Cannabidiol increases gramicidin current in human embryonic kidney cells: An observational study.

Gramicidin is a monomeric protein that is thought to non-selectively conduct cationic currents and water. Linear gramicidin is considered an antibiotic. This function is considered to be mediated by the formation of pores within the lipid membrane, thereby killing bacterial cells. The main non-psychoactive active constituent of the cannabis plant, cannabidiol (CBD), has recently gained interest, and is proposed to possess various potential therapeutic properties, including being an antibiotic. We previously determined that CBD's activity on ion channels could be, in part, mediated by altering membrane biophysical properties, including elasticity. In this study, our goal was to determine the empirical effects of CBD on gramicidin currents in human embryonic kidney (HEK) cells, seeking to infer potential direct compound-protein interactions. Our results indicate that gramicidin, when applied to the extracellular HEK cell membrane, followed by CBD perfusion, increases the gramicidin current.

Introduction

Linear gramicidins are a family of antibiotics whose function is determined by increasing the cationic permeability of the membrane [1, 2]. Increased permeability is achieved by the formation of bilayer-spanning channels via dimerization of two hemi-channels. Relative to the channels formed by other antibiotics, gramicidin (gA) is well-behaved, and forms channels that are cation selective. Gramicidin channels are also among the best-understood of these types of channels. Atomic-resolution structures have been provided, and a wealth of functional experiments have yielded important insights into gA function [3, 4]. Gramicidin channel monomers that reside in each membrane leaflet must dimerize with monomers in the other leaflet to form a continuous pore. This dimerization change is necessary and sufficient for cationic currents to be conducted through gA. The channel association/dissociation and the related energetic cost comes from membrane deformation. The pore diameter is ~4 Å, sufficient to allow the pore to also conduct alkali metals, protons, and water [2, 5–7].

The rate of gramicidin channel dimerization is directly related to membrane stiffness or elasticity [4, 8]. This property has been the foundation of functional assays designed to determine the effects of various compounds on membrane dynamics. For example, compounds that reduce the membrane stiffness or thickness (e.g. detergents) enhance the probability of gramicidin dimerization, which in turn increases the cationic gramicidin signal [8-11].

Amphiphiles are among the compounds characterized using the gramicidin-based assays [10]. Amphiphilic compounds are a set of molecules possessing both lipophilic and hydrophilic properties. These molecules often display non-selective modulatory effects on seemingly unrelated targets, a by-product of amphiphiles modulating membrane elasticity [8-10]. Modulation is achieved when amphiphiles localize at the solution–bilayer interface, which is made possible by the compounds’ polar group residing at the interface with the hydrophobic region, which then inserts into the bilayer core. Partitioning into the lipid bilayer alters membrane elasticity, and changes phase preference and membrane curvature [8-10].

One compound with amphiphilic properties is cannabidiol (CBD), the primary non-psychotropic constituent of Cannabis sativa [12]. CBD is a clinically and experimentally substantiated therapeutic compound with efficacy against a variety of conditions, including seizure disorders (for which CBD is FDA-approved), pain, and muscle spasms [13-19]. Furthermore, CBD has been suggested to have antibiotic properties [20, 21]. Unlike the psychotropic Δ9-tetrahydracannabinol (THC), CBD has little to no affinity for endocannabinoid receptors [22, 23]. However, many studies have shown that CBD interacts with a wide range of other targets, including a diverse array of ion channels [13, 14, 16, 17, 19, 24–26]. We previously characterized the full inhibitory effects of CBD on voltage-gated sodium channels (Nav) and deciphered the mechanism through which CBD inhibits Nav currents [17, 24, 25, 27]. We further found that an important component of this mechanism involves CBD altering membrane elasticity, which was measured using a gramicidin-based fluorescence assay (GFA) [11, 24].

GFA is based on the gramicidin permeability to Tl+, a quencher of the water-soluble fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), encapsulated in large unilamellar vesicles (LUVs) doped with gramicidin. The rate of Tl+ influx, measured as the rate of fluorescence quench, indicates the time-averaged number of gramicidin channels in the LUV membrane [11]. Molecules that alter the thickness and elasticity of the LUV membrane also alter the lipid bilayer contribution to the free energy of dimerization and, thus, the free energy of dimerization [28]. Our previous findings in LUVs suggested that CBD decreases gramicidin signals in that assay [24].

