A pharmacological characterization of Cannabis sativa chemovar extracts.

BACKGROUND: Cannabis contains Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) as the primary constituents responsible for pharmacological activity. However, there are numerous additional chemically-related structures to Δ9-THC and CBD that are pharmacologically active and may influence the pharmacological properties of Δ9-THC and CBD. This study chemically characterized the cannabinoid constituents in a series of cannabis chemovar extracts and investigated the potential cannabinoid entourage effect in two behavioral assays. METHODS: Six chemovar extracts were compared to pure Δ9-THC, CBD and morphine for effects on the following behavioral assays in mice: hot plate and tail suspension. The battery of behavioral tests was conducted post intravenous administration of cannabis chemovar extract. Cannabinoid profiles of extracts were analyzed using high performance liquid chromatography. Cannabis extracts were administered at equal doses of Δ9-THC to investigate the role of their cannabinoid profiles in modulating the effects of Δ9-THC. Dose response curves were fit using a log[inhibitor] vs response three parameter model and differences between group means were determined using a one-way ANOVA followed by a post hoc test. RESULTS: Cannabis chemovars tested in this study exhibited substantially different cannabinoid profiles. All chemovars produced dose-dependent immobility in the tail suspension assay and dose-dependent antinociception in the hot plate assay. The maximum antinociceptive effect and ED50 was comparable between cannabis chemovars and Δ9-THC. Two cannabis chemovars produced significantly greater immobility in the tail suspension test, with no significant differences in ED50. CONCLUSIONS: Commercially available cannabis chemovars vary widely in cannabinoid content, but when equalized for Δ9-THC content, they produce similar behavioral effects with two exceptions. These findings provide only limited support for the entourage hypothesis. Further studies are necessary to characterize the nature of these pharmacological differences between cannabis chemovars and pure Δ9-THC.

Crystal structures of Ca2+-calmodulin bound to NaV C-terminal regions suggest role for EF-hand domain in binding and inactivation.

Voltage-gated sodium (NaV) and calcium channels (CaV) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the C-terminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca2+-occupied CaM, bound to the CT of NaV1.5. The structure shows that the C-terminal lobe binds to a site ∼90° rotated relative to a previous site reported for an apoCaM complex with the NaV1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca2+-occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca2+-free and Ca2+-occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related NaV1.4 channel has been shown to undergo Ca2+-dependent inactivation (CDI) akin to CaVs. We present a crystal structure of Ca2+/CaM bound to the NaV1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EF-hand domain and the IQ domain as a possible conformational switch that underlies CDI.

The voltage-gated sodium channel EF-hands form an interaction with the III-IV linker that is disturbed by disease-causing mutations.

Voltage-gated sodium channels (NaV) are responsible for the rapid depolarization of many excitable cells. They readily inactivate, a process where currents diminish after milliseconds of channel opening. They are also targets for a multitude of disease-causing mutations, many of which have been shown to affect inactivation. A cluster of disease mutations, linked to Long-QT and Brugada syndromes, is located in a C-terminal EF-hand like domain of NaV1.5, the predominant cardiac sodium channel isoform. Previous studies have suggested interactions with the III-IV linker, a cytosolic element directly involved in inactivation. Here we validate and map the interaction interface using isothermal titration calorimetry (ITC) and NMR spectroscopy. We investigated the impact of various disease mutations on the stability of the domain, and found that mutations that cause misfolding of the EF-hand domain result in hyperpolarizing shifts in the steady-state inactivation curve. Conversely, mutations in the III-IV linker that disrupt the interaction with the EF-hand domain also result in large hyperpolarization shifts, supporting the interaction between both elements in intact channels. Disrupting the interaction also causes large late currents, pointing to a dual role of the interaction in reducing the population of channels entering inactivation and in stabilizing the inactivated state.

Expression-dependent pharmacology of transient receptor potential vanilloid subtype 1 channels in Xenopus laevis oocytes.

