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• W2891640176 abstract "Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and nonpsychoactive compounds are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated antiepileptic effects has been from case reports or smaller surveys. The mechanisms for CBD's anticonvulsant effects are unclear and likely involve noncannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating the therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1–1.7 currents, with an IC50 of 1.9–3.8 μm, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of ∼3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade, became more potent at depolarized potentials, and also slowed recovery from inactivation, supporting the idea that CBD binding preferentially stabilizes inactivated Nav channel states. We also found that CBD inhibits other voltage-dependent currents from diverse channels, including bacterial homomeric Nav channel (NaChBac) and voltage-gated potassium channel subunit Kv2.1. Lastly, the CBD block of Nav was temperature-dependent, with potency increasing at lower temperatures. We conclude that CBD's mode of action likely involves 1) compound partitioning in lipid membranes, which alters membrane fluidity affecting gating, and 2) undetermined direct interactions with sodium and potassium channels, whose combined effects are loss of channel excitability. Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and nonpsychoactive compounds are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated antiepileptic effects has been from case reports or smaller surveys. The mechanisms for CBD's anticonvulsant effects are unclear and likely involve noncannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating the therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1–1.7 currents, with an IC50 of 1.9–3.8 μm, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of ∼3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade, became more potent at depolarized potentials, and also slowed recovery from inactivation, supporting the idea that CBD binding preferentially stabilizes inactivated Nav channel states. We also found that CBD inhibits other voltage-dependent currents from diverse channels, including bacterial homomeric Nav channel (NaChBac) and voltage-gated potassium channel subunit Kv2.1. Lastly, the CBD block of Nav was temperature-dependent, with potency increasing at lower temperatures. We conclude that CBD's mode of action likely involves 1) compound partitioning in lipid membranes, which alters membrane fluidity affecting gating, and 2) undetermined direct interactions with sodium and potassium channels, whose combined effects are loss of channel excitability. The cannabis plant is composed of over 100 compounds known as phytocannabinoids (1Lerner M. Marihuana: tetrahydrocannabinol and related compounds.Science. 1963; 140 (17819834): 175-17610.1126/science.140.3563.175Crossref PubMed Scopus (10) Google Scholar). Among these phytocannabinoids, CBD, 2The abbreviations used are: CBDcannabidiolNavvoltage-gated sodium channelTHCΔ9-tetrahydrocannabinolKvvoltage-gated potassium channelGOFgain-of-functionLOFloss-of-functionTTCtetracaineSSFIsteady-state fast inactivation. is of great interest because of its lack of potency on CB1 and CB2 receptors that are thought to mediate psychotropic activity. Interactions with these receptors by yet another cannabinoid, THC, at submicromolar concentrations cause the well known cannabis effects (2Pertwee R.G. The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: Δ9-tetrahydrocannabinol, cannabidiol and Δ9-tetrahydrocannabivarin.Br. J. Pharmacol. 