Modulation of Recombinant Human T-type calcium Channels by Δ9-tetrahydrocannabinolic acid in vitro
Department of Biomedical Sciences, Macquarie University, Sydney, NSW, Australia
#Corresponding Author: Mark Connor, Department of Biomedical Sciences, Macquarie University, NSW, Australia. Phone: +61298502719. Email: mark.connor@mq.edu.auAbstract
Introduction
Low 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 Methods
We 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.
Results
THCA 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.
Discussion
THCA 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.
Article notes
Competing Interest Statement
The authors have declared no competing interest.
Introduction
Cannabis sativa has been used for thousands of years as a medicinal plant for the relief of pain and seizures1–3. There is a growing body of evidence suggesting cannabinoids are beneficial for a range of clinical conditions including pain4 inflammation 5 epilepsy 6–8, sleep disorders9, symptoms of multiple sclerosis10, and other conditions 11,12. Phytocannabinoids, derived from diterpenes in Cannabis, have a range of distinct pharmacological actions 13. The best characterised 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 non-psychotomimetic and proposed to have potential therapeutic effects in a broad range of neurological disorders 16–18 and which has been shown to inhibit signalling via at both CB1 and CB2 receptors 16,19,20 Cannabinoids can also interact with a wide variety of ion channels including Transient Receptor Potential (TRP) channels, ligand gated channels and voltage dependent channels 21. 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 22. THC and CBD inhibit evoked currents through recombinant 5-HT3 receptors independently of cannabinoid receptors 23; and THC caused significant inhibition of native receptor in mammalian neurons 24. THC and CBD also potentiate glycine receptor function through an allosteric mechanism25.
Voltage gated ion channels also modulated by phytocannabinoids. CBD and cannabigerol (CBG) are able to inhibit voltage-gated Na (NaV) channels in vitro 26,27 which has been suggested to contribute to anti-epileptic effects. A wide range of cannabinoids have been shown to modulate T type ICa channels, including endogenous cannabinoids anandamide and N-arachidonoyl dopamine 28, endogenous lipoamino acids such as N-arachidonoyl 5-HT and N-arachidonoyl glycine, as well as the phytocannabinoids THC and CBD 29–31. These effects are thought to be mediated by direct interaction of the ligands with channels, as the experiments were done in cells do not express cannabinoid receptors.
Voltage-dependent Ca2+ channels are categorized into three families: L-type channels (CaV1), the neuronal N-, P/Q- and R-type channels (CaV2) and the T-type channels (CaV3) 32. T-type Ca2+ channels (CaV3), can activate upon small depolarizations of the plasma membrane and are present in many excitable cells 33 where they are critical for neuronal firing and neurotransmitter release and physiological processes such as slow wave sleep 34–36. 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 37–39. T-type calcium are encoded by three CaV3 subunits (CaV3.1, CaV3.2, and CaV3.3). Much smaller membrane depolarizations are required for opening, and at typical neuronal resting membrane potentials a significant number of T-type channels are inactivated. They markedly differ in some of their electrophysiological properties 40,41. The most notable of these are that CaV3.1 and CaV3.2 have much faster activation and inactivation kinetics, than CaV3.3 42,43.
Δ9-tetrahydrocannabinolic acid (THCA) is the precursor of THC in Cannabis. THCA is acutely decarboxylated to form THC by heating44. Importantly, THCA has low affinity at CB1 receptor 45 but interestingly, THCA has been reported to have neuroprotective, anti-inflammatory, and immunomodulatory effects 44, raising the possibility of therapeutic activity without unwanted psychotropic effects.
Previous work from our lab have shown that THC and CBD modulate T-type calcium channels 46, however, there is no information surrounding the effects of other phytocannabinoids including THCA on these channels. The aim of this work was to characterize THCA modulatory effects on the T-type calcium channels and compare its effects with THC. If THCA could also modulate CaV3 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 (ThermoFisher) were stably transfected with pcDNA5/FRT/TO vector encoding human CaV3.1 (NM 018896.4), CaV3.2 (NM 021098.2), or CaV3.3 (NM 021096.3) (GenScript). The integration of this vector to the Flp-In site was mediated by pOG44, Flp-recombinase expression vector pOG44, which was co-transfected 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 were performed using 150μg/mL Hygromycin B Gold (InvivoGen) as per kill curve (data not shown). Flp-In T-Rex 293 HEK cells (expressing CaV3.1, CaV3.2, or CaV3.3) do not express CB1 or CB2 receptors47. Cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, and 1% penicillin-streptomycin. HEK-CaV3.1, CaV3.2, and CaV3.3 were passaged in media with 15μg/ml Blasticidin (InvivoGen) and 100μg/ml Hygromycin. Cells were maintained in 5% CO2 at 37°C in a humidified atmosphere. Channel expression was induced by adding 2μg/mL tetracycline.
