.5 CsMeSO₄,11 EGTA, 10 HEPES adjusted to pH 7.3 with CsOH. Immediately before use, internal solution was added to a concentrated aliquot of GTP and ATP to yield final concentrations of 0.6 and 2 mM, respectively. All recordings were measured using an Axopatch 200B amplifier in combination with Clampex 9.2 software (Molecular Devices, Sunnyvale, CA). All data were sampled at 5–10 kHz and filtered at 1 kHz. All currents were leak subtracted using P/N4 protocol.

THC and THCA were prepared daily from concentrated DMSO stocks and diluted in external solution to appropriate concentrations and applied locally to cells using a custom-built gravity driven microperfusion system. Before running drugs in test of activation and inactivation of Ca_V3 channels, external control solution was applied about 5 min in each experiment to observe in the absence of drugs; vehicle controls themselves have no effects on Ca_V3 channel kinetics. All solutions did not exceed 0.1% DMSO, and this concentration of vehicle had no effect on current amplitude or on half activation and half-inactivation potentials (Table 1).

Table 1.

The Effects of Δ⁹-Tetrahydrocannabinolic Acid and Δ⁹-Tetrahydrocannabinol on the Parameters of Steady-State Activation and Inactivation of Ca_V3 Channels

Drug	CaV3	Change in V0.5
		Activation	Inactivation
THCA	3.1	−7±2****	−8±3****
THCA	3.2	−4.8±2**	−6±2***
THCA	3.3	3±1	−4±1*
THC	3.1	−7±2****	−8±1****
THC	3.2	−5±1***	−9±2****
THC	3.3	−1±0.7	−8±1****
No drug	3.1	0.7±0.2	−1±0.2
No drug	3.2	−1±0.4	−0.6±0.2
No drug	3.3	−0.5±0.2	−1±0.5

Cells expressing recombinant Ca_V3 channels were voltage clamped at −100 mV then stepped to potential above −75 mV (activation) stepped every 5 mV. The results of peak currents were fitted to a Boltzmann sigmoidal equation. Changes in the voltage for half activation/inactivation (V_0.5) of the curve are reported in Table. No drug represents time dependent changes under our recording conditions.

Unpaired Student's t test: ^****p<0.0001; ^***p<0.001; ^**p<0.01; ^*p<0.02.

This voltage step was repeated at 12-sec intervals (1 sweep) for at least 3 min to achieve a stable peak I_Ca. Perfusion was then switched to 10 μM drugs until maximum inhibition was attained (determined when no more I_Ca inhibition was observed after three successive sweeps). Finally, drug was “washed out” by switching perfusion back to control solution consisting of external buffer with vehicle control.

To test whether THCA used contained an appreciable amount of THC, we examined the activity of THCA in a fluorescent assay of CB₁-dependent activation of inwardly rectifying K channels (described in detail in Ref.).⁴⁹ In these experiments, THC (1 μM) produced a change in fluorescence of 12.8%±1.2%. In parallel experiments, THCA (1 μM) did not significantly alter the fluorescence (1.0%±0.6%). pEC₅₀ for THC in this assay is about 300 nM,⁴⁹ and 100 nM THC produces a robust change in fluorescence⁵⁰; the lack of effect of THCA at 1 μM suggests that there was no significant contamination of THCA with THC.

Drugs and reagents

The THC and THCA used in this study were a kind gift from University of Sydney's Lambert Institute for Cannabinoid Therapeutics. Drugs (30 mM) were aliquoted and stored as concentrated stocks in DMSO and stored at −30°C.

Statistics

Data are reported as the mean and standard error of at least six independent experiments. Concentration response curves, steady-state inactivation, and activation were generated by fitting data to a Boltzmann sigmoidal equation in GraphPad Prism 8. Statistical significance for comparing the V_0.5 values of activation and inactivation was determined using unpaired Student's t-test comparing values of V_0.5 calculated for individual experiments. To compare the changes in the time to peak and time constant deactivation, one-way ANOVA was performed, followed by a Tukey post-test. All values are reported as mean±standard errors and were fitted with a modified Boltzmann equation: I=[Gmax*(V_m-E_rev)]/[1+exp((V_0.5act-V_m)/k_a)], where V_m is the test potential, V_{0.5 act} is the half-activation potential, E_rev is the reversal potential, and Gmax is the maximum slope conductance. Steady-state inactivation curves were fitted using Boltzmann equation: I=1/(1 + exp ((V_m - V_h)/k)), where V_h is the half-inactivation potential and k_a is the slope factor.

