1. Neuroscience

Cannabidiol activates neuronal Kv7 channels

  1. Han-Xiong Bear Zhang
  2. Laurel Heckman
  3. Zachary Niday
  4. Sooyeon Jo
  5. Akie Fujita
  6. Jaehoon Shim
  7. Roshan Pandey
  8. Hoor Al Jandal
  9. Selwyn Jayakar
  10. Lee B Barrett
  11. Jennifer Smith
  12. Clifford J Woolf  Is a corresponding author
  13. Bruce P Bean  Is a corresponding author
  1. Department of Neurobiology, Harvard Medical School, United States
  2. F.M. Kirby Neurobiology Research Center, Boston Children's Hospital, United States
  3. ICCB-Longwood Screening Facility and Department of Immunology, Harvard Medical School, United States
https://doi.org/10.7554/eLife.73246
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Cite this article
  1. Han-Xiong Bear Zhang
  2. Laurel Heckman
  3. Zachary Niday
  4. Sooyeon Jo
  5. Akie Fujita
  6. Jaehoon Shim
  7. Roshan Pandey
  8. Hoor Al Jandal
  9. Selwyn Jayakar
  10. Lee B Barrett
  11. Jennifer Smith
  12. Clifford J Woolf
  13. Bruce P Bean
(2022)
Cannabidiol activates neuronal Kv7 channels
eLife 11:e73246.
https://doi.org/10.7554/eLife.73246
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Abstract

Cannabidiol (CBD), a chemical found in the Cannabis sativa plant, is a clinically effective antiepileptic drug whose mechanism of action is unknown. Using a fluorescence-based thallium flux assay, we performed a large-scale screen and found enhancement of flux through heterologously expressed human Kv7.2/7.3 channels by CBD. Patch-clamp recordings showed that CBD acts at submicromolar concentrations to shift the voltage dependence of Kv7.2/7.3 channels in the hyperpolarizing direction, producing a dramatic enhancement of current at voltages near –50 mV. CBD enhanced native M-current in mouse superior cervical ganglion starting at concentrations of 30 nM and also enhanced M-current in rat hippocampal neurons. The potent enhancement of Kv2/7.3 channels by CBD may contribute to its effectiveness as an antiepileptic drug by reducing neuronal hyperexcitability.

Editor's evaluation

Cannabidiol (CBD) has attracted great interest as a potential therapy for epilepsies and has been shown to be effective in several syndromic forms of pediatric epilepsy. This study finds that clinically relevant concentrations of CBD enhance neuronal M-current, a potassium current whose activation is antiepileptic. These findings open up the possibility that activation of M-current could underlie anti-epileptic efficacy of CBD.

https://doi.org/10.7554/eLife.73246.sa0

Introduction

Cannabidiol (CBD), a phytocannabinoid present in marijuana (Mechoulam et al., 1970), has been shown in recent clinical trials to be an effective agent for treating some forms of epilepsy in children, including Dravet syndrome (Devinsky et al., 2017; Devinsky et al., 2018b; Devinsky et al., 2019; Miller et al., 2020) and Lennox–Gastaut syndrome (Devinsky et al., 2018a; Thiele et al., 2019). How CBD ameliorates epileptic activity is unclear (Rosenberg et al., 2015; Rosenberg et al., 2017; Franco and Perucca, 2019). Unlike Δ(9)-tetrahydrocannabinol (THC), the other major phytocannabinoid in marijuana, CBD does not activate CB1 or CB2 G-protein-coupled receptors (Pertwee, 2005). At micromolar concentrations, CBD has inhibitory effects on a wide range of proteins, including many receptors and channels (Ibeas Bih et al., 2015; Watkins, 2019). Like many classic antiepileptic agents, CBD inhibits voltage-dependent sodium channels in a state-dependent manner, with reported half-maximal concentrations of ~2–10 μM (Hill et al., 2014; Patel et al., 2016; Ghovanloo et al., 2018; Mason and Cummins, 2020). However, as CBD reduction of overall epileptiform activity can be detected in brain slice preparations at much lower concentrations (Jones et al., 2010), the importance of sodium channel inhibition for CBD’s anticonvulsant effects remains uncertain (Hill et al., 2014). Other molecular targets that could mediate antiepileptic actions of CBD have been described, notably antagonism of the lipid-activated G-protein-coupled receptor GPR55 (Ryberg et al., 2007; Sylantyev et al., 2013; Kaplan et al., 2017), and electrophysiological effects correlated with GPR55 antagonism have been described at concentrations of CBD as low as 200 nM (Sylantyev et al., 2013).

The most potent effect of CBD on a well-defined electrophysiological function so far reported is an inhibition of endocannabinoid modulation of synaptic transmission (Straiker et al., 2018). This effect of CBD is mediated by a negative allosteric effect on CB1 receptors, with CBD acting at a site distinct from the primary binding site (Laprairie et al., 2015). Electrophysiologically, this inhibitory negative allosteric effect is detectable at 100 nM and is substantial at 500 nM (Straiker et al., 2018). Here, we report that CBD acts at concentrations as low as 30 nM to activate neuronal M-current, a non-inactivating potassium current mediated by Kv7 channels that activate at subthreshold voltages. CBD shifts the voltage dependence of activation of these channels in the hyperpolarizing direction, resulting in a significant activation of Kv7 current at subthreshold voltages. These results suggest that the activation of neuronal M-current may be one mechanism by which CBD exerts its antiepileptic action.

Results

CBD activates heterologously expressed Kv7.2/7.3 channels

We discovered the ability of CBD to activate Kv7.2/7.3 channels in a screen using fluorescence signals from thallium entry evoked by depolarization of a Chinese hamster ovary (CHO) cell line stably expressing human Kv7.2 and Kv7.3 channels. In a screen of a library of 154 compounds chosen from structures with known or possible ion channel modulating activity (Figure 1—source data 1), CBD was the only compound to produce a substantial enhancement of the fluorescence signal, except for retigabine and flupirtine, both known activators of Kv7.2/7.3 channels.

We then tested the action of CBD on the Kv7.2/7.3 cell line using whole-cell patch-clamp recordings and saw a dramatic enhancement of the currents activated by depolarization, with particularly large effects for currents activated near –50 mV. Figure 1A shows an example, where 100 nM CBD produced a doubling of the current activated at –50 mV, while there was little effect at –20 mV, where channels are near-maximally activated in the control situation. 100 nM enhanced the current evoked at –50 mV by an average factor of 2.8 ± 0.4 (n = 20), while 300 nM CBD enhanced the current by a factor of 4.6 ± 0.5 (n = 14).

Figure 1
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Cannabidiol (CBD) enhancement of cloned human Kv7.2/7.3 channel current in Chinese hamster ovary (CHO) cells.