We sought to further characterize CBD effects on gramicidin currents using an electrophysiological HEK cell-based assay. In the present study, we investigated the interactions between gramicidin and CBD over short exposures, using voltage-clamped human embryonic kidney (HEK-293) cells in the absence and presence of gramicidin. In this purely observational study, we report that in contrast to the GFA assay, CBD increases the gramicidin current in HEK cells.

Methods

Cell culture

Suspension Human Embryonic Kidney (HEK-293) cells were used for automated patch-clamp experiments. All cells were incubated at 37 ˚C/5% CO2. All cell culture reagents were purchased from ThermoFisher Scientific, Waltham, MA, unless otherwise noted.

Patch-clamp

Automated patch-clamp recording was performed on untransfected HEK (CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045; ATCC, Manassas, VA, USA) cells. Currents were measured in the whole-cell configuration using a Qube-384 (Sophion A/S, Copenhagen, Denmark) automated voltage-clamp system. Intracellular solution contained (in mM): 120 KF, 10 NaCl, 2 MgCl2, 10 HEPES, adjusted to pH7.2 with CsOH. The extracellular recording solution for the high sodium experiment contained (in mM): 140 NaCl, 3 KCl, 1 MgCl2, 1.5 CaCl2, 10 HEPES, adjusted to pH7.4 with NaOH. For the low sodium experiment the external solution sodium concentration was lowered to 1 mM with N-methyl-D-glucamine (NMDG) as NaCl replacement. Liquid junction potentials calculated to be ~7 mV were not adjusted for. Currents were low-pass-filtered at 5 kHz and recorded at 25 kHz sampling frequency. Series resistance compensation was applied at 100%. The measurements were obtained at room temperature which corresponds to 27 ± 2 ˚C at the recording chamber. Appropriate filters for cell membrane resistance (typically >500 MΩ) and series resistance (<10 MΩ) were used. We made fresh gramicidin stock from powder (5 mg/100 μL) in DMSO (26.56 mM). Gramicidin was dissolved in 100% DMSO, and the final concentration of 26 μM. CBD was purchased from Cayman Chemicals (No. 90080) and gramicidin was obtained from Sigma-Aldrich (CAS 11029-61-1).

The Sophion Qube is an automated electrophysiology instrument that is blinded to cell selections and experimentation, and selection was performed in a randomized manner. All subsequent data filtering and analysis was performed in a non-biased manner, in which automated filters were applied to the entire dataset from a given Qube run.

Electrophysiological data analysis

The analysis of raw patch-clamp recordings was performed using the Sophion Analyzer. Graphing and additional analysis was done using the Prism GraphPad (Version 9) software.

Statistics

A one-factor analysis of variance (ANOVA) or t-test were, when appropriate, were used to compare the mean responses. Post-hoc tests using the Tukey Kramer adjustment compared the mean responses between channel variants across conditions. A level of significance α = 0.05 was used in all overall post-hoc tests, and effects with p-values less than 0.05 were considered to be statistically significant. All values are reported as means ± standard error of means (SEM) for n recordings/samples.

Results

CBD increases gramicidin signal in HEK cells in high extracellular sodium concentrations

Gramicidin channels preferentially conduct cationic (e.g., Na+ and K+) currents upon dimerization and pore formation [1, 2]. We measured cationic currents through dimerized gramicidin channels using whole-cell voltage-clamp of untransfected HEK cells in the absence and presence of 26 μM gramicidin applied to the extracellular side of the membrane. First, we measured gramicidin currents in standard high sodium [Na+ = 140 mM] extracellular solution using a ramp protocol. We clamped the cell membranes at -80 mV, close to the K+ equilibrium potential (EK+). Then, we hyperpolarized the cells to -120 mV and ramped the voltage to +50 mV, which is close to ENa+. We added the gramicidin and compound combinations after about 22 minutes, when the currents were stable, then gramicidin/compound combos were added for a 5-minute interval, and the measurements were taken at the end of the interval ().We show average gramicidin current density from the ratio of current amplitude to the cell membrane capacitance (pA/pF) at -120, -80, 0, and +50 mV (). Our results indicate that, at negative potentials, gramicidin conducts inward currents and, as the membrane potential becomes more positive, the current becomes outward with the reversal potential (Erev) being close to 0 mV, as would be predicted for a non-specific monovalent cationic channel. We also measured the effects of 1 μM and 10 μM CBD [17], and 10 μM Triton X100 (TX100; as positive control [9]) on gramicidin-HEK cells (). TX100 is a detergent, and has been shown to change membrane elasticity and hence to increase gramicidin current amplitude in [9]. Interestingly, our findings indicate that TX100 reduced the cationic gramicidin currents across all potentials (-120 mV: p = 0.0118, -80 mV: p = 0.0123, +50 mV: p = 0.0311) (). CBD had the opposite effect to that of TX100, and slightly increased gramicidin currents at both 1 μM (-120 mV: p = 0.0152, -80 mV: p = 0.0136, +50 mV: p = 0.0368) and 10 μM (p>0.05). Interestingly, although the tendency for CBD to alter gramicidin currents was the same at both concentrations, CBD’s effects were more variable at 10 μM than at 1 μM; this variability resulted in lack of statistical significance at 10 μM (). We speculate the variability at 10 μM may be due to damage to the HEK cell membrane from both gramicidin and CBD over the timescales of voltage-clamp experiments.