Transient receptor potential vanilloid subfamily member 1 channels are polymodal sensors of noxious stimuli and integral players in thermosensation, inflammation and pain signaling. It has been shown previously that under prolonged stimulation, these channels show dynamic pore dilation, providing a pathway for large and otherwise relatively impermeant molecules. Further, we have shown recently that these nonselective cation channels, when activated by capsaicin, are potently and reversibly blocked by external application of quaternary ammonium compounds and local anesthetics. Here we describe a novel phenomenon in transient receptor potential channel pharmacology whereby their expression levels in Xenopus laevis oocytes, as assessed by the magnitude of macroscopic currents, are negatively correlated with extracellular blocker affinity: small current densities give rise to nanomolar blockade by quaternary ammoniums and this affinity decreases linearly as current density increases. Possible mechanisms to explain these data are discussed in light of similar observations in other channels and receptors.

Extracellular quaternary ammonium blockade of transient receptor potential vanilloid subtype 1 channels expressed in Xenopus laevis oocytes.

Transient receptor potential vanilloid subtype 1 (TRPV1) channels are essential nociceptive integrators in primary afferent neurons. These nonselective cation channels are inhibited by local anesthetic compounds through an undefined mechanism. Here, we show that lidocaine inhibits TRPV1 channels expressed in Xenopus laevis oocytes, whereas the neutral local anesthetic, benzocaine, does not, suggesting that a titratable amine is required for high-affinity inhibition. Consistent with this possibility, extracellular tetraethylammonium (TEA) and tetramethylammonium application produces potent, voltage-dependent pore block. Alanine substitutions at Phe649 and Glu648, residues in the putative TRPV1 pore region, significantly abrogated the concentration-dependent TEA inhibition. The results suggest that large cations, shown previously to enter cells through activated transient receptor potential channels, can also act as channel blockers.

The quaternary lidocaine derivative, QX-314, exerts biphasic effects on transient receptor potential vanilloid subtype 1 channels in vitro.

BACKGROUND: Transient receptor potential vanilloid subfamily member 1 (TRPV1) channels are important integrators of noxious stimuli with pronounced expression in nociceptive neurons. The experimental local anesthetic, QX-314, a quaternary (i.e., permanently charged) lidocaine derivative, recently has been shown to interact with and permeate these channels to produce nociceptive and sensory blockade in animals in vivo. However, little is known about the specific interactions between QX-314 and TRPV1 channels. Thus, the authors examined the mechanistic basis by which QX-314 acts on TRPV1 channels. METHODS: The authors conducted an in vitro laboratory study in which they expressed TRPV1 and TRPV4 channels in Xenopus laevis oocytes and recorded cation currents with the two-electrode voltage clamp method. They used confocal microscopy for Ca²âº imaging in TRPV1 transient transfected tsA201 cells. Drugs were bath-applied by gravity perfusion. Statistical analyses were performed using Student t test, ANOVA, and post tests as appropriate (P < 0.05). RESULTS: QX-314 activated TRPV1 channels at 10, 30, and 60 mM (0.4 ± 0.1%, 3.5 ± 1.3%, and 21.5 ± 6.9% of normalized peak activation, respectively; mean ± SEM; n = 12) but not TRPV4 channels (P < 0.001). Activation by QX-314 was blocked by the TRPV1 antagonist, capsazepine (100 µM). QX-314 (60 mM) activation and blockade by capsazepine was also demonstrated in Ca²âº imaging studies on TRPV1-expressing tsA201 cells. At subactivating concentrations (less than 1 mM), QX-314 potently inhibited capsaicin-evoked TRPV1 currents with an IC50 of 8.0 ± 0.6 µM. CONCLUSIONS: The results of this study show that the quaternary lidocaine derivative QX-314 exerts biphasic effects on TRPV1 channels, inhibiting capsaicin-evoked TRPV1 currents at lower (micromolar) concentrations and activating TRPV1 channels at higher (millimolar) concentrations. These findings provide novel insights into the interactions between QX-314 and TRPV1 and may provide an explanation for the irritant properties of intrathecal QX-314 in mice in vivo.