(A) hKv7.2/7.3 current evoked by staircase depolarizations before and after application of 100 nM CBD. (B) Collected results (mean ± SEM) for current at –50 mV after application of 100 nM (n = 20) or 300 nM CBD (n = 14) for 4–6 min, normalized to current before CBD application, using the protocol in (A). ‘No CBD’ values (n = 11) are for 6 min dummy applications of solution containing only vehicle (DMSO). (C) Voltage-dependent activation of hKv7.2/7.3 channels measured in a cell before and after application of 300 nM CBD. Relative conductance at each voltage was measured from the initial tail current at a step to –50 mV following 1 s depolarizations to voltages between –100 mV and +20 mV from a holding potential of –80 mV. Solid lines: fits to data points of fourth power Boltzmann function, [1/ (1 + exp(-(V – Vhn)/k))]4, where V is test pulse voltage, Vhn is voltage of half-maximal activation for single ‘n’ particle, and k is slope factor for activation of n particles. Control: Vhn = –54.4 mV, k = 12.8 mV (midpoint of function = –33.1); 300 nM CBD: Vhn = –67.9 mV, k = 11.8 mV (midpoint of function –48.3 mV). (D) Concentration-dependent shift of activation midpoint by CBD. Measurements of the midpoint were made before and 10 min after exposure to CBD at various concentrations. mean ± SEM, n = 9 for 30 nM CBD, n = 21 for 100 nM CBD, n = 17 for 300 nM CBD, n = 12 for 500 nM CBD, n = 7 for 1 µM CBD, n = 16 for 3 µM CBD, n = 19 for 10 µM CBD, n = 10 for 20 µM CBD. Value for 0 CBD represents the measurement of a small shift that occurred with dummy applications of DMSO-containing control solution for 10 min (n = 11). Solid line: fit to the Hill equation, ΔVh = −2.5 mV − 17.5 mV/(1 + (EC50/[CBD])^nH), where EC50 = 214 nM and the Hill coefficient nH = 1.3.

Figure 1—source data 1

Screen data and source data for Figure 1.

https://cdn.elifesciences.org/articles/73246/elife-73246-fig1-data1-v2.xlsx

The enhancement of the Kv7.2/7.3-mediated current was produced by a shift of the voltage-dependent activation of the channels in the hyperpolarizing direction (Figure 1C). In collected results, 300 nM CBD shifted the midpoint for channel activation by an average of –13.9 ± 0.9 mV (n = 17). The shift in the voltage dependence of activation reached a maximum of about –20 mV at CBD concentrations of 3–10 μM, with CBD acting with a half-maximal concentration of about 200 nM (Figure 1D).

We next tested whether CBD enhances native Kv7 channels in neurons using measurements of M-current in mouse superior cervical ganglion (SCG) neurons. Using the classic voltage protocol for distinguishing M-current from other potassium currents by virtue of its non-inactivating property and activation at subthreshold voltages (Brown and Adams, 1980), we used a steady holding voltage of –30 mV and hyperpolarizing voltage steps to quantify the M-current from its characteristic slow, voltage-dependent deactivation. Application of CBD at concentrations of 30–300 nM produced a dose-dependent enhancement of M-current (Figure 2), with enhancement of the steady-state outward current at –30 mV and of the slowly deactivating current seen during hyperpolarization to –60 or –70 mV, a defining characteristic of M-current (Figure 2A). It was also clear that CBD shifted the voltage dependence of M-current, resulting in less complete deactivation for a step to –60 mV (Figure 2A). In collected results quantifying the effect of CBD, the enhancement of M-current measured at –50 mV increased from a factor of 1.85 ± 0.19 with 30 nm CBD (n = 14) to a factor of 3.02 ± 0.56 with 300 nm CBD (n = 9).

Figure 2
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Cannabidiol (CBD) enhancement of M-current in mouse sympathetic neurons.

(A) Currents evoked by hyperpolarizations to –60 mV, –70 mV, and –80 mV from a holding potential of –30 mV before (blue) and after (red) application of 100 nM CBD. (B) Collected results (mean ± SEM) for effect of CBD on steady-state M-current at –50 mV. Current was read at the end of a 1 s step from –30 mV to –50 mV, normalized to current before CBD application, following exposure to 10 nM CBD (n = 7), 30 nM CBD (n = 14), 100 nM CBD (n = 8), or 300 nM CBD (n = 9). The maximum effect of CBD was reached in 6–9 min for 10 nM and 30 nM CBD and 2–6 min for 100 nM and 300 nM CBD. ‘No CBD’ values (n = 10) are for 7–9 min dummy applications. Gray circles: individual cells. Black circles: mean ± SEM. Non-paired two-tailed t-tests: 10 nM CBD vs. No CBD, p=0.85; 30 nM CBD vs. No CBD, p=0.024; 100 nM CBD vs. No CBD, p=0.015; 300 nM CBD vs. No CBD, p=0.012.

Figure 2—source data 1

Source data for Figure 2.

https://cdn.elifesciences.org/articles/73246/elife-73246-fig2-data1-v2.xlsx

To test whether CBD enhancement of M-current also occurs in central neurons likely involved in epilepsy, we tested CBD on potassium currents in hippocampal neurons (Figure 3). To facilitate application of well-defined concentrations of CBD without potential problems from absorption into the bulk tissue of brain slices, we used a preparation of cultured rat hippocampal neurons. Using a voltage protocol designed to emphasize M-current (holding the neurons at –30 mV and stepping to –50 mV), CBD enhanced the outward current at both –30 mV and –50 mV in 16 of the 20 cells tested. Consistent with this action of CBD being an enhancement of M-current, which in hippocampal neurons is mediated by Kv7.2, Kv7.3, and Kv7.5 (Shah et al., 2002), there was no increase if CBD was applied in the presence of the Kv7 inhibitor XE-991 (Wang et al., 1998; Brown and Passmore, 2009). In fact, CBD applied after XE-991 produced on average a small (13% ± 4%, n = 15) decrease in current at –50 mV, consistent with a weak inhibitory effect on other, non-M-currents.

Figure 3
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Cannabidiol (CBD) enhancement of Kv7 current in rat hippocampal neurons.

(A) Currents at a holding voltage of –30 mV and during a 500 ms hyperpolarization to –50 mV in control, after application of 1 μM CBD, and after addition of 3 μM XE-991 in the continuing presence of CBD. (B) Collected data with this protocol. Current was measured at the end of the step to –50 mV, normalized to current before application of CBD. Connected open circles indicate data for individual cells (n = 20 for application of CBD, n = 15 for application of CBD followed by XE-991) and closed circles represent mean ± SEM. Paired t-test for currents after CBD compared to control currents, p=0.00017 (n = 20, two-tailed), paired t-test for currents in CBD + XE-991 compared to CBD, p=0.00038 (n = 15, two-tailed). (C) Currents in control, after application of 3 μM XE-991, and after addition of 1 μM CBD in the continuing presence of XE-991. (D) Collected data with symbols as in (B); n = 15 cells for application of XE-991 followed by CBD. Paired t-test for currents after XE-991 compared to control, p=0.00071 (n = 15, two-tailed), paired t-test for currents in XE-991 + CBD compared to XE-991, p=0.0105 (n = 15, two-tailed).

Figure 3—source data 1

Source data for Figure 3.

https://cdn.elifesciences.org/articles/73246/elife-73246-fig3-data1-v2.xlsx

Discussion

Kv7 channel-mediated M-current plays a major role in controlling the excitability of many types of neurons, including neocortical pyramidal neurons (Barrese et al., 2018; Brown and Passmore, 2009; Gunthorpe et al., 2012; Vigil et al., 2020; Jepps et al., 2021). Enhancement of M-current is a clinically proven mechanism of antiepileptic action, as demonstrated by the clinical efficacy of retigabine, an antiepileptic drug that acts by enhancement of current through Kv7 channels (Wickenden et al., 2000; Tatulian et al., 2001; Gunthorpe et al., 2012; Sills and Rogawski, 2020). Our results suggest that the clinical efficacy of CBD could result at least in part by the enhancement of the Kv7-mediated M-current in central neurons. As in the case of retigabine, it remains to be determined exactly which populations of neurons are most sensitive to this enhancement of M-current, and how these effects alter the overall network activity relevant to epileptic activity.