2008; 153 (17828291): 199-21510.1038/sj.bjp.0707442Crossref PubMed Scopus (1215) Google Scholar). Recently, reports of the use of CBD as an anticonvulsant agent have been rapidly increasing (3Devinsky O. Cross J.H. Laux L. Marsh E. Miller I. Nabbout R. Scheffer I.E. Thiele E.A. Wright S. Cannabidiol in Dravet Syndrome Study GroupTrial of cannabidiol for drug-resistant seizures in the Dravet syndrome.N. Engl. J. Med. 2017; 376 (28538134): 2011-202010.1056/NEJMoa1611618Crossref PubMed Scopus (850) Google Scholar); however, to date, there is no consensus on a well defined mode of action for the CBD-mediated antiepileptic effects. cannabidiol voltage-gated sodium channel Δ9-tetrahydrocannabinol voltage-gated potassium channel gain-of-function loss-of-function tetracaine steady-state fast inactivation. Because CBD has a lower affinity for the endocannabinoid receptors than THC (4Straiker A. Dvorakova M. Zimmowitch A. Mackie K. Cannabidiol inhibits endocannabinoid signaling in autaptic hippocampal neurons.Mol. Pharmacol. 2018; 94 (29669714): 743-74810.1124/mol.118.111864Crossref PubMed Scopus (51) Google Scholar), several studies suggest that the anticonvulsant effects of THC and CBD in maximal electroshock (ED50 ∼120 mg/kg, brain concentration = ∼22 μm) and pilocarpine models occur via different mechanisms (5Devinsky O. Cilio M.R. Cross H. Fernandez-Ruiz J. French J. Hill C. Katz R. Di Marzo V. Jutras-Aswad D. Notcutt W.G. Martinez-Orgado J. Robson P.J. Rohrback B.G. Thiele E. Whalley B. et al.Cannabidiol: pharmacology and potential therapeutic role in epilepsy and other neuropsychiatric disorders.Epilepsia. 2014; 55 (24854329): 791-80210.1111/epi.12631Crossref PubMed Scopus (597) Google Scholar, 6Deiana S. Watanabe A. Yamasaki Y. Amada N. Arthur M. Fleming S. Woodcock H. Dorward P. Pigliacampo B. Close S. Platt B. Riedel G. Plasma and brain pharmacokinetic profile of cannabidiol (CBD), cannabidivarin (CBDV), Δ9-tetrahydrocannabivarin (THCV) and cannabigerol (CBG) in rats and mice following oral and intraperitoneal administration and CBD action on obsessive-compulsive behaviour.Psychopharmacology (Berl.). 2012; 219 (21796370): 859-87310.1007/s00213-011-2415-0Crossref PubMed Scopus (224) Google Scholar). Whereas the THC activity is mostly on the CB1 receptor, the anticonvulsant effects of CBD are not. These findings have inspired the growth of CB1- and CB2-independent focused research. Many mechanisms have been proposed for the action of CBD on different systems. CBD acts as an agonist on human TRP channels (3–30 μm), and specifically on TRPV1 (7Ledgerwood C.J. Greenwood S.M. Brett R.R. Pratt J.A. Bushell T.J. Cannabidiol inhibits synaptic transmission in rat hippocampal cultures and slices via multiple receptor pathways.Br. J. Pharmacol. 2011; 162 (20825410): 286-29410.1111/j.1476-5381.2010.01015.xCrossref PubMed Scopus (20) Google Scholar, 8Ibeas Bih C. Chen T. Nunn A.V. Bazelot M. Dallas M. Whalley B.J. Molecular targets of cannabidiol in neurological disorders.Neurotherapeutics. 2015; 12 (26264914): 699-73010.1007/s13311-015-0377-3Crossref PubMed Scopus (330) Google Scholar), which is in part responsible for calcium channel modulation (9Blumenfeld H. Lampert A. Klein J.P. Mission J. Chen M.C. Rivera M. Dib-Hajj S. Brennan A.R. Hains B.C. Waxman S.G. Role of hippocampal sodium channel Nav1.6 in kindling epileptogenesis.Epilepsia. 2009; 50 (18637833): 44-5510.1111/j.1528-1167.2008.01710.xCrossref PubMed Scopus (103) Google Scholar). CBD also inhibits heterologously expressed Cav3.1, Cav3.2, and native neuronal T-type voltage-gated calcium channels (10Ross H.R. Napier I. Connor M. Inhibition of recombinant human T-type calcium channels by Δ9-tetrahydrocannabinol and cannabidiol.J. Biol. Chem. 2008; 283 (18390906): 16124-1613410.1074/jbc.M707104200Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). These low-voltage activated channels also can be blocked by other antiepileptic drugs such as zonisamide (11Porter B.E. Jacobson C. Report of a parent survey of cannabidiol-enriched cannabis use in pediatric treatment-resistant epilepsy.Epilepsy Behav. 2013; 29 (24237632): 574-57710.1016/j.yebeh.2013.08.037Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In addition to Cav channels, CBD inhibits persistent and resurgent sodium currents generated by WT-Nav1.6 channels and the exacerbated versions of these currents in GOF epilepsy-causing Nav1.6-mutants (12Patel R.R. Barbosa C. Brustovetsky T. Brustovetsky N. Cummins T.R. Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol.Brain. 