Electrophysiology
Currents in Flp-In T-REx 293 HEK cells expressing CaV3.1, CaV3.2, or CaV3.3 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) (1MgCl2, 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 mM and 2mM, 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 via a custom-built gravity driven micro perfusion system. Before running drugs in test of activation and inactivation of CaV3 channels, external control solution was applied about 5 minutes in each experiment to observe in the absence of drugs, vehicle controls itself have no effects on CaV3 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 (Table1).
This voltage step was repeated at 12 second intervals (1 sweep) for at least 3 mins to achieve a stable peak ICa. Perfusion was then switched to 10μM drugs until maximum inhibition was attained (determined when no more ICa inhibition was observed after 3 successive sweeps). Finally, drug was “washed out” by switching perfusion back to control solution consisting of external buffer with vehicle control.
In order 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 48). In these experiments, THC (1μM) produced a change in fluorescence of 12.8 ± 1.2 %. In parallel experiments, THCA (1μM) did not significantly alter the fluorescence (1.0 ± 0.6%). pEC50 for THC in this assay is about 300nM 48 and 100nM THC produces a robust change in fluorescence 49, the lack of effect of THCA at 1μM 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. Daily dilutions were made fresh before each use in external recording solution to give a final vehicle concentration of 0.1%.
Statistics
Data are reported as the mean and standard error of at least 6 independent experiments. Concentration response curves, steady state inactivation and activation were generated by fitting data to a Boltzmann sigmoidal equation in Graph Pad Prism 8. Statistical significance for comparing the V0.5 values of activation and inactivation were determined using one-way ANOVA comparing values of V0.5 calculated for individual experiments. In order to compare the changes in the time to peak and decay time of deactivation, unpaired t-test was used. All values are reported as mean ± standard errors and were fitted with a modified Boltzmann equation: I = [Gmax*(Vm-Erev)]/[1+exp((V0.5 act-Vm)/ka)], where Vm is the test potential, V0.5 act is the half-activation potential, Erev is the reversal potential and Gmax is the maximum slope conductance. Steady-state inactivation curves were fitted using Boltzmann equation: I =1/ (1 + exp ((Vm - Vh)/k)), where Vh is the half-inactivation potential and k is the slope factor.
Results
Superfusion of THCA and THC on CaV3 inhibited the peak of the ICa evoked by a step from −100mV to −30 mV (Fig 1). At a concentration of 10 μM, THC or THCA blocked the current amplitude of CaV3.1 almost completely, and inhibited CaV3.2 by 56 ± 2% and 53 ± 4 % respectively (n=6). 10μM THC did not affect CaV3.3 ICa while 10μM THCA inhibited CaV3.3 by 43% ± 2 (Fig 1A). CaV3.1 was inhibited by THC and THCA with pEC50 6 ± 0.7 and 5.6 ± 0.4 respectively (Fig1B). The effects of THCA and THC on CaV3.1, 3.2 and 3.3 currents are illustrated in Fig 2 (THCA) and Fig 3 (THC), the drug effects did not readily reverse on washout.
THC and THCA effects on activation and inactivation kinetics
We examined the voltage-dependence of activation CaV3 channels by repetitively stepping cells from −75mV to 50mV from a holding potential of −100mV. 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 CaV3.1 was affected by THCA, notably it increased current amplitudes for depolarisations between −75mV to −45mV and inhibited current amplitude for depolarisations between −35 and 50mV (Fig 4B). THCA produced a significant hyperpolarizing shift in the half activation potential of CaV3.1; these shifts were not seen with time-matched vehicle controls (Table 1). Steady-state inactivation, where cells were voltage clamped at potentials between (−110 mV and −20 mV) for 2s before current were evoked by stepping them to test potentials of −30mV, showed that THCA also caused large shifts in steady-state inactivation of CaV3.1 (Fig 4C). Activation and inactivation changes for cells exposed to vehicle alone for 5 min were less than −1mV (Table1). Using the same protocols, it was found that THCA also shifted CaV3.2 half activation to negative potentials and caused a larger shift in half inactivation of CaV3.2 (Fig 4D). THCA caused small positive shift and significant negative shift in half activation and inactivation of CaV3.3 (Fig 4E).
