Use of cannabinoid compounds for inhibiting potassium ion channels

Abstract

Disclosed herein are methods of inhibiting potassium ion channels, e.g., TWIK-related arachidonic acid-activated K+ channels, with cannabinoid compounds.

Description

USE OF CANNABINOID COMPOUNDS FOR INHIBITING POTASSIUM ION CHANNELS

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Patent Application No. 63 / 699,238, filed September 26, 2024, which is herein incorporated by reference in its entirety.

[0003] ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0004] This invention was made with Government support under Grant No. GM 145869, awarded by National Institutes of Health. The Government has certain rights in the invention.

[0005] BACKGROUND OF THE INVENTION

[0006] 1. FIELD OF THE INVENTION

[0007] The field generally relates to inhibiting or reducing K+channel activity with cannabinoid compounds.

[0008] 2. DESCRIPTION OF THE RELATED ART

[0009] Phytocannabinoids are hydrophobic phenolic compounds produced by cannabis plants. Over one hundred phytocannabinoids have been identified, with delta-9- tetrahydrocannabinol (THC) and cannabidiol (CBD) among the most prevalent. THC is the primary psychoactive cannabinoid that underlies the euphoria-inducing effects of cannabis. The effects of THC are largely mediated by partial agonism of CB1 and CB2 G-protein coupled receptors (ECso about 50 nM). CBD is the predominant nonpsychoactive cannabinoid and is an effective antiepileptic approved by regulatory agencies in multiple jurisdictions for the treatment of seizures in Lennox-Gastaut syndrome, Dravet’s syndrome, and tuberous sclerosis complex. In contrast to THC, the molecular basis for the antiepileptic effects of CBD is unknown. CBD is not a CB1 or CB2 receptor agonist; rather, it negatively modulates GPR55 and CB1 receptors (ICso about 500 nM). Like other classic anticonvulsants, CBD inhibits voltage-gated sodium channels (ICso about 2-10 pM). However, the relevance of these targets is debated, as CBD’s antiepileptic effects are observed in slice recordings at concentrations <100 nM and in treated patients at about 50-100 nM (as measured in plasma or estimated in brain tissue). CBD also modulates other ion channels, for example activating TRPV1-4 (ECso about 1-4 pM), TRPA1 (ECso 110 nM), and Kv7.2 / 7.3 (ECso 200 nM), and inhibiting BK (ICso 280 nM), but the importance of these channels to CBD pharmacology is similarly unverified.

[0010] TRAAK (TWIK-related arachidonic acid-activated K+channel) is a mechanosensitive potassium-selective ion channel belonging to the two-pore domain (K2P) K+ion channel family. TRAAK is localized to axon initial segments and nodes of Ranvier, where it controls the resting membrane potential and facilitates action potential repolarization, thereby enabling high frequency spiking. Gain-of-function TRAAK mutations cause epileptic and neurodevelopmental disorders in humans, with four variants reported in FHEIG (facial dimorphism, hypertrichosis, epilepsy, intellectual disability, and gingival outgrowth) syndrome and one in Rolandic epilepsy.

[0011] TRAAK displays low leak activity under resting conditions and is highly activated by increasing membrane tension. Conformational equilibrium between up and down states of transmembrane helix 4 (TM4) from each subunit of the dimeric channel underlies mechanical activation. At low tension, the TM4 down state predominates, creating membrane-facing fenestrations above TM4 that connect the channel cavity to the surrounding membrane inner leaflet. In this conformation, lipid acyl chains access the channel cavity through the open fenestrations to block conduction. Leak activity is due to rare spontaneous delipidation of the TM4 down state, resulting in brief openings. Increased membrane tension promotes upward movement of the TM4s due to changes in the shape of the channel that are energetically favored by tension. This movement of TM4 seals the lateral fenestrations, preventing lipid block, and results in longer duration and higher conductance mechanically-gated openings. Similar TM4 up and down states have been observed in the related mechanosensitive K2Ps TREK-1 and TREK-2, though gating in these channels also involves conformational changes in the K+-coordinating selectivity filter.

[0012] SUMMARY OF THE INVENTION

[0013] In some embodiments, the present invention is directed to a method of reducing or inhibiting a potassium ion channel, which comprises administering to the potassium ion channel a cannabinoid compound that has the following structural formula:wherein R1 is H, OH, Ra, or RaOH, wherein Ra is an alkyl having 1-5, preferably 1-3, carbon atoms; R2 is H or an alkyl having 1-3 carbon atoms; R3 is an alkyl or alkenyl having 1-10, preferably 2-10, more preferably 3-10, carbon atoms; R4 and R5 are each independently H, an alkyl or alkenyl having 1-5, preferably 1-3, carbon atoms, or R4 and R5 together are a bond or a carbon atom and thereby form a 5- or 6-membered ring, which may be substituted or unsubstituted; and wherein Ring A may be substituted or unsubstituted and 0-3 ring atom bonds may be a double bond. In some embodiments, the cannabinoid compound is selected from the group consisting ofR4 is 2A3-prop-l-ene; and / or R5 is H. In some embodiments, the cannabinoid compound is selected from the group consisting ofembodiments, R3 is an alkyl having 3-8 carbon atoms. In some embodiments, R3 is an alkyl having 5-7 carbon atoms. In some embodiments, the potassium ion channel is a mechanosensitive potassium-selective ion channel belonging to the two-pore domain (K2P) K+ion channel family. In some embodiments, the potassium ion channel is a TWIK -related arachidonic acid-activated K+channel. In some embodiments, the subject suffers from epilepsy (e.g., Rolandic epilepsy) and / or seizures. In some embodiments, the subject has a gain-of-function TWIK-related arachidonic acid-activated K+channel mutation (e.g., TRAAK G165E, TRAAK P259L, TRAAK A270P, TRAAK A198E, TRAAK G158D mutations).

[0014] In some embodiments, the present invention is directed to a method of reducing elevated TWIK-related arachidonic acid-activated K+channel activity in a subject, which comprises administering to the potassium ion channel a cannabinoid compound that has the following structural formula:wherein R1 is H, OH, Ra, or RaOH, wherein Ra is an alkyl having 1-5, preferably 1-3, carbon atoms; R2 is H or an alkyl having 1-3 carbon atoms; R3 is an alkyl or alkenyl having 1-10, preferably 2-10, more preferably 3-10, carbon atoms; R4 and R5 are each independently H, an alkyl or alkenyl having 1-5, preferably 1-3, carbon atoms, or R4 and R5 together are a bond or a carbon atom and thereby form a 5- or 6-membered ring, which may be substituted or unsubstituted; and wherein Ring A may be substituted or unsubstituted and 0-3 ring atom bonds may be a double bond. In some embodiments, the cannabinoid compound is selected from the group consisting ofR4 is 2A3-prop-l-ene; and / or R5 is H. In some embodiments, the cannabinoid compound is selected from the group consisting ofembodiments, R3 is an alkyl having 3-8 carbon atoms. In some embodiments, R3 is an alkyl having 5-7 carbon atoms. In some embodiments, the potassium ion channel is a mechanosensitive potassium-selective ion channel belonging to the two-pore domain (K2P) K+ion channel family. In some embodiments, the potassium ion channel is a TWIK -related arachidonic acid-activated K+channel. In some embodiments, the subject suffers from epilepsy (e.g., Rolandic epilepsy) and / or seizures. In some embodiments, the subject has a gain-of-function TWIK-related arachidonic acid-activated K+channel mutation (e.g., TRAAK G165E, TRAAK P259L, TRAAK A270P, TRAAK A198E, TRAAK G158D mutations).