2016; 139 (27267376): 2164-218110.1093/brain/aww129Crossref PubMed Scopus (84) Google Scholar). A recent human clinical trial has indicated that CBD (dosed at 20 mg/kg) is efficacious against drug-resistant seizures in Dravet syndrome (3Devinsky O. Cross J.H. Laux L. Marsh E. Miller I. Nabbout R. Scheffer I.E. Thiele E.A. Wright S. Cannabidiol in Dravet Syndrome Study GroupTrial of cannabidiol for drug-resistant seizures in the Dravet syndrome.N. Engl. J. Med. 2017; 376 (28538134): 2011-202010.1056/NEJMoa1611618Crossref PubMed Scopus (850) Google Scholar). Previous investigations of the cannabis-mediated blockade of Nav channels have determined that CBD (10 μm) significantly decreases action potential frequency in rat CA1 hippocampal neurons and Nav current density in human neuroblastoma cells and mouse cortical neurons (13Hill A.J. Jones N.A. Smith I. Hill C.L. Williams C.M. Stephens G.J. Whalley B.J. Voltage-gated sodium (NaV) channel blockade by plant cannabinoids does not confer anticonvulsant effects per se.Neurosci. Lett. 2014; 566 (24642454): 269-27410.1016/j.neulet.2014.03.013Crossref PubMed Scopus (61) Google Scholar). Other studies have used dissociated hippocampal cultures derived from embryonic day 18 rats to measure toxicity and neuroprotective responses to CBD. These studies indicate that the EC50 of CBD is within the 1–4 μm range while causing neurotoxicity at 33 μm (14Kinney W.A. McDonnell M.E. Zhong H.M. Liu C. Yang L. Ling W. Qian T. Chen Y. Cai Z. Petkanas D. Brenneman D.E. Discovery of KLS-13019, a cannabidiol-derived neuroprotective agent, with improved potency, safety, and permeability.ACS Med. Chem. Lett. 2016; 7 (27096053): 424-42810.1021/acsmedchemlett.6b00009Crossref PubMed Scopus (29) Google Scholar). The transient sodium current through Nav channels initiates action potentials in neurons, skeletal muscles, and cardiac muscles. Any changes to the gating properties of these channels, and subsequently the current passed through them during an action potential, can cause extremely life-limiting conditions that can sometimes be lethal. Both GOF and LOF in sodium channels disrupt electrical signaling (15Ghovanloo M.R. Aimar K. Ghadiry-Tavi R. Yu A. Ruben P.C. Physiology and pathophysiology of sodium channel inactivation.Curr. Top. Membr. 2016; 78 (27586293): 479-50910.1016/bs.ctm.2016.04.001Crossref PubMed Scopus (26) Google Scholar, 16Ghovanloo M.-R. Abdelsayed M. Peters C.H. Ruben P.C. A mixed periodic paralysis and myotonia mutant, P1158S, imparts pH-sensitivity in skeletal muscle voltage-gated sodium channels.Sci. Rep. 2018; 8 (29674667): 630410.1038/s41598-018-24719-yCrossref PubMed Scopus (9) Google Scholar, 17Ghovanloo M.-R. Abdelsayed M. Ruben P.C. Effects of amiodarone and N-desethylamiodarone on cardiac voltage-gated sodium channels.Front. Pharmacol. 2016; 7 (26973526): 39Crossref PubMed Scopus (16) Google Scholar, 18Catterall W.A. Voltage-gated sodium channels at 60: structure, function and pathophysiology.J. Physiol. 2012; 590 (22473783): 2577-258910.1113/jphysiol.2011.224204Crossref PubMed Scopus (475) Google Scholar). In the primary sodium channel isoforms of the central nervous system, Nav1.1, 1.2, 1.3, and 1.6, both GOF and LOF elicit epilepsy syndromes (15Ghovanloo M.R. Aimar K. Ghadiry-Tavi R. Yu A. Ruben P.C. Physiology and pathophysiology of sodium channel inactivation.Curr. Top. Membr. 2016; 78 (27586293): 479-50910.1016/bs.ctm.2016.04.001Crossref PubMed Scopus (26) Google Scholar, 19Estacion M. Gasser A. Dib-Hajj S.D. Waxman S.G. A sodium channel mutation linked to epilepsy increases ramp and persistent current of Nav1.3 and induces hyperexcitability in hippocampal neurons.Exp. Neurol. 2010; 224 (20420834): 362-36810.1016/j.expneurol.2010.04.012Crossref PubMed Scopus (75) Google Scholar, 20Catterall W.A. Sodium channel mutations and epilepsy.in: Noebels J.L. Avoli M. Rogawski M.A. Olsen R.W. Delgado-Escueta A.V. Jasper's Basic Mechanisms of the Epilepsies. 4th Ed. National Center for Biotechnology Information, Bethesda, MD2012Crossref Google Scholar, 21Veeramah K.R. O'Brien J.E. Meisler M.H. Cheng X. Dib-Hajj S.D. Waxman S.G. Talwar D. Girirajan S. Eichler E.E. Restifo L.L. Erickson R.P. Hammer M.F. De novo pathogenic SCN8A mutation identified by whole-genome sequencing of a family quartet affected by infantile epileptic encephalopathy and SUDEP.Am. J. Hum. Genet. 