The effects of phytocannabinoids and synthetic cannabinoids on T-type calcium channels in vitro

T-type calcium channels are expressed in many excitable cells and a wide range of tissues. They activate upon small depolarizations of the membrane, allowing a surge of calcium entry into the cells that often create the beginnings of an action potential. They are classified by three subtypes, CaV3.1, CaV3.2, and CaV3.3 and have distinct electrophysiological and molecular properties. T-type Calcium Channels play a critical activity in many physiological and medical conditions including epilepsy, and pain. 

Cannabinoids have shown to be used medicinally in the treatment of many conditions. The studies presented in this thesis were undertaken to further the understanding the molecular pharmacology of cannabinoids, and how they can modulate T-type calcium channels. Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) have modulated T-type current however information is limited surrounding the effects of other phytocannabinoids or Synthetic Cannabinoids (SCs) on these channels. The aim of this thesis is to look at how different cannabinoids modulate T-type Ca+2 channels. To do this study, fluorescence-based and electrophysiology assays were employed to screen a library of phytocannabinoids and SCs to observe their ability to modulate T-type calcium channels expressed in HEK293 cells. Most potent compounds were considered to examine in detail using patch clamp recordings. 

Initial FLIPR screening of phytocannabinoids on all three channels showed that most of these compounds inhibited CaV3.1 and CaV3.2 with less inhibition on CaV3.3. Cannabigerolic acid (CBGA) and cannabidivarin (CBDV) were the potent compounds inhibiting CaV3.1 and CaV3.2. Δ9- tetrahydrocannabinol acid (THCA) showed the effective inhibition on CaV3.3. THC showed a complex response on the channels in FLIPR assay, so this drug potentiated calcium entry by the activation of CaV3.1 and CaV3.2. THC and THCA inhibited CaV3.1 and CaV3.2 current amplitudes at relevant concentrations. THC and THCA shifted half activation and inactivation of CaV3.1 and 3.2 significantly to negative potentials. THC and THCA differed in modulation of CaV3.3 gating. THC shifted the half inactivation of CaV3.3 to negative potentials significantly but THCA had small effects on CaV3.3 kinetics. THC prolonged deactivation of CaV3.1 and CaV3.2 however, THCA prolonged CaV3.1 deactivation kinetics. CBGA potently inhibited calcium current of CaV3 channels and affected half activation and inactivation of CaV3.1/CaV3.2 significantly but it did not affect half activation of CaV3.3. CBG also inhibited CaV3.1 calcium conductance and it caused a huge negative shift in the inactivation of CaV3.1. CBDV reduced CaV3.1 current amplitude also shifted half inactivation CaV3.1 to negative directions. The effects of novel SCs tested on T-type calcium channels indicated that most of these compounds had potent activity on CaV3.1 and CaV3.2 rather than CaV3.3. The initial screening experiments for the inhibition of T-type calcium channels using FLIPR assay was cross validated with electrophysiology. MDMB-CHMICA and AMB-CHMINACA potently blocked CaV3.2 with IC50’s of 1.5 and 0.74 μM, respectively. Both SCs (1 μM) and THC and CBD (10 μM) showed significant increase in channel block of CaV3.2 in a prolonged slow inactivated state, suggesting that they preferentially bind to this conformation. 

Phytocannabinoids and SCs both showed inhibitory modulation effects on T-type calcium channels with different potencies depending on channels subtypes. Most of compounds affected steady state inactivation at physiological membrane potentials, with some also affecting channel activation. These results extend our knowledge of cannabinoids beyond previously reported effects of THC and CBD and suggest that they have future potential for creating drugs with high target specificity, improved therapeutic properties in pain and epilepsy via T-type calcium channel modulation under the right conditions.