[0015] In some embodiments, the present invention is directed to a cannabinoid compound for use in the treatment of a disease or disorder caused by elevated TWIK- related arachidonic acid-activated K+channel activity, wherein the cannabinoid compound has the following structural formula:wherein R1 is H, OH, Ra, or RaOH, wherein Ra is an alkyl having 1-5, preferably 1-3, carbon atoms; R2 is H or an alkyl having 1-3 carbon atoms; R3 is an alkyl or alkenyl having 1-10, preferably 2-10, more preferably 3-10, carbon atoms; R4 and R5 are each independently H, an alkyl or alkenyl having 1-5, preferably 1-3, carbon atoms, or R4 and R5 together are a bond or a carbon atom and thereby form a 5- or 6-membered ring, which may be substituted or unsubstituted; and wherein Ring A may be substituted orunsubstituted and 0-3 ring atom bonds may be a double bond. In some embodiments, the cannabinoid compound is selected from the group consisting of. , is H or CH3;R4 is 2A3-prop-l-ene; and / or R5 is H. In some embodiments, the cannabinoid compound is selected from the group consisting ofembodiments, R3 is an alkyl having 3-8 carbon atoms. In some embodiments, R3 is an alkyl having 5-7 carbon atoms. In some embodiments, the potassium ion channel is a mechanosensitive potassium-selective ion channel belonging to the two-pore domain (K2P) K+ion channel family. In some embodiments, the potassium ion channel is a TWIK -related arachidonic acid-activated K+channel. In some embodiments, the disease or disorder is epilepsy (e.g., Rolandic epilepsy) and / or seizures. In some embodiments, the elevated TWIK-related arachidonic acid-activated K+channel activity is the result of a gain-of-function TWIK-related arachidonic acid-activated K+channel mutation (e.g., TRAAK G165E, TRAAK P259L, TRAAK A270P, TRAAK A198E, TRAAK G158D mutations).

[0016] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description explain the principles of the invention.

[0017] DESCRIPTION OF THE DRAWINGS

[0018] This invention is further understood by reference to the drawings wherein:

[0019] FIG. lA to FIG. I : CBD inhibits mechanosensitive K+ channels. (FIG. 1A)Structure of cannabidiol (CBD). (FIG. IB) Representative excised patch recording from a TRAAK-expressing cell perfused with increasing concentrations of CBD. Currents are recorded at 0 mV in a ten-fold K+ gradient. (FIG. 1C) Representative current-voltage relationship from TRAAK containing excised patch treated with increasing concentrations of CBD. Alternating shapes were used for visual clarity. (FIG. ID) TRAAK, (FIG. IE) TREK-1, (FIG. IF) TREK-2, and (FIG. 1G) overlaid dose-response curves. Data from individual patches are plotted with different shapes. Solid lines arefits to four parameter logistic curves and 95% confidence intervals shaded. (FIG. 1H) Comparison of fit IC50 values (TRAAK: 145 ± 4 nM, TREK-1 : 1.53 ± 0.06 pM, and TREK -2: 1.27 ± 0.04 pM (mean ± sem, n=9, 6, and 5 patches, respectively)).Differences assessed with Brown-Forsythe and Welch Annova with Dunnet correction for multiple comparisons, ****p<0.0001, *p=0.0295, n.s. not significant. (FIG. II) Representative current-voltage relationship and (FIG. 1 J) average percent inhibition from whole-cell recordings of TRAAK in response to 1 and 10 pM CBD at 0 mV.

[0020] FIG. 2A to FIG. 2E: Potency of TRAAK inhibition is correlated with cannabinoid hydrophobicity. Representative recording (left) and dose response curve (right) from excised patch of TRAAK treated with increasing concentrations of (FIG. 2A) CBN, (FIG. 2B) CBDP, and (FIG. 2C) CBDV. Data from individual patches are plotted with different shapes. Temporally discontinuous portions of the recording are indicated with gaps separating current trace. (FIG. 2D) Overlaid dose-response curves (from left to right, CBDP, CBD 2nd at 10'8and 3rd at 10'6, CBN 3rd at 10'7and 2nd between 10'7and 10'6, CBDV), and (FIG. 2E) comparison of fit IC50 values (CBD: 145 ±4 nM CBN: 107 ± 5 nM, CBDP: 17 ± 3 nM, and CBDV: 5.58 ± 0.34 pM (mean ± sem, n=9, 4, 4, and 4 patches, respectively). Differences assessed with Brown-Forsythe and Welch Annova with Dunnet correction for multiple comparisons. **p<0.01, ****p<0.0001, n.s. not significant.

[0021] FIG. 3A to FIG. 3G: CBD inhibits gain-of-function TRAAK mutants. Inhibition of (FIG. 3 A) TRAAK A270P, (FIG. 3B) TRAAK A198E, (FIG. 3C) TRAAK G165R, (FIG. 3D) TRAAK G165E, (FIG. 3E) TRAAK P259L, and (FIG. 3F) TRAAK G158D by CBD. Top, middle, and bottom are representative current-voltage relationship and current recording from an excised patch, and dose response curve, respectively. Data from individual patches are plotted with different shapes. (FIG. 3F) Overlaid doseresponse curves and (FIG. 3G) comparison of fit IC50 values (wildtype: 145 ± 4 nM, P259L: 156 ± 8 nM, G165E: 162 ± 8 nM, G165R: 356 ± 18 nM, A198E: 496 ± 26 nM, A270P: 1.12 ± 0.04 pM, and G158D: 1.03 ± 0.22 pM (mean ± sem, n=9, 4, 4, 6, 7, 8 and5 patches, respectively). Differences assessed with Brown-Forsythe and Welch Annova with Dunnet correction for multiple comparisons, ****p<0.0001, n.s. not significant.

[0022] FIG. 4A to FIG. 4E: Mechanical activation of TRAAK reduces CBD efficacy.(FIG. 4A) Representative excised patch recording of TRAAK in response to CBD before and after treatment with methyl-P-cyclodextrin to chemically increase membrane tension. (FIG. 4B) Comparison of percent inhibition by 1 pM CBD before and aftertreatment with methyl-P-cyclodextrin (m-P-CD). Percent inhibition was calculated relative to the current prior to delivery of each dose of 1 pM CBD. (FIG. 4C) Currents (top) recorded in response to steps of negative pressure application (bottom) at 500 nM (left) and (FIG. 4D) 1 pM CBD (right). (FIG. 4E) Current versus pressure relationships from a representative recording before CBD treatment, during treatment with increasing concentrations of CBD, and after washout.

[0023] FIG. 5: Observed structural states of mechanosensitive K2Ps. Model for CBD inhibition of mechanosensitive K+channels. CBD binds in the channel cavity to sterically occlude ion conduction. CBD preferentially binds to TM4-down states over TM4-up states.

[0024] FIG. 6A and FIG. 6B: Comparison of CBD inhibition on disease-causing TRAAK mutants. (FIG. 6A) Overlaid dose-response curves. From left to right at top middle section of the curves are Wild-type, P259 which is slightly overlapped by G165E, G165R, A198E, A270P, G158D. .(FIG. 6B) comparison of fit ICso values (wildtype: 145 ± 4 nM, P259L: 156 ± 8 nM, G165E: 162 ± 8 nM, G165R: 356 ± 18 nM, A198E: 496 ± 26 nM, A270P: 1.12 ± 0.04 pM, and G158D: 1.03 ± 0.22 pM (mean ± sem, n=9, 4, 4, 6, 7, 8 and 5 patches, respectively). Differences assessed with Brown-Forsythe and Welch Annova with Dunnet correction for multiple comparisons, ****p<0.0001, n.s. not significant.

[0025] FIG. 7A to FIG. 7F: CBD inhibition reduces mechanical activation of TRAAK. (FIG. 7A to FIG. 7E) Example current traces from TRAAK-containing excised patches in response to negative pressure at varying concentrations of CBD ranging from (FIG. 7A) 0 pM, (FIG. 7B) 500 nM, (FIG. 7C) 1 pM, (FIG. 7D) 10 pM, and (FIG. 7E) washout following 10 pM CBD. (FIG. 7F) Example plot of data from 0-10 pM.

[0026] FIG. 8 A to FIG. 8C: CBD treatment does not alter membrane tension. (FIG. 8 A) Example images of membrane patches with -10 and -50 mmHg in 0 pM CBD (top) and 50 pM CBD (bottom). (FIG. 8B) Example plot of tension measurement from one patch in 0 pM CBD and 50 pM CBD, * correspond to the images shown in (FIG. 8 A). (FIG. 8C) Comparison of tension generated at matching pressure steps. Data from 10 paired recordings from three different patches are shown (p = 0.58, two-tailed paired t-test, not significant).

[0027] FIG. 9 A and FIG. 9B: CBD inhibition of Danio rerio TREK-1. (FIG. 9 A) Representative current-voltage relationship from a Danio rerio TREK-1 containing patch treated with increasing concentrations of CBD. (FIG. 9B) Dose response curve. Datafrom individual patches are plotted with different shapes. Solid lines are fits to four parameter logistic curves with 95% confidence intervals shaded. IC50 = 11.1 ± 1.3 pM (mean ± sem, n=4 patches).