2012; 90 (22365152): 502-51010.1016/j.ajhg.2012.01.006Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). These include relatively mild epilepsies, like benign familial neonatal-infantile seizures, and more severe forms, such as Dravet syndrome (22Heron S.E. Crossland K.M. Andermann E. Phillips H.A. Hall A.J. Bleasel A. Shevell M. Mercho S. Seni M.-H. Guiot M.-C. Mulley J.C. Berkovic S.F. Scheffer I.E. Sodium-channel defects in benign familial neonatal-infantile seizures.Lancet. 2002; 360 (12243921): 851-85210.1016/S0140-6736(02)09968-3Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 23Scalmani P. Rusconi R. Armatura E. Zara F. Avanzini G. Franceschetti S. Mantegazza M. Effects in neocortical neurons of mutations of the Nav1.2 Na+ channel causing benign familial neonatal-infantile seizures.J. Neurosci. 2006; 26 (17021166): 10100-1010910.1523/JNEUROSCI.2476-06.2006Crossref PubMed Scopus (95) Google Scholar, 24Dravet C. The core Dravet syndrome phenotype.Epilepsia. 2011; 52 (21463272): 3-910.1111/j.1528-1167.2011.02994.xCrossref PubMed Scopus (360) Google Scholar) and early-infantile epileptic encephalopathy-13 (25O'Brien J.E. Meisler M.H. Sodium channel SCN8A (Nav1.6): properties and de novo mutations in epileptic encephalopathy and intellectual disability.Front. Genet. 2013; 4 (24194747): 213Crossref PubMed Google Scholar). Compounds that inhibit Nav current have been used extensively for clinical treatment of all of the above hyperexcitability disorders (26George A.L. Inherited disorders of voltage-gated sodium channels.J. Clin. Invest. 2005; 115 (16075039): 1990-199910.1172/JCI25505Crossref PubMed Scopus (286) Google Scholar). In this study, we sought to characterize, in detail, the biophysical effects of CBD on peripheral and central nervous system sodium channel isoforms. Previous reports suggested that Nav currents are inhibited by micromolar concentrations of CBD but lacked detailed concentration-response data and defined potencies. We sought to determine whether CBD has any selectivity across the sodium-channel superfamily. Thus, we measured the concentration dependence of inhibition of inactivated-state sodium channels by CBD. Our results demonstrate that CBD inhibits hNav1.1–1.7 with low micromolar potency (Fig. 1, A and D). To construct the concentration response, individual cells were exposed to single concentrations of CBD; then normalized inhibition at each concentration was pooled and fit with a Hill Langmuir equation, providing IC50 and Hill slopes (Fig. 1A). Representative current traces at approximately IC50 concentrations for each subtype tested are shown in (Fig. 1C). Interestingly, the sodium-current inhibition has very steep Hill slopes of >2 across all subtypes (Fig. 1, A and D). This indicates that CBD does not likely inhibit the channel currents through a 1:1 binding mechanism but instead suggests multiple interactions contribute to this inhibition. Although there is some variation in the IC50 values obtained on each subtype, the steep Hill slopes make IC50 values extremely sensitive to small changes in concentration, which could account in part for this variation of ∼2-fold in IC50. Similar to the hNav channels, CBD also inhibited the mouse Nav1.6 (mNav1.6) current, suggesting that these human IC50 values hold in rodent isoforms (Fig. 1, A and D). The chemical structures of CBD and THC are very similar, with the sole difference being the closure of a ring on THC as opposed to a free hydroxyl group in CBD. Given that this difference is the structural basis for the functional differences between CBD and THC, we tested THC against hNav1.2. Our results suggest that although the potency of the sodium-current inhibition between THC and CBD is similar, the Hill slope associated with THC is less steep (Fig. 1, B and D). This may indicate that THC has some differences in the mechanism of sodium-current inhibition than CBD. We next examined the effects of CBD on Nav channel activation by measuring peak channel conductance at membrane potentials between −120 and +30 mV. We show the effects of 3.3 μm CBD on peak sodium-current densities (Fig. 