High sodium voltage-clamp, gramicidin (gA).

(A) Shows the averaged cationic current densities of gramicidin in the presence/absence of CBD at 1 and 10 μM, and TX100 at 10 μM, on the left (in pA/pF, ECS: -120 mV = -13.6 ± 6.3, -80 mV = -10.1 ± 5.0, 0 mV = 5.2 ± 0.5, +50 mV = 30.0 ± 11.0, n = 36; gA: -120 mV = -547.8 ± 66.2, -80 mV = -357 ± 44, 0 mV = 56.5 ± 8.1, +50 mV = 441.1 ± 60.5, n = 47; 1 μM CBD: -120 mV = -820.1 ± 83.2, -80 mV = -543 ± 55.8, 0 mV = 57.7 ± 5.2, +50 mV = 607.4 ± 72.2, n = 56; 10 μM CBD: -120 mV = -687.0 ± 70.3, -80 mV = -452.8 ± 47.4, 0 mV = 57.1 ± 7.1, +50 mV = 649.2 ± 118.8, n = 49; 10 μM TX100: -120 mV = -330.8 ± 43.8 ±, -80 mV = -216.5 ± 30.4, 0 mV = 37.3 ± 4.0, +50 mV = 259.4 ± 32.8, n = 48). The ramp voltage protocol is shown on the right. (B) Shows a cartoon diagram of how gramicidin monomers are thought to dimerize and form channels. (C) Shows quantification of the data shown in (A), stars indicate statistical significance. (D) Shows the associated current traces.

CBD increases gramicidin signal in HEK cells in low extracellular sodium concentrations

The presence of a gramicidin dependent current indicates ion flux across the cell membrane. Gramicidin pores are analogous to puncturing cation-selective holes through the cell membrane. Gramicidin induced currents in the previous high [Na+] experiment resulted in a Erev close to 0 mV. This raises the possibility of a potential nonselective leak current component induced by gramicidin, but not carried by gramicidin, as a confounding variable. To ensure that we were recording gramicidin pore currents, we performed the same experiment with lower extracellular sodium [Na+ = 1 mM]. This experiment resulted in the same overall trends of altered gramicidin currents densities as the high [Na+] experiment, for both CBD and TX100 (). As expected, reducing [Na+] lowered the gramicidin Erev to ~-80 mV (close to EK+). These results confirm our results from the high Na+ experiment and further suggest that, when both Na+ and K+ are present at high concentrations, gramicidin permeability is not highly selective for K+ over Na+ bringing the gramicidin Erev to ~0 mV. Overall, these results show that CBD increases gramicidin currents during short exposures and suggests, therefore, that CBD could be altering membrane elasticity or gramicidin channel conductance directly.

Low sodium voltage-clamp.

Shows the averaged cationic current densities of gramicidin in the presence/absence of CBD at 1 and 10 μM, and TX100 at 10 μM (in pA/pF, ECS: -120 mV = -4.1 ± 0.9, -80 mV = -3.5 ± 0.7, 0 mV = 4.1 ± 0.4, +50 mV = 18.8 ± 1.7, n = 33; gA: -120 mV = -67.6 ± 7.7, -80 mV = -23.0 ± 2.9, 0 mV = 189.8 ± 20.6, +50 mV = 437.4 ± 45.6, n = 45; 1 μM CBD: -120 mV = -90.9 ± 9.0, -80 mV = -30.6 ± 2.9, 0 mV = 261.7 ± 28.7, +50 mV = 604.0 ± 64.8, n = 55; 10 μM CBD: -120 mV = -83.6 ± 14.1, -80 mV = -27.8 ± 5.2, 0 mV = 280.8 ± 74.4, +50 mV = 624.9 ± 149.6, n = 60; 10 μM TX100: -120 mV = -26.3 ± 3.5, -80 mV = -9.7 ± 1.3, 0 mV = 74.2 ± 11.3, +50 mV = 182.4 ± 29.0, n = 50). (B) Shows the ramp voltage protocol. (C) Shows quantification of the data shown in (A), stars indicate statistical significance. (D) Shows the associated current traces.