[0028] FIG. 10A to FIG. 10D: Cryo-EM data processing pipeline for TREK-1 :CBD. (FIG. 10 A, FIG. 10B) Representative micrograph and initial cryo-EM data processing from each dataset, (FIG. 10C) cryo-EM data processing pipeline from merged particle stack, and (FIG. 10D) representative 2D class averages and final reconstructed map from final particle stack.

[0029] FIG. HA to FIG. 1 ID: Cryo-EM structure validation for TREK-1 :CBD. Final sharpened map colored by local resolution and viewed from (FIG. 11 A) the membrane plane and (FIG. 1 IB) the cytoplasmic side view angle distribution, (FIG. 11C) View angle distribution of final particle stack. (FIG. 1 ID) Fourier shell correlation (FSC) versus resolution between half maps from final refinement. At 0.7, the curves from left to right are: No Mask, Spherical, Loose, Corrected, Tight.

[0030] Color versions of the Figures may be found in U.S. Application No. 63 / 699,238, and also Docter, et al. Cannabinoid inhibition of mechanosensitive K+ channels. bioRxiv. 2024 Dec 10:2024.12.09.627564. doi: 10.1101 / 2024.12.09.627564, which is herein incorporated by reference in its entirety.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] Cannabidiol (CBD) is a prominent non-psychoactive small molecule produced by cannabis plants. While CBD is used clinically as an antiepileptic, its relevant molecular targets are unknown. Here, we show CBD and other cannabinoids are potent inhibitors of the mechanosensitive two-pore domain K+(K2P) channels TRAAK (KCNK4), TREK-1 (KCNK2), and TREK-2 (KCNK10), which are involved in action potential propagation. More planar, hydrophobic cannabinoids are better channel inhibitors, with cannabinol (CBN) and cannabidiphorol (CBDP) showing higher — and cannabidivarin (CBDV) lower — affinity for TRAAK than CBD. Four distinct gain-of-function TRAAK mutants that cause epilepsy and the neurodevelopmental syndrome FHEIG retain sensitivity to cannabinoid inhibition. A TREK- 1 : CBD cryo-EM structure shows that CBD binds in the channel cavity to sterically occlude ion conduction. These results demonstrate that cannabinoids are potent inhibitors of mechanosensitive K2Ps and reveal the mechanistic basis for channel block. The antiepileptic effects of CBD arise from K2P inhibition and indicate that cannabinoids may effectively treat TRAAK channelopathies in humans.

[0033] Given the physiological contribution of TRAAK to action potential propagation, implication of TRAAK in epileptic disorders, and demonstrated channel inhibition by hydrophobic lipid acyl chains, we asked whether CBD modulates TRAAK — and related K2P channel — activity. Here, we show cannabinoids are potent blockers of mechanosensitive K2Ps and elucidate the mechanistic basis of channel inhibition.

[0034] We first investigated cannabinoid inhibition of TRAAK channels in excised patches. CBD potently inhibited TRAAK, with effects evident at 1-10 nM, full inhibition at about 1 pM, and an ICso about 145 nM (FIGS. 1 A-D). Preliminary experiments using plastic perfusion components resulted in lower apparent potency, similar to effects reported in a study of CBD activation of Kv7.2 / 7.3 channels and attributed to cannabinoid absorption to plastics. Final experiments were therefore performed with all glass components. We next asked if the related mechanosensitive K2Ps TREK-1 and TREK-2 were similarly cannabinoid-sensitive. All three channels were inhibited by CBD, but TREK-1 and TREK-2 were about 10-fold less sensitive than TRAAK (ICsos 1.5 and 1.3 pM, respectively, FIGS. 1D-H). TREK-2 showed a steeper response profile and larger Hill-coefficient (1.3, 1.5, and 3.8 for TRAAK, TREK-1, and TREK -2, respectively), which suggests TREK -2 may have a unique CBD binding mode and / or stoichiometry.

[0035] In excised inside-out patch experiments, CBD has direct access to the intracellular side of the channels (FIGS. 1 A-H). To test whether CBD can inhibit TRAAK from extracellular side, we conducted whole-cell recordings from cultured cells. TRAAK remained strongly inhibited by extracellular CBD (FIG. II, J). While conducting these experiments, we found bath perfusion readily activated TRAAK, likely due to shear forces that increase membrane tension. We used focal perfusion and low solution flow rates to minimize mechanical channel activation, but cannot exclude residual mechanical effects contributing to the lower apparent sensitivity of TRAAK to extracellular compared to intracellular CBD (FIGS. ID, J).

[0036] CBD consists of a bicyclic head group and hydrocarbon tail (FIG. 1 A). To identify the chemical features of CBD important for channel inhibition, we evaluated several related phytocannabinoids with select structural differences. Cannabinol (CBN), an oxidized derivative of A9-THC, has with the same hydrocarbon tail as CBD but a different, tricyclic head structure (FIG. 2A). The additional aromatization in CBN increases head planarity and eliminates rotational freedom of motion relative to CBD. CBN is about 33% more potent than CBD (IC50 107 nM), suggesting that the entropiccost of ordering the bicyclic head of CBD limits affinity. Cannabidiphorol (CBDP) and cannabidivarin (CBDV) share the same bicyclic head structure as CBD, but possess hydrocarbon tails with two additional or two fewer methylene units, respectively. These changes in hydrocarbon tail length have dramatic effects on potency. Compared to CBD, the more hydrophobic CBDP is about 10-fold more potent (IC50 17 ± 3 nM) while the less hydrophobic CBDV is about 40-fold less potent (5.58 ± 0.34 pM, FIGS. 2B-D). CBDV still fully inhibits TRAAK channels at high (20 pM) concentration. This demonstrates that increasing planarity or hydrophobicity among related cannabinoids increases inhibitor potency.

[0037] Gain-of-function TRAAK mutations have been identified in human disease and functional studies of K2Ps. The mechanistic basis for channel activation by several of these mutations is known and involves stabilization of either leak- or mechanically gated-like open states. We evaluated five TRAAK gain-of-function mutants to better understand CBD’s mechanism of action and to explore the susceptibility of diseasecausing channel variants to cannabinoid inhibition.

[0038] We first considered the FHEIG-inducing mutations TRAAKA27OP and TRAAKAI98E. These mutants are highly activated under low-tension basal conditions, with open probabilities well over 0.9, because they mimic the effect of high membrane tension on wild-type channels. Both mutations shift the conformational equilibrium from the TM4-down, lipid-blocked / leak open state to TM4-up, mechanically gated-like open conformations. We reasoned that if CBD inhibits TRAAK by competing with lipid acyl chains to block the channel cavity in the TM4-down state, these mutants will show diminished sensitivity to inhibition. Indeed, TRAAKA27OP and TRAAKAI98E were about 8-fold and about 4-fold less sensitive than wild-type TRAAK to CBD (IC50 = 1.12 ± 0.04 pM and 496 ± 26 nM, respectively, FIGS. 3 A,B, FIGS. 6A,B). For both mutants, high (10 pM) concentrations of CBD fully inhibited channel activity.

[0039] We next considered three mutants, TRAAKGI65R, TRAAKGI65E, and TRAAKP259L, recently identified in cases of FHEIG (G165R, G165E) and Rolandic epilepsy (P259L). All three mutants retained high sensitivity to CBD (FIGS. 3C-E), with TRAAKGI65E and TRAAKP259L statistically indistinguishable (IC50 = 162 ± 8 and 156 ± 8 nM, respectively) and TRAAKGI65R only modestly reduced compared to wild-type (IC50 = 356 ± 18 nM) (FIGS. 6A,B). This suggests these mutations activate TRAAK to a lesser degree or in a manner distinct from the FHEIG-causing mutations TRAAKA27OP and TRAAKAI98E.

[0040] Finally, we considered TRAAKGISSD, a pan-activating K2P mutation. This mutation is activated under basal conditions with an open probability of about 0.7. G158 points into the channel cavity towards the lipid binding site; introduction of the negatively charged aspartic acid electrostatically disfavors lipid block, shifting the conformational equilibrium towards a TM4-down, leak-like open state under basal conditions. We reasoned that if CBD, like lipids, binds in the channel cavity to block conduction, this mutant would be less sensitive to inhibition. The response of TRAAKoi58Dto CBD treatment was found to be different from wild-type TRAAK in two ways. First, the channel was about 7.1-fold less sensitive to CBD with an ECso = 1.03 ± 0.22 pM (FIGS. 3E-G). Second, CBD showed lower efficacy against TRAAKGISSD than wild-type TRAAK or other disease-causing variants, with inhibition plateauing at about 45% of initial current even at the highest CBD concentration tested (10 pM, FIG. 3F, FIGS. 6A,B). These results suggest that CBD has reduced affinity for TRAAKGISSD — due to electrosteric repulsion between the mutated residue and the cannabinoid — and only partially occludes its conduction pathway — potentially because of an altered CBD binding pose.