Interestingly, the effect of CBD in enhancing the neuronal M-current is the opposite of the effect of cannabinoids that act as agonists at the CB1 receptor, which inhibit M-current in hippocampal neurons (Schweitzer, 2000). Thus, the fact that CBD is not a CB1 agonist – and actually acts as an allosteric antagonist at CB1 receptors (Laprairie et al., 2015; Straiker et al., 2018) – may be an important aspect of its mechanism of action. The opposite effects on M-current of CBD and CB1 agonists like THC fit well with the history of the development of CBD as an antiepileptic drug, which began with anecdotal evidence that extracts from a particular strain of cannabis with high CBD and low THC (‘Charlotte’s Web’) were an effective adjunctive therapy for a child with Dravet syndrome (Maa and Figi, 2014; Rosenberg et al., 2015; Williams and Stephens, 2020).

In doing our experiments, an important step was discovering that use of plastic containers and plastic tubing could greatly reduce the apparent effects of solutions with CBD at submicromolar concentrations, likely reflecting loss of CBD by absorption into plastic as occurs with other cannabinoids with similarly high lipophilicity (Christophersen, 1986; Hippalgaonkar et al., 2011). This issue complicates a comparison of the concentration dependence with which CBD affects various targets potentially relevant for its antiepileptic activity. Although published data for CBD inhibition of sodium channels have typically reported half-blocking concentrations of 2–10 μM, we have found that when using glass reservoirs and tubing to apply well-defined concentrations of CBD to isolated neurons, substantial inhibition of steady-state ‘persistent’ sodium current can be seen with 30–100 nM CBD (unpublished results). CBD at 100 nM also significantly depresses endocannabinoid modulation of synaptic transmission (Straiker et al., 2018), suggesting that the overall effects of submicromolar concentrations of CBD on neuronal excitability could involve multiple actions. Further work will be required to evaluate the relative importance of actions on various targets to CBD’s antiepileptic action.

Our results add to recent experiments demonstrating that Kv7.2/7.3 channels are susceptible to enhancement by a wide variety of agents acting by several different mechanisms (De Silva et al., 2018; Manville and Abbott, 2018c; Manville and Abbott, 2018a; Miceli et al., 2018; Wang et al., 2018; Kanyo et al., 2020; Kurata, 2020; Li et al., 2020). Such agents include endogenous compounds like GABA (Manville et al., 2018b), the ketone body β-hydroxybutyrate (Manville et al., 2020), and arachidonic acid metabolites and derivatives (Schweitzer et al., 1990; Schweitzer et al., 1993; Larsson et al., 2020a; Larsson et al., 2020b), as well as a variety of natural products including cilantro (Manville and Abbott, 2019). Further development of Kv7.2/7.3 enhancers for treating epilepsy and other neuronal disorders seems promising (Maljevic and Lerche, 2014; Vigil et al., 2020), especially because retigabine has been withdrawn from clinical use because of a number of off-target side effects (Brickel et al., 2020). Compared to other compounds recently found to enhance Kv7.2/7.3 channels, CBD has the distinction of having already been successfully used in multiple epilepsy clinical trials. However, CBD is far from a perfect drug (Sekar and Pack, 2019) as it requires large dosages and has a complex pharmacokinetic profile that limit its effective oral administration (Millar et al., 2019). Improved knowledge of CBD’s most important molecular targets should allow for the design of novel compounds that retain its key molecular actions but with improved pharmacokinetics and reduced off-target effects.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)Swiss WebsterCharles RiverCat# 024 
Strain, strain background (Rattus norvegicus)Sprague–DawleyCharles RiverCat# 400 
Cell line (Cricetulus griseus)Kv7.2/7.3 CHO cell lineMayflower BioscienceBSYS-KV7.2/3-CHO-C CHO (Chinese hamster [C. griseus ] ovary) cell line stably transfected with recombinant human Kv7.2/7.3 ion channels
Commercial assay or kitMycoAlert PLUS Mycoplasma Detection KitLonzaLT07-703 
Commercial assay or kitFluxOR II Green Potassium Ion Channel AssayInvitrogenLT07-703 
Chemical compound, drugCannabidiolCayman ChemicalCat# 90080, CAS 13956-29-1 
Chemical compound, drugHam’s F12-Glutamax-l mediumGibcoCat# 31765-035 
Chemical compound, drugPenicillin-streptomycinGibcoCat# 15140-122 
Chemical compound, drugPuromycinInvivoGenCat# ant-pr-1 
Chemical compound, drugPapainWorthington BiochemicalCat# LS003126 
Chemical compound, drugL-15GibcoCat# 11415-064 
Chemical compound, drugNeurobasal A MediumGibcoCat# 10888-022 
Chemical compound, drugB-27GibcoCat# 17504-010 
Chemical compound, drugPenicillin-streptomycinSigma-AldrichCat# P4333 
Chemical compound, drugMinimal Essential MediumAmerican Tissue Type CollectionCat# DMEM 30-2002 
Chemical compound, drugHank’s Balanced Salt SolutionGibcoCat# 14170-112 
Chemical compound, drugDMEM/F12GibcoCat# 11330-032 
Chemical compound, drugTetrodotoxin w/citrateAbcamAb120055 
Software, algorithmClampexMolecular DevicesVersion 10.3.1.5https://www.moleculardevices.com
Software, algorithmIgor ProWaveMetricsVersion 6.12Ahttps://www.wavemetrics.com
Software, algorithmDataAccessBruxton Corporation http://www.bruxton.com/DataAccess/index.html

Thallium flux assay

Cell culture

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CHO cells coexpressing human Kv7.2 and Kv7.3 channels (Mayflower Bioscience, BSYS-KV7.2/3-CHO-C) were cultured at 37°C in 5% CO2 in a Thermo Scientific incubator in Ham’s F12-Glutamax-l medium (Gibco, Cat# 31765-035) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin/streptomycin solution (Gibco, Cat# 15140-122), and 5 μg/mL puromycin (InvivoGen, Cat# ant-pr-1). The cell line was validated by patch-clamp recording of large voltage-activated currents (>1 nA for depolarizations to 0 mV) that reversed at the potassium equilibrium potential, had the voltage dependence and kinetics previously reported for Kv7.2/7.3 heteromeric channels expressed in CHO cells (Tatulian et al., 2001), and were enhanced by 3 µM retigabine (Tatulian et al., 2001). The cell line was tested for mycoplasma contamination using the Lonza MycoAlert PLUS Mycoplasma Detection Kit (LT07-703, Lonza Pharma & Biotech). Cells were seeded in 15 cm dishes at 200,000 cells per dish, fed twice weekly, and cultivated once weekly. 24 hr before the start of the screen, the culture dishes were trypsinized, and a Countess automated cell counter (Invitrogen) was used to quantify cell numbers before plating them into four Greiner poly-D-lysine-coated 384-well black clear-bottomed microplates at 20,000 cells per well in 40 μL media using a Multidrop Combi Reagent Dispenser. The four microplates were incubated overnight in a Thermo Scientific incubator at 37°C in 90% humidity and 5% CO2.

Compound preparation and handling

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The Panacea Channel Modulator Library, a custom collection of 154 compounds oriented toward known or possible ion channel modulators, was assembled and deposited at the ICCB-Longwood Screening Facility, Harvard Medical School. Compound metadata are listed in Figure 1—source data 1 (assay raw data). Each compound was assayed at four concentrations. The compounds were initially plated as stock solutions in DMSO at concentrations of 0.08 mM, 0.4 mM, 2 mM, and 10 mM, which yielded final assay concentrations of 267 nM, 1.3 μM, 6.7 μM, and 33 μM. Using a custom Seiko compound transfer workstation, 300 nL of experimental compound stock solutions, as well as positive (retigabine at 10 mM in DMSO) and negative (DMSO) controls, were pin transferred into a Greiner Bio-One 384 Deep Well Small Volume polypropylene microplate containing 30 μL of 1× FluxOR chloride-free buffer. This resulted in 16 positive and 16 negative control wells on every assay plate. Each of the two compound microplates was screened in duplicate (four assay plates).