Results

Superfusion of THCA and THC on Ca_V3 inhibited the peak of the I_Ca evoked by a step from −100 mV to −30 mV (Fig. 1). At a concentration of 10 μM, THC or THCA blocked the current amplitude of Ca_V3.1 almost completely and inhibited Ca_V3.2 by 56%±2% and 53%±4%, respectively (n=6). THC (10 μM) did not affect Ca_V3.3 I_Ca, while 10 μM THCA inhibited Ca_V3.3 by 43%±2% (Fig. 1A). Ca_V3.1 was inhibited by THC and THCA with pEC_50s 6±0.7 and 5.6±0.4, respectively (Fig. 1B). The effects of THCA and THC on Ca_V3.1, 3.2, and 3.3 currents are illustrated in Figure 2 (THCA) and Figure 3 (THC); the drug effects did not readily reverse on washout.

FIG. 1.

Effects of 10 μM THCA and THC on T-type calcium channel current and concentration response curve for THCA and THC effects on Ca_V3. (A) Peak I_Ca was elicited by a step from −100 mV to −30 mV; Ca_V3.1 current was almost completely inhibited by10 μM application of THC and THCA. THC (10 μM) and THCA blocked Ca_V3.2 calcium current about 52%±3%. Ca_V3.3 current was not affected by 10 μM THC but THCA decreased Ca_V3.3 calcium current by 43%±4%. (B) Concentration response curves were created to determine the potency of these compounds at Ca_V3.1. Each point represents the mean±SEM of 6 cells. THC, Δ⁹-tetrahydrocannabinol; THCA, Δ⁹-tetrahydrocannabinolic acid.

FIG. 2.

THCA effects on Ca_V3 current amplitude. Each trace represents the current elicited by a voltage step from −100 mV to −30 mV. (A) 1 μM THCA inhibited calcium current of Ca_V3.1. (B) Time course of inhibition and degree of reversibility THCA inhibition of Ca_V3.1 are illustrated. (C) THCA (10 μM) inhibited calcium current of Ca_V3.2. (D) Time course of inhibition and degree of reversibility THCA inhibition of Ca_V3.2 are illustrated. (E) THCA at 10 μM inhibited current amplitude of Ca_V3.3. (F) The inhibition of Ca_V3.3 by 10 μM THCA was not washed out as shown in time course inhibition of Ca_V3.3.

FIG. 3.

THC effects on Ca_V3 current amplitude. Recording of Ca_V3 channel was made as outlined under experimental procedures. Each trace represents the current elicited by a voltage step from −100 mV to −30 mV. (A) 1 μM THC inhibited Ca_V3.1 calcium current. (B) Inhibitory effects of THC on Ca_V3.1 were not washed out using external solution. (C) THC inhibited Ca_V3.2 calcium current at 10 μM. (D) A reversal of THC (10 μM) inhibition of Ca_V3.2 was not seen by washing. (E) THC at 10 μM had little effect on calcium current of Ca_V3.3. (F) Inhibition by THC at 10 μM was not reversible.

THC and THCA effects on activation and inactivation kinetics

We examined the voltage dependence of activation Ca_V3 channels by repetitively stepping cells from −75 mV to 50 mV from a holding potential of −100 mV. After a control I/V relationship was generated, it was repeated after 5 min perfusion of THCA (Fig. 4A). The voltage dependence of activation for Ca_V3.1 was affected by THCA; notably, it increased current amplitudes for depolarizations between −75 and −45 mV and inhibited current amplitude for depolarizations between −35 and 50 mV (Fig. 4B). THCA produced a significant hyperpolarizing shift in the half activation potential of Ca_V3.1; these shifts were not seen with time-matched vehicle controls (Table 1). Steady-state inactivation, where cells were voltage clamped at potentials between (−110 and −20 mV) for 2 sec before current was evoked by stepping them to test potentials of −30 mV, showed that THCA also caused large shifts in steady-state inactivation of Ca_V3.1 (Fig. 4C). Activation and inactivation changes for cells exposed to vehicle alone for 5 min were less than −1 mV (Table 1). Using the same protocols, it was found that THCA also shifted Ca_V3.2 half activation to negative potentials and caused a larger shift in half inactivation of Ca_V3.2 (Fig. 4D). THCA caused a small positive shift and significant negative shift in the half activation and inactivation of Ca_V3.3 (Fig. 4E).

FIG. 4.