[0041] Data to this point are consistent with a model in which cannabinoids bind in the channel cavity to inhibit mechanosensitive K+channels and preferentially bind TM4- down over TM4-up states. A prediction of this model is that increasing membrane tension will reduce CBD inhibition of TRAAK current by promoting TM4-up states. We tested this prediction by using methyl-beta-cyclodextrin (m[3CD) to increase membrane tension in TRAAK-containing patches. m[3CD has been shown to irreversibly increase tension and activate force-gated ion channels by sequestering cholesterol and lipids from the membrane. FIG. 4A shows an experiment in which TRAAK activity and CBD sensitivity were recorded before and after treatment with mpCD. Prior to m[3CD treatment, 1 pM CBD treatment resulted in near complete (91 ± 3%) inhibition (FIGS. 4A,B) that was reversible after washout. Subsequent m[3CD treatment activated channels and activity remained elevated following m[3CD washout, indicating stable high membrane tension in the patch. From this activated state, 1 pM CBD treatment resulted in only partial (43 ± 13 %) inhibition. Approximately 10-fold higher CBD concentrations were required to achieve a similar degree of inhibition to prior to m[3CD treatment (FIG. 4A).

[0042] A second prediction of our model is that CBD inhibition will reduce mechanically activated TRAAK currents by preferentially binding to and stabilizingTM4-down conformations. To test this, we measured current elicited by steps of negative pressure before CBD application and after addition of 0.5, 1, and 10 pM CBD (FIGS. 4C-E, FIGS. 7A-F). We found that as CBD concentration increased, more negative pressure was required to activate TRAAK to the same degree.

[0043] These results support our model for cavity binding and steric occlusion of the conduction path by CBD, but do not fully exclude an alternative explanation in which CBD inhibits TRAAK indirectly by decreasing basal tension or changing other membrane properties to promote closed channel states. We therefore asked if increasing concentrations of CBD alters membrane tension. In a patch recording, the Young- Laplace equation T = APr / 2 relates membrane tension (T) to the measurable pressure difference across the lipid bilayer (AP) and membrane radius of curvature (r). We measured changes in patch radius elicited by pressure steps before and after CBD treatment (FIGS. 8A-C). We found that even 50 pM CBD, a concentration higher than that used in the other electrophysiology experiments herein, had no effect on membrane tension, consistent with direct inhibition of TRAAK and TREK channels by CBD.

[0044] We next pursued a structural approach to gain molecular insight into the basis for cannabinoid inhibition of mechanosensitive K+channels. Cryo-EM structures of Danio rerio TREK-1 in apo, phosphatidylethanolamine(PE)-inhibited, and phosphatidic acid (PA)-activated states are known. Like human TREK-1 (FIG. IE), we found Danio rerio TREK-1 is inhibited by CBD (ICso = 11.1 ± 1.3 pM, FIGS. 9A,B). We determined a cryo-EM structure of Danio rerio TREK-1 in the presence of 100 pM CBD (FIGS. 10A- D, Table 1). The structure was resolved to an overall resolution of 3.94 A, though local resolution for most of the channel is substantially higher and map quality in regions discussed below is comparable to previously reported structures resolved to 2.8-3.5 A (FIGS. 11A-D).Table 1 - Cryo-EM data collection, refinement, and validation statistics dr TREK-1 :CBDPDB ID 9DBREMDB ID 46725EMPIAR IDData collection Collection 1 Collection 2Total movies 9,046 8,470Magnification 105,000xVoltage (kV) 300Electron exposure (eV A2) 50Defocus range (um) -0.5 to -1.8Pixel size (A2) 0.848ProcessingInitial particle images (no.) 261,227 228,680Joint Particle Count 484,500Final particle images (no.) 117,609Map resolution Masked (A, FSC = 0.143) 3.94Symmetry imposed ClRefinementModel resolution (A, FSC = 0.143) 3.9Map-sharpening B factor (A2) 190.5CompositionNumber of atoms 4037Number of protein residues 510Number of ligands 3RMS deviationsBond lengths (A) 0.004Bond angles (A) 0.567ValidationMolProbity score 1.44Clashscore 5.14Ramachandran plotFavored (%) 97.04Allowed (%) 2.96Disallowed (%) 0Rotamer outliers (%) 0Mean B factor (A2)Protein 135.78Ligand 93.61

[0045] A strong, elongated density feature consistent with CBD is evident in the channel cavity directly underneath the selectivity filter. The density is best fit with the CBD aromatic ring centered under the filter and the terpene head group and hydrophobic tail projecting on either side towards the lateral fenestrations. We note this placement is speculative because the resolution is insufficient to unambiguously define CBD binding pose. Several lines of structural evidence support this model for CBD binding in the channel cavity. First, cavity density in the TREK-1 :CBD structure is markedly different than that in the apo TREK-1 structure, which was prepared in the same way apart from the addition of CBD. In apo TREK-1, the cavity density is smaller and biased towards one subunit, extends into the surrounding micelle, and is consistent with a modeled dodecyl maltoside detergent (DDM) molecule. Second, CBD addition resulted in conformational changes to TM4 position. In the TREK- 1 : CBD structure both TM4s are down, while in apo TREK-1 one TM4 is up and one TM4 is down (FIGS. 11 A-D). The symmetric TM4-down TREK-1 :CBD conformation is more similar to the inhibited TREK-1 :PE structure than the apo TREK-1 structure (FIG. 5, FIG. 11 A-D). Third, the TREK-1 :CBD structure shows differences in selectivity filter occupancy (FIGS. 11 A-D). In the presence of CBD, density for K+is evident in sites 2 and 4. In contrast, K+density is evident in sites 1-3 in apo TREK-1, 1-3 in TREK-1 :POPE, and 1-4 in TREK-1 :POPA structures. We conclude CBD inhibits TREK-1 by binding in the channel cavity to sterically block ion passage. Destabilization of K+coordination in the selectivity filter may additionally contribute to channel inhibition.

[0046] Data presented herein indicates CBD inhibits mechanosensitive K+channels by binding in the channel cavity to block ion conduction (FIG. 5). At low tension, TRAAK and TREK channels predominantly adopt TM4-down conformations that expose lateral fenestrations above TM4 to the surrounding lipid membrane. In this conformation, the channels are predominantly closed because lipid acyl chains access the cavity through lateral fenestrations and block ion conduction; spontaneous delipidation results in low leak activity. High tension promotes a TM4-up conformation that closes lateral fenestrations to prevent lipid block, resulting in a high conductance, open channel. We found increasing mechanical activation of TRAAK reduces CBD inhibition, while increasing CBD concentration reduces mechanically activated currents (FIGS. 4A-E). Mutations that promote TM4-up conformations decrease channel sensitivity to CBD (FIGS. 3 A-F). The TREK-1 :CBD structure shows both TM4s down, rather than one up and one down as in the apo TREK-1 structure. This is likely because CBD as modeled would sterically clash with TM4 residues if the helix adopted an up conformation. We conclude CBD preferentially binds to TM4-down channel conformations and effectively competes with abundant membrane lipids for an overlapping cavity site to inhibit channel activity. Whether CBD accesses the channel cavity through membrane-facing fenestrations, through the cytoplasm, or both, remains to be determined.

[0047] CBD modulates distantly-related ion channels in the voltage-gated ion channel superfamily, inhibiting Navs and BK and activating TRPVs, TRPA1, and KCNQs. With an ICso of about 150 nM, TRAAK is among the highest-affinity CBD targets known, while TREK-1 and TREK -2 show roughly 10-fold lower potency, similar to Navs. CBD is the highest affinity TRAAK inhibitor reported to date: about 3-5 fold higher affinity than RU-TRAAK-1 and -2 and greater than 10-fold higher affinity than nonspecific inhibitors ruthenium red and TKDC.