Kv7.2/7.3 assay

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The FluxOR potassium channel assay (Thermo Fisher) was performed using a Hamamatsu FDSS 7000 plate reader essentially as outlined in the product sheet. After the Kv7.2/7.3 CHO cells were incubated in four 384-well assay microplates for 24 hr, a 40 mL solution of FluxOR dye was made by combining 400 μL Powerload concentrate (100×), 40 μL of 13 FluxOR II green reagent (1000× fluorescent dye) in DMSO, 31.2 mL purified water, 4 mL 10× FluxOR assay buffer, 4 mL FluxOR II background suppressor, and 400 μL probenecid (100× in water). Next, media were aspirated from each well of the assay microplates containing Kv7.2/7.3 CHO cells using an Agilent Bravo Liquid Handling system. The assay microplates were then washed two times with FluxOR chloride-free buffer diluted from 5× to 1× (20 μL per well per wash). After the second wash was removed, 7.68 mL of the 40 mL dye solution was dispensed to each 384-well assay microplate (20 μL per well). The assay microplates were incubated in the dye solution at room temperature protected from light for 45 min. Subsequently, 10 μL of diluted compounds in FluxOR chloride-free buffer were added to each assay microplate from the compound dilution plate prepared as described above, resulting in final compound concentrations of 267 nM, 1.3 μM, 6.7 μM, and 33 μM. Assay microplates were incubated in compound and dye for 15 min at room temperature protected from light. For the assay, stimulus buffer was first prepared by mixing 50 mM thallium sulfate (Tl2SO4, 4.8 mL), FluxOR chloride-free buffer (5×, 6.0 mL), and purified water (19.2 mL). Next, 19.2 mL of this stimulus solution (50 μL per well) was loaded into an additional Greiner Bio-One 384 Deep Well Small Volume polypropylene microplate. The four assay plates and plate containing stimulus buffer were then loaded onto a Hamamatsu FDSS 7000Ex plate reader and liquid handler. For each assay microplate, 10 μL of stimulus buffer was added per well after 50 s for a final concentration of 4 mM Tl+ in the assay plate. Fluorescence was measured for 600 data points (~3 min) at 4 Hz. FDSSv3.3.1 software was used for baseline correction and data analysis. All results from the screen are shown in Figure 1—source data 1 (assay raw data).

Electrophysiology with CHO Kv7.2/7.3 cell line

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Cells were maintained and passaged in a humidified 37°C incubator in sterile culture flasks containing Ham’s F12-Glutamax-l medium (Gibco, Cat# 31765-035) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin/streptomycin solution (Gibco, Cat# 15140-122), and 5 µg/mL puromycin (InvivoGen, Cat#ant-pr-1), and cells were passaged at a confluence of about 50–80%. For electrophysiological recordings, cells were seeded onto 12 mm cover slips (Fisherbrand, Cat# 12-545-80). Whole-cell patch-clamp recordings were made using a Multiclamp 700B Amplifier (Molecular Devices). Electrodes were pulled from borosilicate capillaries (VWR International, Cat# 53432-921) on a Sutter P-97 puller (Sutter Instruments), and shanks were wrapped with Parafilm (American National Can Company) to allow optimal series resistance compensation without oscillation. The resistances of the pipettes were 1.8–3.5 MΩ when filled with the intracellular solution consisting of 140 mm KCl, 10 mM NaCl, 2 mM MgCl2, 1 mm EGTA, 0.2 mm CaCl2, 10 mM HEPES, 14 mM creatine phosphate (Tris salt), 4 mM MgATP, and 0.3 mM GTP (Tris salt), pH adjusted to 7.4 with KOH. Seals were formed in Tyrode’s solution consisting of 155 mM NaCl, 3.5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4 adjusted with NaOH. After establishing whole-cell recording, cell capacitance was nulled and series resistance was partially (~70%) compensated. The cell was then lifted and placed in front of an array of quartz fiber flow pipes (250 μm internal diameter, 350 μm external diameter, Polymicro Technologies, Cat# TSG250350) attached with styrene butadiene glue (Amazing Goop, Eclectic Products) to a rectangular aluminum rod (cross section 1.5 cm × 0.5 cm) whose temperature was controlled by resistive heating elements and a feedback-controlled temperature controller (Warner Instruments, TC-344B). Solutions were changed (in ~1 s) by moving the cell from one pipe to another. Recordings were made at 37°C.

Voltage commands were delivered and current signals were recorded using a Digidata 1321A data acquisition system (Molecular Devices) controlled by pCLAMP 10.3 software (Molecular Devices). Current and voltage records were filtered at 5 kHz and digitized at 100 kHz. Analysis was performed with Igor Pro 6.12 (WaveMetrics, Lake Oswego, OR) using DataAccess (Bruxton Software) to import pClamp data.

The effects of CBD on Kv7 current in the cell line were quantified in two ways: by the enhancement of current evoked at –50 mV during stair-step protocols like that in Figure 1A and by the shift in midpoint of activation curves as in Figure 1C. In both cases, current records were corrected for linear capacitative and leak current by subtracting scaled responses to signal-averaged 5 mV hyperpolarizations delivered from –80 mV. Calculation of the enhancement of current at –50 mV during the stair-step protocol was confined to cells in which the current was at least 20 pA in control in order to minimize any error resulting from imperfect leak correction. For determining activation curves, the voltage dependence of activation was measured from the initial tail current at a step to –50 mV following 1 s depolarizations to voltages between –100 mV and +40 mV from a holding potential of –80 mV. Tail current was averaged over a 1 ms interval starting at a time when the immediate jump in current had settled, typically 0.8–1.6 ms after the voltage step. Plots of normalized tail current versus test voltage could be fit well by a Boltzmann function raised to the fourth power. The midpoint of activation was measured in a fit-independent manner by calculating the test voltage at which tail current reached half of its maximal value (reached at voltages between 0 to +40 mV) using linear interpolation between the test voltages straddling the midpoint. Calculation of shifts of activation midpoint by CBD was confined to cells in which the maximal tail current at –50 mV was at least 100 pA and in which the activation curve in CBD was fit well by a Boltzmann function raised to the fourth power.

CBD (Cayman Chemical, Cat# 90080, CAS 13956-29-1) was prepared as a 10 mM stock solution in DMSO, which was diluted in the external Tyrode’s solution to the final concentration. DMSO was added to the control solution at the same concentration as in the CBD solution. In early experiments, CBD-containing solutions were prepared in polystyrene test tubes and applied to cells from reservoirs made from 10 mM polypropylene syringe bodies. Realizing that phytocannabinoids have exceptionally high lipophilicity (Thomas et al., 1990) and can apparently partition into plastic (Christophersen, 1986; Hippalgaonkar et al., 2011), we then switched to using glass reservoirs from which solutions flowed through hollow quartz fibers to be applied to cells. We found that using glass reservoirs and tubing resulted in larger and more reproducible effects of CBD concentrations of 1 μM and below. The reported data for these concentrations are confined to experiments using glass reservoirs and tubing. The effects of concentrations of 3 μM and above were not less when using plastic reservoirs, and the collected data for concentrations of 3–20 μM include experiments done with both plastic and glass reservoirs.