THCA effects on the activation and inactivation of Ca_V3 channels. (A) Current-Voltage (I−V) relationship showing the activation of Ca_V3.1 from a holding membrane potential of −100mV in the absence and presence of 1 μM THCA. The peak current amplitude is plotted, (B) example traces of this experiment illustrating the effects of 1 μM THCA at testing membrane potential of −51 mV and −22 mV: current is enhanced at lower test potentials then inhibited at more depolarized potentials. (C) 1 μM THCA affected half activation and inactivation of Ca_V3.1 expressed in HEK293 to negative potentials. (D) Steady-state activation and inactivation of Ca_V3.2 expressed in HEK293 in the presence and absence of THCA showed a significant shift in inactivation of Ca_V3.2; however, 10 μM THCA created slight shift in activation of Ca_V3.2. (E) THCA caused a small positive shift in activation kinetics of Ca_V3.3 and a small negative shift in inactivation of Ca_V3.3. Each data point represents the mean±SEM of six cells.

THC (1 μM) also affected steady-state inactivation and activation of Ca_V3.1. THC shifted half activation and inactivation of Ca_V3.1 to more negative voltages (Fig. 5C). THC shifted half activation of Ca_V3.2 to negative potentials and caused significant negative shift in inactivation of Ca_V3.2 (Fig. 5D). THC at 10 μM had no effect on the half activation of Ca_V3.3; however, THC negatively shifted the half inactivation of Ca_V3.3 significantly (Fig. 5E).

FIG. 5.

Effects of THC on the voltage dependence of Ca_V3 activation and inactivation. (A) Current-Voltage (I-V) relationship showing the activation of Ca_V3.1 from a holding membrane potential of −100mV in the absence and presence of 1 μM THC. (B) The peak current amplitude is plotted at testing membrane potential of −51 mV and −22 mV. Example traces of this experiment illustrating the effects of 1 μM THC: current is enhanced at lower test potentials then inhibited at more depolarized potentials. (C) THC effect on Ca_V3 channel kinetics when HEK293 cells were voltage clamped at −100 mV, depolarized to 50 mV from −75 mV showed that 1 μM THC shifted activation and inactivation of Ca_V3.1 to negative potentials significantly. (D) 10 μM THC effects on activation and inactivation kinetics of Ca_V3.2 indicated that steady-state inactivation was shifted to negative potentials significantly; however, THC caused −5mV shift in activation kinetics of Ca_V3.2. (E) 10 μM THC effects on activation and inactivation kinetics of Ca_V3.3; THC had no effects on steady-state activation; however, THC caused significant shift in inactivation kinetics of Ca_V3.3. Each data point represents the mean±SEM of six cells.

Effects of THC and THCA on time to peak and kinetics of current deactivation of Ca_V3 channels

THC and THCA caused no significant changes on time to peak on any of the T-type channels at any voltage (Fig. 6A–F). The effects of THC and THCA on deactivation of currents elicited during the standard I/V protocol were measured by fitting a monophasic exponential to the inward “tail” currents that resulted immediately following the voltage step. THCA (1 μM) slowed deactivation of Ca_V3.1 (Fig. 7A, C); however, the deactivation of both Ca_V3.2 (Fig. 7E) and Ca_V3.3 (not shown) was unaffected by THCA at 10 μM. THC slowed deactivation of Ca_V3.1 (1 μM, Fig. 7B, D) and Ca_V3.2 (10 μM, Fig. 7F), but THC did not change deactivation of Ca_V3.3 (not shown).

FIG. 6.

THCA and THC effects on time to peak of Ca_V3 channels. The plots illustrate the time to peak of current Ca_V3 before and after 5 min superfusion of THC and THCA. THCA had no significant effects on time to peak of (A) Ca_V3.1, (B) Ca_V3.2, and (C) Ca_V3.3. No shift was seen to those in parallel THC experiments where solvent alone was super fused for (D) Ca_V3.1, (E) Ca_V3.2, and (F) Ca_V3.3. Each point represents the mean±SEM of at least six cells (ANOVA t-test p>0.05).

FIG. 7.

THCA and THC effects on Ca_V3 time constant deactivations and tail current. Cells expressing Ca_V3 channels were stepped repetitively from −75 to 50 mV. (A) THCA showed significant change in time constant deactivation of Ca_V3.1 (One-way ANOVA p<0.0001). (B) THC had significant changes in time constant deactivation of Ca_V3.1 across a range of membrane potentials. (C) 1 μM THCA prolonged deactivation of Ca_V3.1 showing in example trace of tail current from I-V current relationships. (D) Example traces of tail current for Ca_V3.1 showed that 1 μM THC slowed deactivation of Ca_V3.1. (E) Representative traces illustrated that THCA at 10 μM did not affect Ca_V3.2, and (F) 10 μM THC slowed deactivation of Ca_V3.2.