[0048] Structures of TRPV2, Navl .7, and KCNQ7.2 / 7.3 in complex with CBD have been reported. Notably, CBD binds all these channels in similar fenestration site(s) that connect channel cavities to the surrounding membrane. CBD binding within all four fenestrations of TRPV2 and KCNQ channels stabilizes their open states, while binding within one fenestration of Nav1.7 (together with a second CBD molecule binding to the IFM motif) stabilizes the inactivated state. In bacterial NavM, CBD binds deeper within all four fenestrations, partially entering the channel cavity. The mechanism of action of CBD on mechanosensitive K+channels is fundamentally different. CBD does not bind within fenestrations, but rather fully accesses the channel cavity to bind directlyunderneath the selectivity filter. Differences in these binding sites suggests structural insights could guide the design of cannabinoid derivatives with higher specificity to particular ion channels.

[0049] CBD inhibition of mechanosensitive K2Ps could have clinical applications that warrant further exploration. The molecular target of CBD that underlies its antiepileptic effects remains unknown, but TRAAK is a compelling candidate for several reasons. TRAAK activity controls spike propagation in myelinated neurons, where it localizes to axon initial segments and nodes of Ranvier. In these specialized neuronal compartments, TRAAK colocalizes with Navchannels, the target of most classic anticonvulsants, and KCNQ channels, the target of the only approved K+channel-specific antiepileptic. Inhibition of TRAAK or TREK-1 reduces Navavailability by preventing recovery from inactivation, thereby limiting spike velocity and frequency, which could diminish the ectopic excitability characteristic of epileptic disorders. In addition, gain-of-function mutations in TRAAK cause epilepsy in humans, either in a form of Rolandic epilepsy or the neurodevelopmental disorder FHEIG. While some patients respond to anti-seizure medications including carbamazepine, oxcarbazepine, and valproate, others are refractory to treatment. There are currently no specific TRAAK-targeting drugs known. We show multiple disease-causing variants (TRAAKGI65E, TRAAKP259L, TRAAKGI65R, TRAAKA27OP, and TRAAKAI98E) retain sensitivity to inhibition by CBD. These results suggest CBD could be pursued as a targeted treatment of diseases caused by gain-of- function TRAAK mutations in patients.

[0050] The experiments herein indicate that one or more cannabinoid compounds may reduce elevated TWIK-related arachidonic acid-activated K+channel activity in subjects. Thus, the experiments herein indicate that one or more cannabinoid compounds may be effective in treating a subject for a disease or disorder caused by elevated TWIK-related arachidonic acid-activated K+channel activity. Such diseases and disorders include epilepsy, seizures, and abnormal neurological disorders caused by a gain-of-function TWIK-related arachidonic acid-activated K+channel mutation (e.g., TRAAKG165E, TRAAKG165R, TRAAKP259L, TRAAKA270P, and TRAAKA198E mutations).

[0051] As used herein, a “cannabinoid compound” refers to a compound that has the following structural formula:R1 is H, OH, Ra, or RaOH, wherein Ra is an alkyl having 1-5, preferably 1-3, carbon atoms;R2 is H or an alkyl having 1-3 carbon atoms;R3 is an alkyl or alkenyl having 1-10, preferably 2-10, more preferably 3-10, carbon atoms;R4 and R5 are each independently H, an alkyl or alkenyl having 1-5, preferably 1-3, carbon atoms, or R4 and R5 together are a bond or a carbon atom and thereby form a 5- or 6-membered ring, which may be substituted or unsubstituted; and wherein Ring A may be substituted or unsubstituted and 0-3 ring atom bonds may be a double bond. Exemplary cannabinoid compounds include cannabinol (CBN), cannabidiphorol (CBDP), and cannabidivarin (CBDV).

[0052] The cannabinoid compounds may be provided in the form of a composition, e.g., a pharmaceutical composition comprising, consisting essentially of, or consisting of one or more cannabinoid compounds as described herein. In some embodiments, the cannabinoid compounds are substantially purified, e.g., provided in the form of an extract. As used herein, a “substantially purified” compound refers to a compound that is removed from its natural environment and / or is at least about 60% free, preferably about 75% free, and more preferably about 90% free, and most preferably about 95- 100% free from other macromolecular components or compounds with which the compound is associated with in nature or from its synthesis.

[0053] The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a subject. A composition generally comprises an effective amount of an active agent and a diluent and / or carrier. A pharmaceutical composition generally comprises a therapeutically effective amount of an active agent and a pharmaceutically acceptable carrier.

[0054] As used herein, an “effective amount” refers to a dosage or amount sufficient to produce a desired result. The desired result may comprise an objective or subjective change as compared to a control in, for example, in vitro assays, and other laboratory experiments. As used herein, a “therapeutically effective amount” refers to an amount that may be used to treat, prevent, or inhibit a given disease or condition in a subject as compared to a control, such as a placebo. Again, the skilled artisan will appreciate that certain factors may influence the amount required to effectively treat a subject, including the degree of the condition or symptom to be treated, previous treatments, the general health and age of the subject, and the like. Nevertheless, effective amounts and therapeutically effective amounts may be readily determined by methods in the art.

[0055] The one or more cannabinoid compounds may be administered, preferably in the form of pharmaceutical compositions, to a subject. Preferably the subject is mammalian, more preferably, the subject is human. Preferred pharmaceutical compositions are those comprising at least one cannabinoid compound in a therapeutically effective amount and a pharmaceutically acceptable vehicle. It should be noted that treatment of a subject with a therapeutically effective amount may be administered as a single dose or as a series of several doses. The dosages used for treatment may increase or decrease over the course of a given treatment. Optimal dosages for a given set of conditions may be ascertained by those skilled in the art using dosage-determination tests and / or diagnostic assays in the art. Dosage-determination tests and / or diagnostic assays may be used to monitor and adjust dosages during the course of treatment.

[0056] Pharmaceutical compositions may include one or more of the following: a pharmaceutically acceptable vehicle, pH buffered solutions, adjuvants (e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. The compositions and formulations may be optimized for increased stability and efficacy using methods in the art. See, e.g., Carra et al., (2007) Vaccine 25:4149-4158.

[0057] As used herein, a “pharmaceutically acceptable vehicle” or “pharmaceutically acceptable carrier” are used interchangeably and refer to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration and comply with the applicable standards and regulations, e.g., the pharmacopeial standards set forth in the United States Pharmacopeia and the National Formulary (USP-NF) book, forpharmaceutical administration. Thus, for example, unsterile water is excluded as a pharmaceutically acceptable carrier for, at least, intravenous administration. Pharmaceutically acceptable vehicles include those known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th ed (2000) Lippincott Williams & Wilkins, Baltimore, MD.

[0058] A “pharmaceutically acceptable solvate” refers to a solvate form of a specified compound that retains the biological effectiveness of such compound. Examples of solvates include compounds of the invention in combination with water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine, or acetone. Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates”. For example, a complex with water is known as a “hydrate”. Solvates of compounds of formulas I and II are within the scope of the invention. It will also be appreciated by those skilled in organic chemistry that many organic compounds can exist in more than one crystalline form. For example, crystalline form may vary from solvate to solvate. Thus, all crystalline forms of the compounds described herein and the pharmaceutically acceptable solvates thereof are contemplated herein.

[0059] The term “pharmaceutically acceptable salts” refers to salt forms that are pharmacologically acceptable and substantially non-toxic to the subject being treated with the compound of the invention. Pharmaceutically acceptable salts include conventional acid-addition salts or base-addition salts formed from suitable non-toxic organic or inorganic acids or inorganic bases. Exemplary acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, isethionic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, 2-acetoxybenzoic acid, acetic acid, phenylacetic acid, propionic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, ascorbic acid, maleic acid, hydroxymaleic acid, glutamic acid, salicylic acid, sulfanilic acid, and fumaric acid. Exemplary base-addition salts include those derived from ammonium hydroxides (e.g., a quaternary ammonium hydroxide such as tetramethylammonium hydroxide), those derived from inorganic bases such as alkali oralkaline earth-metal e.g., sodium, potassium, lithium, calcium, or magnesium) hydroxides, and those derived from non-toxic organic bases such as basic amino acids.