Preparation of SCG neurons

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SCG were removed from adult Swiss Webster mice of either sex (postnatal day 56), cut in half, and treated for 20 min at 37°C with 20 U/mL papain (Worthington Biochemical, Cat# LS003126) in a calcium- and magnesium-free (CMF) Hank’s buffer (Gibco, Cat# 14170-112) containing 137 mM NaCl, 5.36 mM KCl, 0.33 mM Na2HP4, 0.44 mM KH2PO4, 4.2 mM NaHCO3, 5.55 mM glucose, and 0.03 mM phenol red. The ganglia were then treated for 20 min at 37°C with 3 mg/mL collagenase (type I; Roche Diagnostics, Cat# 10103586001) and 4 mg/mL Dispase II (Roche Diagnostics, Cat# 37045800) in CMF Hank’s buffer. Cells were dispersed by trituration with a fire-polished glass Pasteur pipette in a solution composed of two media combined in a 1:1 ratio: Leibovitz’s L-15 medium (Gibco, Cat# 11415-064) supplemented with 5 mM HEPES and DMEM/F12 medium (Gibco, Cat# 11330-032) and plated onto coverslips. Then cells were incubated at 37°C (5% CO2) for 2 hr, after which Neurobasal medium (Gibco, Cat# 10888-022) containing B-27 supplement (Gibco, Cat# A3582801), and penicillin and streptomycin (Sigma-Aldrich, Cat# P4333) was added to the dishes. Cells were stored at room temperature and used within 48 hr.

Electrophysiology with SCG neurons

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Whole-cell patch-clamp recordings were made using a Multiclamp 700B Amplifier (Molecular Devices) interfaced to a Digidata 1321A data acquisition system (Molecular Devices) controlled by pCLAMP 10.3 software (Molecular Devices). Electrodes were 2–4 MΩ when filled with the intracellular solution consisting of 140 mM K aspartate, 13.5 mM NaCl, 1.6 mM MgCl2, 5 mM EGTA, 9 mM HEPES, 14 mM creatine phosphate (Tris salt), 4 mM MgATP, 0.3 mM Tris-GTP, pH 7.2 adjusted with KOH, with shanks wrapped with Parafilm to allow optimal series resistance compensation (70–80%). Seals were formed in Tyrode’s solution consisting of 155 mM NaCl, 3.5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4 adjusted with NaOH, and cells were lifted in front of quartz fiber flow pipes attached to a temperature-controlled aluminum rod. M-current was recorded with external Tyrode’s solution containing 1 μM TTX and 10 μM CdCl2 and quantified by measuring the current at the end of a 1 s step to –50 mV from a steady holding potential of –30 mV, after subtracting linear leak current determined by extrapolation of current measured at voltages between –80 mV and –90 mV. However, the traces in Figure 2A show raw records with no correction of capacitative current or leak current. Recordings were made at 37°C.

Voltage commands were delivered and current signals were recorded using a Digidata 1321A data acquisition system (Molecular Devices) controlled by pCLAMP 10.3 software (Molecular Devices). Current and voltage records were filtered at 5 kHz and digitized at 50 kHz. For display, current records were smoothed by binomial (Gaussian) smoothing using a smooth factor of 101 sampling intervals, equivalent to low-pass filtering with a time constant of about 80 μs. Analysis was performed with Igor Pro 6.12 (WaveMetrics) using DataAccess (Bruxton Software) to import pClamp data.

Preparation of rat hippocampal neurons

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Primary cultures of hippocampal neurons were prepared from rat embryos (E19–E20). Pregnant female Sprague–Dawley rats were anesthetized with isoflurane. The skin was washed with 70% ethanol, the peritoneal cavity was opened, and embryos were transferred into ice-cold preparation solution Ca2+/Mg2+-free HBSS (Gibco, Cat# 14170-112) with 5 mM HEPES (Gibco, Cat# 15630-080) and 1 mM sodium pyruvate (Gibco, Cat# 11360-070) in a 100 mm Petri dish on ice. Heads and brains were sequentially dissected from embryos, with the ice-cold preparation solution exchanged during each step. Under a dissecting microscope, the meninges were stripped away from the cerebral hemispheres and dorsal hippocampi were dissected with a fine scissor. The hippocampal pieces were transferred into a pre-warmed preparation solution containing 37U papain (Worthington, Cat# LS003126), 5 mM L-cysteine (Sigma, Cat# C7352), and 1080U DNase I (Sigma, Cat# DN-25), incubated at 37°C for 15 min, and then washed three times with enzyme-free warmed preparation solution. The preparation solution was then exchanged for a titration medium (EMEM, ATCC, Cat# 30-2003), 5% FBS (Gibco, Ca# 16140-071), and 1× penicillin/streptomycin (P/S, Gibco, Cat# 15140-122), and the hippocampal pieces were titrated using Pasteur pipettes fire-polished to two different tip sizes. After determining cell density using a hematocytometer, a maintenance medium (Neurobasal media [Gibco, Cat# 21103-049], 2% B27 [Gibco, Cat# 17504-044], 5 mM glutamine [Gibco Cat# 25030-081], and 1× P/S) was added into cell suspension to make cell density of 1–1.5 × 105/mL. Five poly-D-lysine (Sigma, Cat# P-7405)-coated coverslips (Fisherbrand, Cat# 12-545-80) were placed in 35 mm dishes and 2–3 × 105 cells were plated in each 35 mm dish (≥4–6 × 104 cells/coverslip). Neurons were maintained for 13–17 days in vitro (DIV). Every 2–3 days, half of the medium was removed from the 35 mm dishes and replaced with the same volume of the fresh maintenance solution.

All experiments using animals were performed according to an institutional IACUC-approved protocol.

Electrophysiology with rat hippocampal neurons

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Recordings were made from neurons after 13–17 DIV. Neurons with three processes and a pyramidal shape were selected for recording. To avoid problems arising from absorption of CBD to plasticware, recordings were made in an all-glass chamber made by attaching a glass ring (18 mm outer diameter, 3 mm height, Thomas Scientific 6705R24) to a glass-bottom microwell dish (MatTek# P35G-1.5-20C). Whole-cell recordings were obtained using patch pipettes with resistances of 2.2–2.5 MΩ when filled with the internal solution, consisting of 140 mM K-gluconate, 9 mM NaCl, 1.8 mM MgCl2, 0.09 mM EGTA, 9 mM HEPES, 14 mM creatine phosphate (Tris salt), 4 mM MgATP, and 0.3 mM Tris-GTP, pH adjusted to 7.2 with KOH. The shank of electrode was wrapped with Parafilm to allow optimal series resistance compensation. Seals were obtained and the whole-cell configuration established in Tyrode’s solution consisting of 155 NaCl, 3.5 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH adjusted to 7.4 with NaOH, with added 1 μM TTX. Reported membrane potentials are corrected for a liquid junction potential of –13 mV between the K-gluconate-based internal solution and the Tyrode’s solution in which current was zeroed at the start of the experiment. The amplifier was tuned for partial compensation of series resistance (typically 40–70% of a total series resistance of 4–10 MΩ), and tuning was periodically readjusted during the experiment. Currents were recorded with a Multiclamp 700B Amplifier (Molecular Devices), filtered at 5 kHz with a low-pass Bessel filter, and digitized using a Digidata 1322 A data acquisition interface controlled by pCLAMP 9.2 software (Molecular Devices). Recordings were made at 30°C.

M-current was evoked by 500 ms steps to –50 mV from a steady holding potential of –30 mV. Stock solutions of 10 mM CBD in DMSO and 20 mM XE-991 in DMSO were made in glass vials and diluted into Tyrode’s solution (in glass vials) as 20 μM CBD or 60 μM XE-991 on the day of recording. Aliquots of these solutions were applied directly into the glass chamber and mixed with a 100 μL pipettor to make final concentrations of 1 μM CBD or 3 μM XE-991, respectively. To minimize any residual effect of CBD from the previous recording, the glass chamber was rinsed with 70% ethanol for three times and distilled water for three times before putting a new coverslip into the chamber.