2A) and a plot of conductance (Fig. 2B). Approximately 90% of the sodium conductance was inhibited. The normalized conductance is plotted against membrane potential, showing that CBD does not induce large changes in either the midpoint (V½) or apparent valence (slope, k) of activation of the available sodium channels (p > 0.05) (Fig. 2C). Therefore, exposure to CBD at this concentration prevents channels from conducting; however, this exposure does not alter the voltage dependence of activation. We next measured the voltage dependence of fast inactivation. The normalized current amplitudes at the test pulse is shown as a function of prepulse voltages (Fig. 2D). The current at the test pulse was inhibited by more than 90%; however, unlike activation, the voltage dependence of steady-state fast inactivation of the remaining current was hyperpolarized by 8.1 mV (p = 0.0002). This indicates CBD increased the propensity for channels to inactivate over the 500-ms prepulse in channels that were not inhibited from opening from rest, suggesting that CBD stabilizes the inactivated state of sodium channels. It was previously shown that 1 μm CBD inhibits the persistent and resurgent sodium currents in form of epilepsy caused by hNav1.6 GOF mutation, N1768D, which displays a noninactivating component (12Patel R.R. Barbosa C. Brustovetsky T. Brustovetsky N. Cummins T.R. Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol.Brain. 2016; 139 (27267376): 2164-218110.1093/brain/aww129Crossref PubMed Scopus (84) Google Scholar). We also found that CBD inhibits the resurgent current induced by including 200 μm β4-peptide to the intracellular solution. Fig. 2E shows that 5 μm CBD inhibits the majority of resurgent currents, which is consistent with CBD preventing channels from opening (Fig. 2A). Next, we sought to establish the concentration dependence of CBD inhibition of the persistent current of the inactivation-deficient N1768D mutant. We found that persistent currents were inhibited at slightly lower concentrations than the peak currents, suggesting there may be interactions between CBD and the open or inactivated states over the course of a 100-ms depolarization (Fig. 2F). We used a protocol to examine state-dependent inhibition across a range of holding potentials where channel inactivation varies (27Kuo C.C. Bean B.P. Slow binding of phenytoin to inactivated sodium channels in rat hippocampal neurons.Mol. Pharmacol. 1994; 46 (7969051): 716-725PubMed Google Scholar). We first held channels at a holding potential of −100 mV where channels are almost all in the resting state, whereas pulsing the channels 180 times at 1 Hz to allow CBD to reach equilibrium. Then we depolarized the holding potential by 10 mV three more times and repeated the pulse train at each voltage (Fig. 3A). We show the fractional block of sodium currents from the last pulse (180th) from each holding potential (Fig. 3B). Fig. 3C shows a plot of the inverse of the apparent IC50 fit with a four-state binding model that used parameters obtained from the Boltzmann fit of the voltage dependence of steady-state fast inactivation. This established that the apparent potency is directly related to the proportion of inactivated channels at different holding potentials. Our results demonstrate that CBD inhibits the sodium current from both rest and inactivated states; however, the potency of CBD is ∼10-fold greater for inhibiting inactivated compared with resting states (Fig. 3C). To assess the time dependence and degree of stabilization of the inactivated state, we then measured the recovery from inactivation of hNav1.6 in the presence of 3.7 μm CBD (Fig. 1A). This was done after depolarizing prepulse durations of 300 ms and 10 s, from a holding potential of −120 mV. These prepulse durations correspond to inactivation recovery from fast and slow inactivated states, respectively. The mean normalized recovery following the prepulse in CBD and control conditions were plotted and fit with a biexponential function (Fig. 3, D and E). The τSlow and fraction of the recovery fit with τSlow are plotted in Fig. 3F, which shows that CBD increases the fraction of recovery that is slow and