[0060] A “pharmaceutically acceptable prodrug” is a compound that may be converted under physiological conditions or by solvolysis to the specified compound or to a pharmaceutically acceptable salt of such compound. A “pharmaceutically active metabolite” refers to a pharmacologically active product produced through metabolism in the body of a specified compound or salt thereof. Prodrugs and active metabolites of a compound may be identified using routine techniques known in the art. See, e.g., Bertolini, G, etal., (1997) J Med Chem 40:2011-2016; Shan, D, etal., J Pharm Sci, 86(7):765-767; Bagshawe K., (1995) Drug Dev Res 34:220-230; Bodor, N, (1984) Advances in Drug Res 13:224-331; Bundgaard, H, Design ofProdrugs (Elsevier Press, 1985) and Larsen, I K, Design and Application ofProdrugs, Drug Design and Development (Krogsgaard-Larsen, et al., eds., Harwood Academic Publishers, 1991).

[0061] The pharmaceutical compositions may be provided in dosage unit forms. As used herein, a “dosage unit form” refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the one or more cannabinoid compound calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the given cannabinoid compound and desired therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0062] Toxicity and therapeutic efficacy of cannabinoid compounds according to the instant invention and compositions thereof can be determined using cell cultures and / or experimental animals and pharmaceutical procedures in the art. For example, one may determine the lethal dose, LCso (the dose expressed as concentration x exposure time that is lethal to 50% of the population) or the LDso (the dose lethal to 50% of the population), and the EDso (the dose therapeutically effective in 50% of the population) by methods in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. cannabinoid compounds which exhibit large therapeutic indices are preferred. While cannabinoid compounds that result in toxic sideeffects may be used, care should be taken to design a delivery system that targets such compounds to the site of treatment to minimize potential damage to uninfected cells and, thereby, reduce side-effects.

[0063] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. Preferred dosages provide a range of circulating concentrations that include the EDso with little or no toxicity. The dosage may vary depending upon the dosage form employed and the route of administration utilized. Therapeutically effective amounts and dosages of one or more cannabinoid compounds can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the ICso (z.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. Additionally, a dosage suitable for a given subject can be determined by an attending physician or qualified medical practitioner, based on various clinical factors.

[0064] The following examples are intended to illustrate but not to limit the invention.

[0065] EXAMPLES

[0066] Electrophysiology

[0067] For patch clamp recording from Xenopus oocytes, genes encoding full-length Homo sapiens TRAAK (UniProt Accession No. Q9NYG8-2), TREK-1 (UniProt Accession No. 095069), and TREK-2 (UniProt Accession No. P57789) were codon optimized for eukaryotic expression, synthesized (Genewiz), and cloned into a modified pGEMHE vector using Xhol and EcoRl restriction sites. The transcribed messages encode H. sapiens TRAAK amino acids 1-393, TREK-1 amino acids 1-426, or TREK- 2 amino acids 1-538 with an additional three amino acids (SNS) at the C terminus. TRAAK mutants were introduced by polymerase chain reaction (PCR). Linearized DNA was transcribed in vitro using T7 polymerase. Complementary RNA (0.1 to 10 ng for TRAAK, TREK-1, TREK-2, and mutants) in 50 nL H2O was injected into Xenopus laevis oocytes extracted from anesthetized frogs. Currents were recorded at 25°C from inside-out patches excised from oocytes 1 to 5 days after RNA injection. The pipette solution contained 15 mM KC1, 135 mM NaCl, 2 mM MgCh, and 10 mM HEPES (pH = 7.4 with KOH) and the bath solution contained 150 mM KC1 and 10 mM HEPES (pH = 7.1 with KOH). Borosilicate glass pipettes were pulled to 2-5 MQ resistance.

[0068] For patch clamp recording from cultured cells, dr TREK-1 or / r.sTRAAK genes were cloned into a modified pCEH vector to generate C-terminally EGFP-tagged constructs. HEK293T were cultured in DMEM (Gibco, Thermo Fisher Scientific) with 10% FBS and 100 U ml-1 penicillin and 100 pg ml-1 streptomycin. Trypsinized cells were deposited on 12-mm glass coverslips in a six-well dish one to two days before transfection. For transfection, t / / TREK- l or / r.sTRAAK plasmids were mixed with FuGENE 6 in OptiMEM at a 1 :3 ratio with 1 pg DNA per well per construct and applied to cells in growth medium with antibiotics. Patching was conducted from 24 to 60 h post-transfection. Coverslips were placed in a perfusion chamber at room temperature in isotonic bath solution: 135 mM NaCl, 15 mM KC1, 1 mM CaCh, 1 mM MgCh, 10 mM HEPES, adjusted to a final pH of 7.5 with NaOH. Cells were chosen by a combination of the presence GFP fluorescence and cell morphology consistent with healthy interphase cells. Borosilicate glass pipettes were pulled to a resistance of 1.2-1.8 MQ for patch electrophysiology and 1.8-2.8 MQ for whole cell electrophysiology. Pipettes were filled with pipette solution: 150 mM KCL, 5 mM EGTA, 3 mM MgCh, and 10 mM HEPES, adjusted to pH 7.5 with KOH.

[0069] Patches were evaluated with voltage-step protocols to confirm TRAAK expression and minimal non-specific leak based on reversal potential. Currents were recorded using an Axopatch 200B amplifier (Molecular Devices) at a bandwidth of 1 kHz and digitized with an Digidata 1550B (Molecular Devices) at 100-500 kHz using pClampl0.7 software. Pressure was applied with a second-generation high-speed pressure clamp device (HSPC-2-SB, ALA Scientific Instruments). Aggregated data were from patches from n > 2 different cells on different days. No relevant differences were observed between patches from different cells. In an effort to minimize variability associated with differences in resting membrane tension, we targeted patches with low basal curvature and low resting channel activity and ensured measured pressure difference across the patch was as close to zero as possible at rest to prevent mechanical activation of channels. Data were analyzed using Clampfit 10.7, Excel, and Graphpad Prism software. For voltage families, the data were decimated lOOx and plotted in Prism. Statistical analysis was conducted with Dunnett’s multiple comparisons test after an ordinary one-way ANOVA using Prism software.

[0070] To avoid cannabinoid absorption to plastic, cannabinoid solutions were made in glass vials and diluted by weight, perfusion was performed using glass tubing pulled from borosilicate glass Pasteur pipettes (Fisherbrand, Cat# 13-678-20C), and recordingswere performed in glass chambers. Channel activity was continuously recorded during perfusion steps. Patches were exposed to each cannabinoid concentration until a stable current level was reached (0.5-2 minutes). CBD (Cayman Chemical Cat# 90080) was prepared as a 10 mM stock solution in DMSO, CBN (Cayman Chemical Cat# 25495) was prepared as a 80 mM stock solution in DMSO, CBDV (Phytolab Cat# 85955) was prepared as a 10 mM stock solution in DMSO, and CBDP (Cayman Chemical Cat# 33611) was purchased as a 2.9 mM stock solution in methanol. Cannabinoids were serially diluted in bath solution to achieve working concentrations of 50 pM to 10 nM. Bath volume in the recording chamber was minimized by suction and drug-containing solutions were delivered three times to ensure establishment of desired concentration in the recording chamber. Cannabinoids were applied from low to high concentration. The recording chamber and perfusion system were washed extensively with methanol after every trial.

[0071] The effects of cannabinoids on channel activity were quantified by the reduction in channel current evoked at 0 mV during a gap-free recording or during 200 ms long voltage families with steps ranging from -100 to +100 mV. Currents were normalized to those prior to cannabinoid application to facilitate comparisons across different patches and cells. Dose response curves were four-parametric Boltzmann fits to aggregated data. For voltage-step recordings, current was measured at 0 mV from each recording. Data were aggregated for all recordings with and without drug present and were normalized to the baseline activity before drug administration. Comparisons between constructs and drug isoforms were conducted with ordinary one-way ANOVA and Tukey multiplecomparison tests.

[0072] Patch imaging and membrane tension calculation

[0073] A construct encoding EGFP fused to the CAAX-containing C-terminal tail of H. sapiens H-Ras (NP 005334 amino acids 170-189) through a GGRS linker was cloned into a pCS2+ vector using Gibson assembly. Linearized DNA was transcribed in vitro using T7 polymerase. 3-10 ng complementary RNA in 50 nL H2O was injected into Xenopus laevis oocytes. Excised patches were illuminated with an LED light engine (SpectraX, Lumencor) through a GFP filter (450 / 50 nm excitation, 506 nm dichroic mirror, 500 nm longpass emission filter) and water immersion objective lens (x60, NA1.0). Movies were recorded at 120 Hz with an infrared camera (IR-2000, DAGE- MTI). Images were preprocessed within FIJI (ImageJ). Image contrast was enhanced to facilitate analysis. Video files were loaded into FIJI and converted into a JPEG stack.Frames were time matched to stimuli by multiplying frame rate and time. Tension was calculated using python scripts as previously reported.