Data availability

Source data is provided for data in all figures, in the Source Data files for each figure.

References

    1. Ibeas Bih C
    2. Chen T
    3. Nunn AVW
    4. Bazelot M
    5. Dallas M
    6. Whalley BJ
    (2015) Molecular Targets of Cannabidiol in Neurological Disorders
    Neurotherapeutics : The Journal of the American Society for Experimental NeuroTherapeutics 12:699–730.
    https://doi.org/10.1007/s13311-015-0377-3
    1. Schweitzer P
    (2000)
    Cannabinoids decrease the K(+) M-current in hippocampal CA1 neurons
    The Journal of Neuroscience 20:51–58.
    1. Thomas BF
    2. Compton DR
    3. Martin BR
    (1990)
    Characterization of the lipophilicity of natural and synthetic analogs of delta 9-tetrahydrocannabinol and its relationship to pharmacological potency
    The Journal of Pharmacology and Experimental Therapeutics 255:624–630.

Decision letter

  1. Jon T Sack
    Reviewing Editor; University of California Davis School of Medicine, United States
  2. Kenton J Swartz
    Senior Editor; National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
  3. Jon T Sack
    Reviewer; University of California Davis School of Medicine, United States
  4. Ken Mackie
    Reviewer; INSERM-Indiana University, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for submitting your article "Cannabidiol activates neuronal Kv7 channels" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Jon T Sack as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kenton Swartz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ken Mackie (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

We think you have made a finding important for the fields of M-current, epilepsy, and CBD research, that can be effectively communicated as an eLife Short Report. We imagine the significance of your finding could be enhanced by addressing:

1) The CBD EC50 for M-current in hippocampal neurons.

2) CBD impacts on action potential firing in hippocampal neurons.

3) Whether CBD impacts on Nav currents at low concentrations which are efficacious against M-current (e.g., 100 nM) with the same solution delivery system.

A straightforward way to address such revision could be through inclusion of a few key additional results while keeping the scope of the study limited and consistent with a Short Report.

We look forward to a revised manuscript, and also to see what future research reveals with CBD and M-current.

Reviewer #1:

This research presents clear evidence that cannabidiol activates Kv7.2/7.3 channels and neuronal M-current. A strength of this work is the finding that CBD activates Kv7 channels a heterologous system and endogenous M-currents (presumptive Kv7 channels) in neurons from rats and mice. The observation of consistent effects suggests across cell-types and species that this result is robust and will translate to Kv7 M-current in other cell types. Another strength is the identification of the basic mechanism by which CBD acts: shifting the voltage dependence of Kv7 current activation to more negative voltages. This study finds that 100 nM concentration of CBD can activate Kv7 M-current, a concentration that is reported to be ineffective against most other CBD-modulated proteins, though a direct comparison of effective concentrations against other CBD targets is not tested here. Results are interpreted with appropriately nuanced discussion considering the promiscuous effects of CBD on membrane proteins and the range of other molecules that modulate Kv7 channels.

A claim of this manuscript is that CBD acts at lower concentrations on Kv7 than it acts on its many other targets. As nicely described in the methods, careful solution handling is needed to observe sensitivity of Kv7 channels to 100 nM CBD. I noted that DRG neuron Nav current is inhibited by 300 nM CBD in your recent study. Clarifying how distinct the concentration-response of Kv7 and Nav currents are with the same solution handling seems important to substantiate this claim.

Reviewer #2:

The strengths of this study are its identification of a novel potential mechanism for the anti-epileptic actions of CBD, the evaluation across four modalities/cell types (thallium flux, patch clamp in CHO cells over expressing Kv7.2/Kv7.3, mouse super cervical ganglion (SCG) neurons, and cultured mouse hippocampal neurons), and the finding that M current is activated by concentrations of CBD that are similar to those achieved in the plasma of patients administered CBD as an anti-epileptic. The experiences of the authors with plastic versus glass and the adsorption of CBD will be very helpful for others following up on their studies.

The only limitation of this study is that it doesn't explore the mechanism(s) for CBD enhancement of M current (e.g., interactions with PIP2, etc.), however the primary finding of this study is so significant for the field and it will undoubtedly stimulate much additional experimentation, that this is a minor limitation and doesn't negatively affect the impact of the work presented here.

1. Page 3: CBD does affect perception (e.g., https://pubmed.ncbi.nlm.nih.gov/32247649/ ), so it is incorrect to say that it is not psychoactive. It certainly lacks the characteristic psychoactivity of THC.

2. Page 3: "direct primary ligand" is confusing. Would say it's not an orthosteric ligand as some work suggests that there is defined allosteric binding site on CB1 for CBD.

3. Page 5, top: Should "M-current activation" by "M-current deactivation"?

4. Page 12, in the paragraph describing compound preparation, a number of concentrations on my pdf were mM where they should probably be uM.

Reviewer #3:

The manuscript investigated the effects of cannabidiol on KV7.2/KV7.3 channel activity in cell lines and neurons. The authors show that cannabidiol enhances expressed KV7.2/KV7.3 channel activity in cell line at very low doses with a half maximal concentration for activating the current at -50 mV being approximately 200 nM. As KV7.2/KV7.3 channels encode for the M-current in neurons, they also tested the effects of cannabidiol on the M-current in cultured rat SCG neurons and hippocampal neurons. Cannabidiol at 300 nM enhanced the M-current in SCG neurons. However, 1 μm cannabidiol produced a much smaller effect at enhancing the M-current in cultured rat hippocampal neurons (Figure 3).

Overall, the effects of cannabidiol on the M-current are interesting and this may be the potential mechanism of action by which cannabidiol exerts its' anti-epileptic effects. The study is, though, very limited and could be extended to include the effects of cannabidiol on neuronal activity. It might also be interesting to test whether compounds with similar structures to cannabidiol also enhance the M-current.

1) The authors state that they tested a library of 154 compounds chosen from the 'structures with known or possible ion channel modulating activity' (page 4). Could the authors please expand on where they got this library from and which compounds this library contained?

2) The lesser effect of cannabidiol on the M-current in cultured rat hippocampal neurons compared with SCG neurons suggests that the EC50 for cannabidiol on the M-current in hippocampal neurons differs from that in SCG neurons. The authors ought to determine the EC50 values for the compound on the M-current in hippocampal neurons and SCG neurons. If they differ, the authors should attempt to explain why this might be the case.

3) If the EC50 values for cannabidiol differ in hippocampal neurons compared with that obtained for expressed KV7.2/KV7.3 currents in cell lines or the M-current in SCG neurons, it raises questions on whether cannabidiol may exert its anti-epileptic effects via activation of the M-current. Thus, the authors should investigate whether low concentrations of cannabidiol (100 nM) that have been reported to reduce epileptiform activity in brain slices reduce hippocampal neuronal action potential firing and if so, whether this is by activating the M-current. This would be a really important and essential experiment to determine if cannabidiol exerts its anti-epileptic effect by enhancing the M-current.

https://doi.org/10.7554/eLife.73246.sa1

Author response

Reviewer #1:

[…] A claim of this manuscript is that CBD acts at lower concentrations on Kv7 than it acts on its many other targets. As nicely described in the methods, careful solution handling is needed to observe sensitivity of Kv7 channels to 100 nM CBD. I noted that DRG neuron Nav current is inhibited by 300 nM CBD in your recent study. Clarifying how distinct the concentration-response of Kv7 and Nav currents are with the same solution handling seems important to substantiate this claim.