the time constant of the slow component of recovery from inactivation from 300 ms to 10 s. This indicates that CBD slows the recovery from inactivation, supporting the hypothesis that CBD stabilizes the inactivated states (Fig. 3, D–F). Binding kinetics are typically responsive to changes in temperature, with higher temperatures increasing the rates of compound equilibration. To further investigate the mode of CBD interaction with sodium channels, we measured the observed rates of equilibration (time constant observed, τobs) of inhibition by fitting single exponential decays to inhibition of currents at three different temperatures. We examined the kinetics at concentrations above the IC50 to ensure a clear inhibition signal window to define the τobs (Fig. 4, A–C). Channels were held at −45 mV and pulsed at 1 Hz following a recovery pulse to −150 mV for 60 ms, and CBD was rapidly applied to the cells at different concentrations (Fig. 4A). The fraction of inhibition was normalized against the response in vehicle and plotted against the time elapsed after CBD addition, which was set at t = 0. The inhibition was then fit with a single exponential function to obtain τ and plotted against concentration. We show that τobs saturated at a minimum with increasing concentrations, counter to the prediction of a single two-state ligand-binding reaction, which predicts a continually increasing τobs with increasing compound concentrations (Fig. 4D). This suggests a rate-limiting step in the inhibitory pathway that is not dependent on the concentration of CBD in bulk solution as would be expected for binding to a specific inhibitory site on the channel. Interestingly, the kinetics of CBD inhibition were also found to be more rapid at cooler temperatures (Fig. 4D). This is the opposite of what is expected for a classic inhibitor in a two-state binding model, where the binding and unbinding rate constants, kon and koff, are intrinsically temperature-dependent. These results further suggest that CBD does not inhibit conductance through a direct interaction with a single specific binding site on the channel. We also found that the potency of CBD is increased at the lower temperatures (Fig. 4, E and F). Because CBD inhibition shares a characteristic of classic pore blockers (preference for the inactivated state), we created a pore mutation in the local-anesthetic receptor site in hNav1.1 (F1763A) to determine whether the CBD potency was affected. The Phe1763 residue is part of a well established binding site for many of the most common local anesthetics, including tetracaine (TTC) (28Ragsdale D.S. McPhee J.C. Scheuer T. Catterall W.A. Molecular determinants of state-dependent block of Na+ channels by local anesthetics.Science. 1994; 265 (8085162): 1724-172810.1126/science.8085162Crossref PubMed Scopus (734) Google Scholar). To avoid any impacts on potency being caused by the shifts in stability of inactivation in this mutant (WT-hNav1.1 inactivation V½ = −62.0 ± 0.4 mV, slope = 7.2 ± 0.4, n = 3; F1763A inactivation V½ = −49.3 ± 0.1 mV, slope = 6.4 ± 0.1, n = 17), we measured inhibition from a holding potential of −45 mV where both channels were >50% inactivated. To validate the F1763A-mutant channels, we also measured the potency of TTC and compared the results against WT-hNav1.1, which showed a drop in potency (Fig. 5A). For CBD, the F1763A-mutant channels only caused a slight drop in potency of ∼2-fold (Fig. 5B). Molecular docking using a homology model of hNav1.1 based on the eukaryotic cockroach cryo-EM structure (Navpas) (Fig. S1A) suggested that CBD may in fact bind close to Phe1763. This suggests that Phe1763 is not a primary determinant of CBD inhibition but does not rule out the possibility that CBD may interact with other pore residues. To determine whether CBD could also inhibit other nonhuman Nav channels, we tested it on the bacterial homomeric Nav channel (NaChBac) (Fig. 5C). Interestingly, we found that NaChBac is also blocked by CBD, although with a slightly greater potency (Fig. 5C). Unlike mammalian Nav channels, NaChBac does not inactivate quickly (29Ren D. Navarro B. Xu H. Yue L. Shi Q. Clapham D.E. A prokaryotic voltage-gated sodium channel.Science. 