[0074] TREK-1 expression and purification

[0075] Danio rerio TREK-1 (UniProt Accession No. Q9NYG8-2) was cloned for expression in Pichia pastoris as previously described with modifications described here. The construct used for purification included an additional 26 amino acid N-terminal sequence from human TRAAK compared to Q9NYG8 that improved heterologous expression. The final construct is C-terminally truncated by 119 amino acids, incorporates two mutations to remove N-linked glycosylation sites (N104Q / N108Q), and is expressed as a C-terminal PreScission protease-cleavable EGFP-lOx His fusion protein. As a result, there is an additional amino acid sequence of “SNSLEVLFQ” at the C terminus of the final purified protein after protease cleavage.

[0076] Starter cultures of recombinant Pichia were grown in YPD with 0.5 mg / mL Zeocin and grown at 30°C with shaking at 250 rpm overnight (12-14 h). Four 1 L flasks of BMGY with 25 pg / mL Zeocin were inoculated with 10 mL of starter culture each. Cells grew for approximately 24 hours at 30°C at 250 rpm. 1 L cell cultures were pelleted by centrifugation at 8000 g for 10 minutes and resuspended in 1 L BMMY with 25 pg / mL Zeocin. Cells were harvested 40-60 h after induction with methanol and flash frozen in liquid N2. About 60 g of frozen Pichia cells expressing t / / TREK- l were disrupted by milling (Retsch model MM301) 5 times for 3 min at 25 Hz. All subsequent purification steps were carried out at 4°C. Milled Pichia cells were thawed in 200 mL of Lysis Buffer containing 50 mM TRIS, 150 mM KC1, 1 mM EDTA pH 8. Protease inhibitors (Final Concentrations: E64 (1 pM), pepstatin A (1 pg / mL), soy trypsin inhibitor (10 pg / mL), benzamidine (1 mM), aprotinin (1 pg / mL), leupeptin (1 pg / mL), AEBSF (1 mM), PMSF (1 mM)), benzonase (10 pL) and DNAse (10 pL) were added to the lysis buffer immediately before use. Cells were lysed by sonication and centrifuged at 150,000 x g for 45 minutes. The supernatant was discarded, and residual nucleic acid was removed from the top of the membrane pellet using DPBS. Membrane pellets were scooped into a Dounce homogenizer containing extraction buffer (50 mM TRIS, 150 mM KCl, 1 mM EDTA, 1.5% n-Dodecyl-P-D-Maltopyranoside (DDM, Anatrace, Maumee, OH), 0.3% cholesteryl hemisuccinate Tris salt (CHS, Anatrace, Maumee, OH) pH 8). A stock solution of 10% DDM, 2% CHS was dissolved and clarified by bath sonication in 200 mM HEPES pH 8 prior to addition to buffer to the indicated final concentration. Membrane pellets were then homogenized in extraction buffer and thismixture (150 mL final volume) was gently stirred at 4°C for 2 hours. The extraction mixture was centrifuged at 33,000 x g for 45 minutes and the supernatant, containing solubilized membrane protein, was bound to 4 mL of Sepharose resin coupled to anti- GFP nanobody for 2 hours at 4°C. The resin was then collected in a column and washed with 10 mL of buffer 1 (20 mM TRIS, 150 mM KC1, 1 mM EDTA, 0.025% DDM, 0.005% CHS, pH 8), 40 mL of buffer 2 (20 mM TRIS, 500 mM KC1, 1 mM EDTA, 0.025% DDM, 0.005% CHS, pH 8), and 10 mL of buffer 1. The resin was then resuspended in 6 mL of buffer 1 with 0.5 mg of PPX protease and rocked gently in the capped column overnight (about 12-14 h). Cleaved TREK-1 was then eluted with an additional 12 mL of wash buffer, spin concentrated to about 1 mL with Amicon Ultra spin concentrator 100 kDa cutoff (Millipore) and loaded onto a Superose S200 increase column (GE Healthcare, Chicago, IL) on an NGC system (Bio-Rad, Hercules, CA) equilibrated in an elution buffer (20 mM TRIS, 150 mM KC1, 1 mM EDTA, 0.025% DDM, pH 8). Peak fractions containing TREK-1 protein were then collected and spin concentrated prior to sample freezing.

[0077] Cryo-electron microscopy

[0078] TREK-1 in DDM detergent was prepared at a final concentration of 3.1 (purification 1) or 4.4 (purification 2) mg / mL. CBD was spiked into the sample from a stock concentration of 10 mM in DMSO to a final concentration of 100 pM. The sample was incubated on ice for 30 minutes then clarified by a 10-minute 21,000 x g spin at 4°C prior to grid preparation. 3.4 pl of protein was applied to freshly glow discharged Holey Carbon, 300 mesh R 1.2 / 1.3 gold grids (C-flat, Electron Microscopy Sciences, USA (purification 1), Quantifoil, GroBldbichau, Germany (purification 2)) and plunge frozen in liquid ethane using a FEI Vitrobot Mark IV (ThermoFisher Scientific) set to 4°C, 100% humidity, 1 blot force, wait time of about 5 seconds, and 3 second blot time. Grids were clipped and stored in liquid nitrogen.

[0079] Both datasets were collected on a Titan Krios G3i electron micro-scope (Thermo Fisher) operated at 300 kV and equipped with a Gatan BioQuantum Imaging Filter with a slit width of 20 eV. Dose-fractionated images (about 50 electrons per A over 50 frames) were recorded on a K3 direct electron detector (Gatan) at a pixel size of 0.848 A. 242 movies were collected in an 11 x 11 hole pattern with two targets per hole around a central hole position using image shift. Defocus was varied from -0.5 to -1.8 pm using SerialEM.

[0080] Motion correction was performed on 9,046 micrographs from collection 1 and 8,470 micrographs from collection 2 using patch motion correction in cryoSPARC v4.4.1. Contrast transfer function (CTF) parameters were fit with patch CTF. For collection 1, template-free auto-picking of particles was performed with a blob picker with a minimum particle size of 120 A and a maximum particle size of 200 A on 5,481 movies CTF fit to 5.0 A or better, yielding an initial set of 2,315,705 particles. These were extracted at a 300-pixel box size for two-dimensional (2D) classification. Iterative rounds of 2D classification yielded a set of 45,022 particles that were used to train a Topaz model. Topaz-trained particle-picking yielded 1,176,701 particles which were manually curated through iterative rounds of 2D classing and multiple multi-class Ab- initios in which particle subsets that yielded the best 3D structure were chosen. Final ab- initio and non-uniform refinements (Cl, 4 extra passes, 15A initial resolution) yielded a 5.14 A map from 261,227 particles.

[0081] For collection 2, the topaz model from collection 1 was utilized to conduct topaztrained particle picking on 7,968 movies CTF fit to 7 A or better. Topaz picking and extraction a box size of 300A yielded an initial set of 1,673,778 particles. These particles were curated through iterative rounds of 2D classification to a subset of 129,260 particles. These particles were then re-extracted from 3729 micrographs CTF fit to between 2.4 A and 7 A for a subset of 92,936 particles that were then used to train a new topaz model on the 3729 micrographs CTF fit between 2.4 A and 7 A. The second round of Topaz training, picking, and extraction yielded 750,776 particles. These classes were manually curated through a single round of 2D classification before a 3 -class Ab-initio. The class with the clearest protein density was chosen yielding a subset of 316,175 particles with clear protein density. These particles were subjected to iterative rounds of 2D and 3D classification. Final ab-initio and non-uniform refinements (Cl, 4 extra passes, 15 A initial resolution) yielded a 5.86 A map from 228,680 particles.