The reviewer makes a very good point. After submission of the manuscript, we have been doing further experiments on different kinds of sodium channels in a variety of preparations using glass reservoirs and tubing. We have found that CBD inhibits different kinds of sodium channels with different potency. The inhibition of sodium channels is strongly state-dependent, so the concentration-response is complicated and dependent on the voltage protocol. Most relevant for potential anti-epileptic action, we see inhibition of subthreshold persistent sodium current in native central neurons (predominantly carried by Nav1.6 channels) by concentrations similar to those that enhance M-current, with substantial effects at CBD concentrations as low as 30 nM in both cases. Therefore, we currently think it likely that the anti-epileptic effects of CBD could involve both M-current enhancement and state-dependent sodium channel inhibition. In the revised paper, we include new data on native M-current in sympathetic neurons with lower concentrations of CBD than we studied initially. The new data demonstrates enhancement by 30 nM CBD. And, we have added a sentence to the Discussion mentioning our unpublished data showing that 30 nM CBD also produces some inhibition of persistent sodium current in some central neurons. We note that submicromolar concentrations of CBD also significantly depress endocannabinoid modulation of synaptic transmission (Straiker et al., 2018), suggesting that overall effects of submicromolar concentrations of CBD on neuronal excitability may well involve multiple actions.

Reviewer #2:

[…] 1. Page 3: CBD does affect perception (e.g., https://pubmed.ncbi.nlm.nih.gov/32247649/ ), so it is incorrect to say that it is not psychoactive. It certainly lacks the characteristic psychoactivity of THC.

Thanks, good point – we have removed the statement that it is not psychoactive.

2. Page 3: "direct primary ligand" is confusing. Would say it's not an orthosteric ligand as some work suggests that there is defined allosteric binding site on CB1 for CBD.

Thanks – we have changed the wording to simply say that is not an activator of CB1 or CB2 receptors.

3. Page 5, top: Should "M-current activation" by "M-current deactivation"?

Thanks, the wording was clumsy. The terminology is tricky because M-current is activated by depolarization but the experimental measurements are done using deactivation. It seems simplest to just say that CBD alters the voltage-dependence of M-current so we have used that terminology.

4. Page 12, in the paragraph describing compound preparation, a number of concentrations on my pdf were mM where they should probably be uM.

Thanks! Those concentrations referred to the stock solution concentrations in the plate of compounds that were then diluted by 300-fold when applied to the assay plate, but that was not clear. We have modified this section to be clearer about the dilutions from the stock solutions to the final concentrations in the experimental wells. We have also made this clear in the description of file of raw data from the assay that we added to Source Data associated with Figure 1.

Reviewer #3:

[…]

1) The authors state that they tested a library of 154 compounds chosen from the 'structures with known or possible ion channel modulating activity' (page 4). Could the authors please expand on where they got this library from and which compounds this library contained?

We have now added a spreadsheet from the screen (as part of Source Data for Figure 1) that includes the list of compounds, along with sources of the compounds, concentrations that were tested, and the raw data from the thallium flux fluorescence measurements for each compound.

2) The lesser effect of cannabidiol on the M-current in cultured rat hippocampal neurons compared with SCG neurons suggests that the EC50 for cannabidiol on the M-current in hippocampal neurons differs from that in SCG neurons. The authors ought to determine the EC50 values for the compound on the M-current in hippocampal neurons and SCG neurons. If they differ, the authors should attempt to explain why this might be the case.

We have spent a good part of the four months since receiving the reviews attempting to better define the dose-response of CBD in both SCG neurons and hippocampal neurons.

In SCG neurons, we originally studied a concentration of 300 nM because it gave a near maximal-effect in the experiments on cloned channels. To define the dose-response of native channels, we have now done new experiments with 10, 30 and 100 nM CBD, along with dummy applications of CBD-free solutions. The data show a clear enhancement of native M-current by 30 nM CBD and increasing effects as the CBD concentration increases. We have added these new data to Figure 2. However, the data do not allow a clear determination of an EC50 value. Unlike the cloned channels where we could define the midpoint of activation and use this as a measure of effect, in the neurons we found it impossible to define complete G-V curves because of the difficulty of distinguishing M-current from other currents at voltages positive to -30 mV. We spent several weeks experimenting with various blocker cocktails to inhibit other channels but were unable to sufficiently block other channels activated positive to -30 mV to enable quantification of pure M-current at these voltages. Therefore, we were unable to quantify the concentration-dependence of CBD’s action by the value of the shift of midpoint, as we did for the cloned channels. We believe the best procedure is to display all of the data for all the cells at each concentration, as we have now done in Figure 2B. At all concentrations of CBD, there is substantial cell-to-cell variability in the degree of enhancement, which we believe may reflect different degrees of activation of the channels at -50 mV in the basal condition. Despite this cell-to-cell variability, the collected results show clearly that 30 nM CBD enhances the current and there is a clear increase of effect with higher CBD concentrations.

Quantification in hippocampal neurons was even more difficult. The reason we did experiments with hippocampal neurons in culture instead of in slice is that in early experiments studying excitability of hippocampal CA1 neurons in brain slice experiments, we found that concentrations of CBD at concentrations as high as 2 μm had almost no effect on firing when applied to slices, even though they strongly inhibit firing of acutely-isolated neurons. We believe this is because the tissue in the slice soaks up the highly lipophilic CBD and prevents effective application to the cell being recorded from, even at levels of several uM. We note that published experiments with CBD in brain slice preparations have generally used far higher concentrations of CBD to see electrophysiological effects, very likely reflecting this problem. We turned to experiments with cultured hippocampal neurons because this allowed application of well-defined CBD concentrations. However, a limitation of cultured neurons is that there a large cell-to-cell variability in the size of the XE-991 sensitive current, and some cells have none. You can see this variability in the data in Figure 3B and 3D, where you can see that a significant fraction of cells had very little or no current inhibited by XE-991. An even bigger challenge with the cultured neurons was obtaining whole-cell recordings that were stable for more than ~ 5-10 minutes before there were changes in leak current. With low concentrations of CBD, it takes ~7-8 minutes for the effects to reach steady-state. A third limitation of quantifying effects on M-current in hippocampal neurons, even in brain slice recordings of M-current under control conditions, is that because the channels are in the axon initial segment, the currents recorded in the soma are relatively small and often have their apparent voltage-dependence shifted in the depolarizing direction (e.g. Yamada-Hanff and Bean, J Neurosci. 33:15011-15021, 2013) so that to separate them from other currents it is necessary to use XE-991, as we did (Figure 3). However, recovery from XE-991 is too slow to allow an “XE-991-subtraction” quantification of both control and CBD-enhanced current, which is why we had to do experiments with a separate population of neurons to test whether XE-991 prevented the enhancing effect of CBD (Figure 3C,D).

Since the reviews, we have tried hard to develop a preparation of hippocampal or cortical neurons that would allow application of low concentrations of CBD for long enough to quantify their effects on M-current, but this has been unsuccessful. We spent several weeks doing experiments with acutely-dissociated hippocampal pyramidal neurons. These allow robust recordings with little change in leak current, but we found that the acutely-isolated hippocampal neurons have little or no M-current, probably because of loss of the axon. We also tried a variety of culture conditions and longer culture times with both mouse and rat hippocampal cultures and also mouse cortical cultures but were ultimately unable to find a preparation where recordings of M-current lasted long enough to allow studies with CBD below 1 uM. So far, the most promising preparation is a preparation of cultured cortical neurons differentiated from human induced pluripotent cells, which appear to have substantial M current based on retigabine (and CBD) responses in multi-electrode array recordings, but as the differentiation procedure has a turn-around time of 8 weeks, it will be many months before we can hope to obtain detailed dose-response data with whole-cell recordings, even if the preparation allows good quantification of M-current with somatic recordings – which is still uncertain since the channels may be primarily in axons.