1μM THC also affected steady state inactivation and activation of CaV3.1. THC shifted half activation and inactivation of CaV3.1 to more negative voltages (Fig 5C). THC shifted half activation of CaV3.2 to negative potentials and caused significant negative shift in inactivation of CaV3.2 (Fig 5D). THC at 10μM had no effect on the half activation of CaV3.3 however THC negatively shifted the half inactivation of CaV3.3 significantly (Fig 5E).
Effects of THC and THCA on time to peak and kinetics of current deactivation of CaV3 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. 1μM THCA slowed deactivation of CaV3.1 (Fig. 7A, C), however, the deactivation of both CaV3.2 (Fig 7E) and CaV3.3 (not shown) were unaffected by THCA at 10μM. THC slowed deactivation of CaV3.1 (1 μM, Figure 7B, D) and CaV3.2 (10 μM, Figure 7F) but THC did not change deactivation of CaV3.3 (not shown).
Discussion
The major finding of this study is that THCA inhibited T-type calcium channels with most potent effects on CaV3.1. THC also most potently affected CaV3.1, and CaV3.2 was moderately inhibited by both drugs at 10μM with less inhibition of CaV3.3. THCA shifted the half activation and inactivation voltages of CaV3.1 and CaV3.2 to more negative potentials, THC behaved in a similar fashion. THCA and THC also slowed the time constant deactivation of CaV3.1 however at 10μM only THC slowed the deactivation of CaV3.2. Both THCA and THC produced modest shifts in CaV3.3 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 CB2 15 and causes a distinctive intoxication via activation of the CB1 50 receptors, however, studies of affinity of THCA for the CB1 receptor have produced different results, but studies where THCA was tested for THC produced by THCA degradation, there was little activity attributable to THCA51,52. Verhoeckx et al examined THC and THCA affinity using radioligand binding assay and determined that THC had greater affinity compared to THCA at CB144. 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 had similar affinity for CB1, did not examine the potential contamination of THCA with THC51. 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 sate inactivation of the CaV3.1 and CaV3.2 channels to more negative potentials, reducing the number of channels that can open when the cell is depolarised, preventing their transition to an inactivated state. THCA had the same effect as THC on CaV3.1 steady state activation, causing a hyperpolarising shift so that when the cells are depolarised, more channels are available for activation. THCA effects on CaV3.2 kinetics were less pronounced than THC, causing a more negative shift in both activation and inactivation of CaV3.2. The effects of THCA and THC on in half activation of CaV3.3 was not significant. Conversely, THCA and THC caused a significant shift in steady state inactivation of CaV3.3. Interestingly, THCA and THC potentiated CaV3.1 current evoked by modest depolarization and then inhibited current amplitudes following stronger depolarisation. These data suggest that THCA and THC may increase the initial depolarizing drive produced by CaV3.1 in some circumstances, despite the overall inhibitory effects on the channels.
The results with THC are in good agreement with previous studies from our lab. In general, THC showed modestly higher potency to inhibit CaV3.2 and CaV3.3 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 (−100mV vs −86mV)28.
Both THC and THCA have been reported to activate TRPA1 and TRPV2 channels and showed the similar antagonist activity on TRPV1 and TRPM821,22,55. 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, 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 ICa is a possible contributor. 58,59
ABBREVIATIONS
- ICa
- Voltage gated calcium channel current
- THC
- Δ9-tetrahydrocannabinol
- THCA
- Δ9-tetrahydrocannabinolic acid
- CBD
- Cannabidiol
- TRP
- Transient Receptor Potential
Acknowledgment
We would like to thank Lambert initiative for gift of THC and THCA. We would also like to thank Shivani Sachdev for performing some of the experiments with THCA and THC on CB1 receptor signalling.
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 MC and CB. SM was supported by Macquarie University International Research Excellence Scholarship. CB was supported by Macquarie University Research Fellowship.