Discussion

In our previous study, using a GFA, it was determined that CBD has the opposite effect to TX100 and that it decreases the rate of dimerized gramicidin channel formation, and hence a smaller gramicidin current. These findings indicate that CBD is a modifier of the bilayer physical properties at the tested concentrations of 1–30 μM [24]. In this study, by electrophysiologically measuring K+ and Na+ currents flowing through the gramicidin channel, the opposite result was observed. CBD increased gramicidin currents and decreased TX100 currents suggesting an alternate mechanism of gramicidin interaction with amphiphiles like CBD or TX100 than in the GFA assay.

Although the gramicidin structure does not indicate an obvious direct binding-site for CBD [3], there is a chance of a direct CBD-gramicidin interaction taking place. Indeed, in almost every report of CBD activity on a given target, a response has been determined, including various ion channels and receptor proteins [25, 29]. Therefore, the opposite result that was observed might suggest direct CBD and gramicidin interactions that in some way increase the probability of conducting pores in these conditions.

The molecular structure of CBD is composed of two oxygen atoms on both sides of a benzene ring, with the other two ends of the ring having a hydrocarbon tail on one end, and a hydrocarbon ring on the other. These features give the CBD molecule an overall shape that is loosely reminiscent of a phospholipid molecule. Phospholipids, in turn, are molecules that specialize in separating various cellular and sub-cellular environments, a function that is dependent on their amphiphilicity. In our previous paper, molecular dynamics (MD) simulations suggested that CBD molecules tend to localize below phospholipid headgroups, but above the tail-end region [24]. Thus, CBD molecules hovered around carbons ~3–7 of aliphatic chains, as per MD and verified by NMR [24]. It is conceivable that interactions between CBD positioned in the leaflet of the HEK membrane may interact with gramicidin hemi-channels to impact pore dimerization formation in a way that offsets any membrane stiffness affects that inhibit gramicidin currents, as we saw in previous GFA studies.

In the GFA assay, gramicidin monomers are incubated for 24 hours with liposomes at 13 ˚C to reach equilibration, and then the effect of compound is investigated by measuring fluorescence quenching rates [11]. In this study, we measured conventional macroscopic cationic currents in HEK cells using standard voltage-clamp, after the cells were extracellularly perfused with gramicidin monomers over the course of minutes at 27 ˚C. Therefore, the experimental setups between the two studies are fundamentally different, and likely investigate different phenomenon pertaining to gramicidin and CBD interactions. However, CBD’s effect on gramicidin currents in this study appear to be relatively weak. A potential explanation for our results could also be that because we used gramicidin in the micromolar concentrations (instead of the nanomolar used in some studies), the gramicidin monomers adopted less canonical conformations. These conformations could be the reason for why CBD’s effect, which is not very strong on gramicidin are such that the currents are increased [30]. Finally, there may also be direct interactions between TX100 and gramicidin monomers in our experiments.

Our goal in this study was to describe the effects of CBD on HEK cells externally treated with gramicidin. Our results suggest that there may be a direct interaction between CBD and gramicidin but the mechanism by which this potentiates gA currents in HEK cells remains unclear. Further studies will be required for instance, using MD simulations to examine potential for direct interactions.

Time course of experiment.

(A) Shows the time course of experiment at high sodium and (B) at low sodium concentrations.

(DOCX)

Click here for additional data file.

21 Apr 2022

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Reviewer #1: This is a nice observational study of cannabidiol impacts on gramicidin in human embryonic kidney cells as a test of whether CBD has effects on bulk bilayer properties in mammalian cells. This is an interesting and important topic, as CBD is poorly soluble with very high partition coefficient and affects a wide array of membrane proteins. Whether CBD's promiscuity is generally due to bilayer effects or protein binding is unclear. The use of gramicidin in patch clamped cells to test for bilayer effects is smart. The study appears to be straightforward and observational as promised. This study will have value to readers interested int he topic of CBD-membrane interactions.