Because the channels underlying most native M-current in both sympathetic neurons and central neurons are likely Kv7.2/7.3 channels, we think it is very likely that the characteristics of CBD enhancement in sympathetic neurons provide a good model for effects on central neurons. However, we agree this has not yet been shown directly and are careful not to imply that it has.

3) If the EC50 values for cannabidiol differ in hippocampal neurons compared with that obtained for expressed KV7.2/KV7.3 currents in cell lines or the M-current in SCG neurons, it raises questions on whether cannabidiol may exert its anti-epileptic effects via activation of the M-current. Thus, the authors should investigate whether low concentrations of cannabidiol (100 nM) that have been reported to reduce epileptiform activity in brain slices reduce hippocampal neuronal action potential firing and if so, whether this is by activating the M-current. This would be a really important and essential experiment to determine if cannabidiol exerts its anti-epileptic effect by enhancing the M-current.

We agree that our data leave open the question of how important activation of M-current is for the overall effect of CBD on epileptic activity. Since our new experiments show activation of native M-current in SCG neurons at 30 nM CBD, we think it is justified to at least suggest that this could be one mechanism of CBD’s anti-epileptic effect. However, as noted above, since submitting the manuscript we have done experiments using glass reservoirs and tubing to study effects of CBD on various kinds of sodium channels and have found that 30 nM CBD can significantly inhibit persistent sodium current in central neurons. Therefore our current working hypothesis is that CBD’s efficacy for epilepsy may reflect combined actions to inhibit sodium current (particularly persistent sodium current and perhaps resurgent sodium current) and to activate M-current. Indeed, these two actions simultaneously would seem ideal for inhibiting firing of cortical pyramidal neurons, where spikes are initiated in the axon initial segment where there is a large persistent sodium current from Nav1.6 channels and also high expression of Kv7.2/7.3 channels that strongly control spike generation. We currently do not have a good experimental preparation that allows dissection of the relative importance of the two mechanisms. We are studying effects on sodium current in acutely dissociated Purkinje and hippocampal neurons, where we can study effects of CBD at submicromolar concentrations, but these acutely dissociated neurons do not have M-current. The cultured hippocampal neurons have M-currents but have small sodium currents and feeble action potential firing compared to slice or acutely dissociated neurons, so they are not well-suited for looking at changes in excitability. We plan to try to develop an all-glass method for local application of CBD in brain slice preparations but we expect it will be challenging with low concentrations of CBD, which take 5-10 minutes to approach steady-state even when applied directly to an acutely isolated cell body with no other tissue around.

In writing the revised version, we have been careful to avoid any implication that activation of M-current is necessarily the only mechanism of CBD’s anti-epileptic action and have added a mention of our on-going experiments showing that low concentrations of CBD also inhibit persistent sodium current..

https://doi.org/10.7554/eLife.73246.sa2

Article and author information

Author details

  1. Han-Xiong Bear Zhang

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing
    Contributed equally with
    Laurel Heckman, Zachary Niday and Sooyeon Jo
    Competing interests
    No competing interests declared

  2. Laurel Heckman

    F.M. Kirby Neurobiology Research Center, Boston Children's Hospital, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing
    Contributed equally with
    Han-Xiong Bear Zhang, Zachary Niday and Sooyeon Jo
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0124-0519

  3. Zachary Niday

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing
    Contributed equally with
    Han-Xiong Bear Zhang, Laurel Heckman and Sooyeon Jo
    Competing interests
    No competing interests declared

  4. Sooyeon Jo

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing
    Contributed equally with
    Han-Xiong Bear Zhang, Laurel Heckman and Zachary Niday
    Competing interests
    No competing interests declared

  5. Akie Fujita

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared

  6. Jaehoon Shim

    F.M. Kirby Neurobiology Research Center, Boston Children's Hospital, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared

  7. Roshan Pandey

    F.M. Kirby Neurobiology Research Center, Boston Children's Hospital, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared

  8. Hoor Al Jandal

    F.M. Kirby Neurobiology Research Center, Boston Children's Hospital, Boston, United States
    Contribution
    Data curation, Formal analysis, Investigation, Writing – review and editing
    Competing interests
    No competing interests declared

  9. Selwyn Jayakar

    F.M. Kirby Neurobiology Research Center, Boston Children's Hospital, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Writing – review and editing
    Competing interests
    No competing interests declared

  10. Lee B Barrett

    F.M. Kirby Neurobiology Research Center, Boston Children's Hospital, Boston, United States
    Contribution
    Data curation, Methodology, Project administration, Supervision, Validation
    Competing interests
    No competing interests declared

  11. Jennifer Smith

    ICCB-Longwood Screening Facility and Department of Immunology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared

  12. Clifford J Woolf

    1. Department of Neurobiology, Harvard Medical School, Boston, United States
    2. F.M. Kirby Neurobiology Research Center, Boston Children's Hospital, Boston, United States
    Contribution
    Conceptualization, Funding acquisition, Methodology, Project administration, Writing – review and editing
    For correspondence
    clifford.woolf@childrens.harvard.edu
    Competing interests
    No competing interests declared

  13. Bruce P Bean

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review and editing
    For correspondence
    bruce_bean@hms.harvard.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5093-3576

Funding

National Institute of Neurological Disorders and Stroke (NS36855)

  • Bruce P Bean

National Institute of Neurological Disorders and Stroke (NS110860)

  • Bruce P Bean

National Institute of Neurological Disorders and Stroke (NS105076)

  • Clifford J Woolf

Eunice Kennedy Shriver National Institute of Child Health and Human Development (U54HD090255)

  • Clifford J Woolf

Defense Advanced Research Projects Agency (HR0011-19-2-0022)

  • Clifford J Woolf
  • Bruce P Bean

Charles R. Broderick III Phytocannabinoid Research Initiative

  • Bruce P Bean
  • Clifford J Woolf

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by the NIH (NS105076, NS36855, NS110860, U54HD090255), DARPA (HR0011-19-2-0022), and the Charles R Broderick III Phytocannabinoid Research Initiative. We are grateful to Dr. Mustafa Sahin and Ms. Candace Tong-Li of the Assay Development and Screening Core Facility of the Boston Children’s Hospital Intellectual & Developmental Disabilities Research Center for support and help.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved Institutional Animal Care and Use Committee (IACUC) protocols of Harvard Medical School ( Protocol 02538).

Senior Editor

  1. Kenton J Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Reviewing Editor

  1. Jon T Sack, University of California Davis School of Medicine, United States

Reviewers

  1. Jon T Sack, University of California Davis School of Medicine, United States
  2. Ken Mackie, INSERM-Indiana University, United States

Version history

  1. Preprint posted: August 21, 2021 (view preprint)
  2. Received: August 21, 2021
  3. Accepted: February 9, 2022
  4. Version of Record published: February 18, 2022 (version 1)
  5. Version of Record updated: February 22, 2022 (version 2)

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© 2022, Zhang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Han-Xiong Bear Zhang
  2. Laurel Heckman
  3. Zachary Niday
  4. Sooyeon Jo
  5. Akie Fujita
  6. Jaehoon Shim
  7. Roshan Pandey
  8. Hoor Al Jandal
  9. Selwyn Jayakar
  10. Lee B Barrett
  11. Jennifer Smith
  12. Clifford J Woolf
  13. Bruce P Bean
(2022)
Cannabidiol activates neuronal Kv7 channels
eLife 11:e73246.
https://doi.org/10.7554/eLife.73246

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