[0082] The 261,227 particles from collection 1 and 228,680 particles from collection 2 were then re-extracted at a 300 A box size and joined into one particle set. Particles too close to the edges of micrographs were discarded, resulting in a joint subset of 484,500 particles. These particles were curated over 3 rounds of 3D classification to remove remaining junk. An ab-initio reconstruction of the remaining 160,072 particles was performed to provide an initial volume and a subsequent non-uniform refinement (Cl, 4 extra passes, 15 A initial resolution) which resulted in a map with a 4.31 A overall resolution. This map was post-processed in cryoSPARC v4.4.1 and used for reference-based motion correction. The resulting ‘shiny’ particles were subject to one round of 2D classification before one 3-class 3D classification resulting in 117,795 particles. These particles were used to generate a new ab initio model. We generated a mask to refine this map in Chimera (UCSF) from the previously reported t / rTREK-1 structure. This mask was imported to cryoSPARC v4.4.1 and modified to a dilation radius of 7 pixels and a soft padding of 25 pixels. Local refinement (Cl, 2 extra passes, 12 A initial resolution) of our volume with this mask resulted in a map with 4.15 A overall resolution. This map was utilized for a second round of reference-based motion correction. An ab initio and local refinement (Cl, 2 extra passes, 12 A initial resolution) of the twice-polished 117,609 particles resulted in a final map of 3.94 A nominal resolution.

[0083] The final non-uniformed local refined and sharpened map from Cryosparc was used for modeling. An initial apo TREK-1 model (PDB: 8DE7) was rigid body fit to the density in Phenix. Model building was performed iteratively with manual adjustment in Coot, global real space refinement in Phenix, and geometry assessment in Molprobity.

[0084] REFERENCES

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Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION -3.1. lUCrJ 7, 253- 267 (2020).Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta cry stall ographica. Section D, Structural biology 74, 531-544 (2018).Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta crystallographica Section D, Biological crystallography 66, 486-501 (2010).Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta crystallographica. Section D, Structural biology 75, 861-877 (2019).Williams, C. J. et al. MolProbity: More and better reference data for improved all-atom structure validation. Protein science : a publication of the Protein Society 27, 293- 315 (2018).

[0086] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.

[0087] As used herein, the terms “subject”, “patient”, and “individual” are used interchangeably to refer to humans and non-human animals. The terms “non-human animal” and “animal” refer to all non-human vertebrates, e.g., non-human mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects and test animals. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0088] As used herein, “and / or” means “and” or “or”. For example, “A and / or B” means “A, B, or both A and B” and “A, B, C, and / or D” means “A, B, C, D, or a combination thereof’ and said “A, B, C, D, or a combination thereof’ means any subset of A, B, C, and D, for example, a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B, and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, and D).

[0089] As used herein, the phrase “one or more of’, e.g., “one or more of A, B, and / or C” means “one or more of A”, “one or more of B”, “one or more of C”, “one or more ofA and one or more of B”, “one or more of B and one or more of C”, “one or more of A and one or more of C” and “one or more of A, one or more of B, and one or more of C”.

[0090] A sentence reciting a string of alternates is to be interpreted as if a string of sentences were provided such that each given alternate was provided in a sentence by itself. For example, the sentence “In some embodiments, the composition comprises A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises A. In some embodiments, the composition comprises B. In some embodiments, the composition comprises C ” As another example, the sentence “In some embodiments, the composition comprises at least A, B, or C” is to be interpreted as if written as the following three separate sentences: “In some embodiments, the composition comprises at least A. In some embodiments, the composition comprises at least B. In some embodiments, the composition comprises at least C ”

[0091] To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

[0092] Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.

Claims

What is claimed is:

1. A method of reducing or inhibiting a potassium ion channel, which comprises administering to the potassium ion channel a cannabinoid compound that has the following structural formula:R1 is H, OH, Ra, or RaOH, wherein Ra is an alkyl having 1-5, preferably 1-3, carbon atoms;R2 is H or an alkyl having 1-3 carbon atoms;R3 is an alkyl or alkenyl having 1-10, preferably 2-10, more preferably 3-10, carbon atoms;R4 and R5 are each independently H, an alkyl or alkenyl having 1-5, preferably 1-3, carbon atoms, or R4 and R5 together are a bond or a carbon atom and thereby form a 5- or 6- membered ring, which may be substituted or unsubstituted; and wherein Ring A may be substituted or unsubstituted and 0-3 ring atom bonds may be a double bond.

2. A method of reducing elevated TWIK-related arachidonic acid-activated K+channel activity in a subject, which comprises administering to the potassium ion channel a cannabinoid compound that has the following structural formula:R1 is H, OH, Ra, or RaOH, wherein Ra is an alkyl having 1-5, preferably 1-3, carbon atoms;R2 is H or an alkyl having 1-3 carbon atoms;R3 is an alkyl or alkenyl having 1-10, preferably 2-10, more preferably 3-10, carbon atoms;R4 and R5 are each independently H, an alkyl or alkenyl having 1-5, preferably 1-3, carbon atoms, or R4 and R5 together are a bond or a carbon atom and thereby form a 5- or 6- membered ring, which may be substituted or unsubstituted; and wherein Ring A may be substituted or unsubstituted and 0-3 ring atom bonds may be a double bond.

3. The method according to claim 1 or claim 2, wherein the cannabinoid compound is selected from the group consisting of4. The method according to any one of claims 1 - 3, whereinR1 is CH3;R2 is H or CH3;R4 is 2A3-prop-l-ene; and / orR5 is H.

5. The method according to any one of claims 1 - 3, wherein the cannabinoid compound is selected from the group consisting of6. The method according to any one of claims 1 - 5, wherein R3 is an alkyl having 3-8 carbon atoms.

7. The method according to any one of claims 1 - 5, wherein R3 is an alkyl having 5-7 carbon atoms.

8. The method according to any one of claims 1 - 7, wherein the potassium ion channel is a mechanosensitive potassium-selective ion channel belonging to the two-pore domain (K2P) K+ion channel family.

9. The method according to any one of claims 1 - 7, wherein the potassium ion channel is a TWIK -related arachidonic acid-activated K+channel.

10. The method according to any one of claims 2 - 9, wherein the subject suffers from epilepsy (e.g., Rolandic epilepsy) and / or seizures.

11. The method according to any one of claims 2 - 9, wherein the subject has a gain-of-function TWIK -related arachidonic acid-activated K+channel mutation (e.g., TRAAK G165E, TRAAK P259L, TRAAK A270P, TRAAK A198E, TRAAK G158D mutations).

12. A cannabinoid compound for use in the treatment of a disease or disorder caused by elevated TWIK -related arachidonic acid-activated K+channel activity, wherein the cannabinoid compound has the following structural formula:R1 is H, OH, Ra, or RaOH, wherein Ra is an alkyl having 1-5, preferably 1-3, carbon atoms;R2 is H or an alkyl having 1-3 carbon atoms;R3 is an alkyl or alkenyl having 1-10, preferably 2-10, more preferably 3-10, carbon atoms;R4 and R5 are each independently H, an alkyl or alkenyl having 1-5, preferably 1-3, carbon atoms, or R4 and R5 together are a bond or a carbon atom and thereby form a 5- or 6- membered ring, which may be substituted or unsubstituted; and wherein Ring A may be substituted or unsubstituted and 0-3 ring atom bonds may be a double bond.

13. The cannabinoid compound according to claim 12, wherein the cannabinoid compound is selected from the group consisting of14. The cannabinoid compound according to claim 12 or claim 13, whereinR1 is CH3;R2 is H or CH3;R4 is 2A3-prop-l-ene; and / orR5 is H.

15. The cannabinoid compound according to claim 12 or claim 13, wherein the cannabinoid compound is selected from the group consisting of16. The cannabinoid compound according to any one of claims 12 - 15, wherein R3 is an alkyl having 3-8 carbon atoms.

17. The cannabinoid compound according to any one of claims 12 - 15, wherein R3 is an alkyl having 5-7 carbon atoms.

18. The cannabinoid compound according to any one of claims 12 - 17, wherein the potassium ion channel is a mechanosensitive potassium-selective ion channel belonging to the two-pore domain (K2P) K+ion channel family.

19. The cannabinoid compound according to any one of claims 12 - 17, wherein the potassium ion channel is a TWIK-related arachidonic acid-activated K+channel.

20. The cannabinoid compound according to any one of claims 12 - 17, wherein the disease or disorder is epilepsy (e.g., Rolandic epilepsy) and / or seizures.

21. The cannabinoid compound according to any one of claims 12 - 17, wherein the elevated TWIK -related arachidonic acid-activated K+channel activity is the result of a gain-of-function TWIK -related arachidonic acid-activated K+channel mutation (e.g., TRAAK G165E, TRAAK P259L, TRAAK A270P, TRAAK A198E, TRAAK G158D mutations).

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