2001; 294 (11743207): 2372-237510.1126/science.1065635Crossref PubMed Scopus (386) Google Scholar). We hypothesized that if the previously observed state dependence of CBD block in hNav channels depends upon the fast-inactivation process, then NaChBac should not show such a state dependence. The potency measured at −100 and −55 mV suggests that state dependence also exists in NaChBac; moreover, this effect may be even more pronounced than in hNav channels (Fig. 5D). This may implicate that CBD is not dependent upon, or selective among, different modes of Nav inactivation. Our findings in the homotetrameric NaChBac raise the question of whether CBD might also inhibit voltage-gated potassium channels, which are also homotetrameric. Our results indicate that CBD also inhibits the Kv2.1 current. Current traces are shown (Fig. 5E). These findings, along with the previous reports of CBD modulation of calcium-channel currents, support the idea that CBD is a polypharmacological inhibitor of voltage-dependent ionic currents (10Ross H.R. Napier I. Connor M. Inhibition of recombinant human T-type calcium channels by Δ9-tetrahydrocannabinol and cannabidiol.J. Biol. Chem. 2008; 283 (18390906): 16124-1613410.1074/jbc.M707104200Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Because CBD nonspecifically inhibits voltage-dependent currents, we sought to establish whether incubation of CBD could affect the channel trafficking to the cell surface over the time scale of our voltage-clamp experiments. To address this question, we assessed membrane-channel distribution in cells incubated overnight with CBD. The results indicate that overnight incubation in 5 μm CBD does not alter sodium channel trafficking and therefore would not affect trafficking on time scales of the voltage-clamp experiments (<20 min of exposure) (Fig. S1, B–D). To determine whether our observations in HEK cells translate to native neuronal voltage-dependent currents, we measured the effects of CBD on human iPSC neurons. First, we established that potency established with manual patch-clamp methodology, using continuous perfusion of compound, correlated with Qube data by measuring the potency of sodium-current inhibition of HEK cells expressing hNav1.2. Fig. 6A shows the mean normalized concentration response plot, which gave an IC50 value similar to the Qube value for hNav1.2. The slightly increased potency in the manual assay can be explained by the temperature dependence data (Fig. 4), because manual voltage clamp was performed at ∼20 °C (room temperature), and the Qube IC50 values were established at 28 °C (Fig. 1A). Consistent with our previous results in HEK cells, both the neuronal sodium and potassium currents were blocked ∼50% by 1 μm CBD as shown by the representative families of current traces in Fig. 6 (D and E). CBD also caused a hyperpolarization of ∼16 mV in the steady-state inactivation in the remaining available Nav channels in iPSCs (p = 0.0031), which was similar to the shifts observed in hNav1.1 in HEK cells (Figs. 2D and 6B). We also measured the rate time constant of open-state fast inactivation at −20 mV, which did not differ significantly before and after CBD perfusion (p > 0.05) (Fig. 6C). To test the effect of IC50 concentrations of CBD on neuronal excitability, we used a modified version of the Hodgkin–Huxley model to simulate a cortical neuron's excitability (30Hodgkin A.L. Huxley A.F. Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo.J. Physiol. 1952; 116 (14946713): 449-47210.1113/jphysiol.1952.sp004717Crossref PubMed Scopus (1354) Google Scholar" @default.
• W2891640176 created "2018-09-27" @default.
• W2891640176 creator A5017211526 @default.
• W2891640176 creator A5029781869 @default.
• W2891640176 creator A5043846225 @default.
• W2891640176 creator A5049605356 @default.
• W2891640176 creator A5088905895 @default.
• W2891640176 creator A5089135560 @default.
• W2891640176 date "2018-10-01" @default.
• W2891640176 modified "2023-10-14" @default.
• W2891640176 title "Inhibitory effects of cannabidiol on voltage-dependent sodium currents" @default.
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