Discussion

The major finding of this study is that THCA inhibited T-type calcium channels with most potent effects on Ca_V3.1. THC also most potently affected Ca_V3.1, and Ca_V3.2 was moderately inhibited by both drugs at 10 μM with less inhibition of Ca_V3.3. THCA shifted the half activation and inactivation voltages of Ca_V3.1 and Ca_V3.2 to more negative potentials; THC behaved in a similar manner. THCA and THC also slowed the time constant deactivation of Ca_V3.1; however, at 10 μM only THC slowed the deactivation of Ca_V3.2. Both THCA and THC produced modest shifts in Ca_V3.3 inactivation without any effects on the deactivation kinetics. The presence of the carboxylic acid moiety in THCA does not result in substantial differences in modulation of T-type calcium channel compared with THC.

THC has higher affinity to cannabinoid receptors CB₁ and CB₂¹⁵ and causes a distinctive intoxication through activation of the CB₁⁵¹ receptors; however, studies of affinity of THCA for the CB₁ receptor have produced different results, but in studies where THCA was tested for THC produced by THCA degradation, there was little activity attributable to THCA.^46,52 Verhoeckx et al. examined THC and THCA affinity using radioligand binding assay and determined that THC had greater affinity compared to THCA at CB₁.⁴⁵ However, Ahmed et al. reported no affinity of THCA on CB₁⁵³ while Husni et al., found some activity on CB₁,⁵⁴ while the one study that reported THC and THCA which had similar affinity for CB₁ did not examine the potential contamination of THCA with THC.⁵² We tested the activity of our THCA in a membrane potential assay in AtT20 cells expressing CB₁ receptors. THC (1 μM) produced a significant hyperpolarization of the cells, as reported many times previously, while THCA did not produce changes in fluorescence, suggesting that in our experiments, THC contamination of the THCA was insignificant.

In current study, THCA like THC shifted steady-state inactivation of the Ca_V3.1 and Ca_V3.2 channels to more negative potentials, reducing the number of channels that can open when the cell is depolarized, preventing their transition to an inactivated state. THCA had the same effect as THC on Ca_V3.1 steady-state activation, causing a hyperpolarizing shift so that when the cells are depolarized, more channels are available for activation. THCA causes a significant negative shift in both the activation and inactivation of Ca_V3.2, which is less pronounced than THC. The effects of THCA and THC on half activation of Ca_V3.3 were not significant. Conversely, THCA and THC caused a significant shift in steady-state inactivation of Ca_V3.3. Interestingly, THCA and THC potentiated Ca_V3.1 current evoked by modest depolarization and then inhibited current amplitudes following stronger depolarization. These data suggest that THCA and THC may increase the initial depolarizing drive produced by Ca_V3.1 in some circumstances, despite the overall inhibitory effects on the channels.

The results with THC are in good agreement with previous studies from our laboratory. In general, THC showed modestly higher potency to inhibit Ca_V3.2 and Ca_V3.3 in the study of Ross et al.; this can be attributed to the subtle different recording conditions where potency was determined in cells voltage clamped at slightly more depolarized potentials (−100 mV vs. −86 mV).⁴⁷

Both THC and THCA have been reported to activate TRPA1 and TRPV2 channels and showed the similar antagonist activity on TRPV1 and TRPM8.^22,23,55 Together with the results of our study, these data show that THCA and THC generally behave in a similar manner for ion channel modulation, but they have very different activity on cannabinoid GPCR. The very limited permeability of THCA to cross the blood–brain barrier suggests a potential role as a drug for treatment of pain and inflammation in the periphery,⁵⁶ and THCA has been shown to reduce inflammation in the gut.⁵⁷ While the mechanism(s) underlying this are still unknown, inhibition of T-type I_Ca is a possible contributor.^58,59 Our study has provided the first characterization of the interactions between THCA and human Ca_V3 channels, providing another potential mechanism of cannabinoid action that needs to be considered when interpreting the actions of these drugs in complex systems.

Abbreviations Used

CBD

cannabidiol

GPCR

G protein-coupled receptors

I _Ca

voltage-gated calcium channel current

THC

Δ⁹-tetrahydrocannabinol

THCA

Δ⁹-tetrahydrocannabinolic acid

TRP

transient receptor potential

Authors' Contribution

S.M. designed, performed, and analyzed experiments and wrote the article. M.C. and C.B. contributed to the conception, design, analysis of experiments, and writing of article. M.S. created the cell lines used in experiments.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported, in part, by a grant from Sydney Vital Translational Cancer Research Center to M.C. and C.B. S.M. was supported by Macquarie University International Research Excellence Scholarship. C.B. was supported by Macquarie University Research Fellowship.

Cite this article as: Mirlohi S, Bladen C, Santiago M, Connor M (2022) Modulation of recombinant human T-type calcium channels by Δ⁹-tetrahydrocannabinolic acid in vitro, Cannabis and Cannabinoid Research 7:1, 34–45, DOI: 10.1089/can.2020.0134.