My major concern is that the data do not strongly support the conclusions. I suggest softening the conclusions. The CBD effect is weak, and I was not convinced that it is not a fluke of serial errors. The statement " CBD ... slightly increased gramicidin currents at ... 10 μM (p>0.05)." is logically flawed because the test indicates that the increase was not significant. The statistic of p<0.05 for 1 μM is not compelling. Additional statistical analyses and discussion of why the statistical test is appropriate could be helpful. Any information about how opportunities for serial errors were minimized between compound and cell prep could be important. A positive control for gA activation is lacking. I think this manuscript would better err on the side of caution and describe the weakness of the CBD effect, and discuss the possibility the effect could not even be real. I suggest changing the conclusion to "CBD does not appear to inhibit gA currents at concentrations where is active against other membrane proteins, and the statistical analyses suggest there may even be an activating effect."

Reporting transparency:

What specific gramicidin & CBD were used? Report source.

Report on care taken handling of gramicidin and CBD (tubes tubing etc). Both stick to surfaces and easily depleted from solution.

Report p-values rather than just p<0.05.

Report if blinding and randomization were implemented and how.

Report the sequence and timing of compound additions to HEK cells.

Please provide a metric for assessing what the maximum tolerated voltage error was. Series resistance compensation was applied at 100% yet the accuracy of the compensation is dependent on the accuracy of the series resistance estimate itself, and these estimates can be noisy. As current densities of up to 10 nA/pF are reported the potential for error seems real.

Please report whether series resistance was reassessed after every compound addition.

Reviewer #2: This manuscript by Ghovanloo Goodchild and Ruben describes the effects of CBD on gramicidin channels incorporated in HEK293 cells, using patch clamp.

Major comments

The description of the methods appears insufficient. In particular, application of gramicidin on only one side of the membrane would be difficult to compare to gramicidin added in the tiny amount used when added to both side of lipid bilayers. Did the authors thought of using a low concentration of gramicidin in the pipette to let the two sides bring a small amount of gramicidin as in the bilayer experiments of Andersen’s lab? How much was the stock in DMSO? How much DMSO was added to get to 26 μM?

It would be useful to see the full temporal course of the experiment. This is because the actions of gramicidin on biological membranes tend to take long to equilibrate when gramicidin is added on only one side (although this would depend on the concentration used and the biological membrane being considered). This will demonstrate that the drugs were always added only after the current is completely stable can the drugs be added. If this is not possible, at least give the time course of increase in conductance after application of gramicidin and how long after gramicidin application were the drugs added, and how long was the waiting time before measurements in the presence of drug.

I believe the large concentrations of gramicidin required to form pores in the whole cell are the reason why opposite than expected results are observed with triton. It would be good to discuss the differences between what is observed in HEK cells and what has been described in lipid bilayers and the possibility that at these massive concentrations part of the gramicidin channels are not as well behaved as in the bilayers. I think that it is good that the authors have the results in both systems. Perhaps what happens is that large part of the channels are not in the conformation normally seen at the low concentrations used in artificial lipid bilayers.

The gramicidin monomer/dimer association dissociation is not a conformational change. The channel associates and dissociates and the energetic cost for this only comes from the membrane deformation, please correct that statement.

About the discussion

I don’t think this results suggest that there is a different mechanism, I think this suggests that the channels formed by gramicidin are not the well behaved channels seen in artificial bilayers. This will require reviewing literature about different structures of gramicidin channels can adopt (I won’t dare to say this is a double stranded gramicidin, see Andersen et al NSB, 1999), but clearly gramicidin channel in artificial membranes form at concentrations of 10 nM not the 26 μM used in this work or to increase permeability in biological membranes. The possibility of the channel adopting strange conformations should be considered here, even if its cation selectivity remains.

As presented more than indicating that CBD acts through a different mechanism than bilayer modification, the data indicates that triton may be interacting directly with the gramicidin channel. I say this, because the observation with CBD is an increase in current, as expected from the GFA. The effect of triton is not.

Minor comments.

3rd line of the discussion, please correct the statement on bilateral modification to

These findings indicate that CBD is a modifier of the bilayer physical properties at the tested concentrations of 1-30 μM ...

The room temperature appears to be very high (27C), I am just confirming this is not a typo occurring twice.

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Submitted filename: This manuscript by Ghovanloo Goodchild and Ruben describes the effects of CBD on gramicidin channels incorporated in HEK293 cells.docx

Click here for additional data file.

29 Jun 2022

Reviewer #1: This is a nice observational study of cannabidiol impacts on gramicidin in human embryonic kidney cells as a test of whether CBD has effects on bulk bilayer properties in mammalian cells. This is an interesting and important topic, as CBD is poorly soluble with very high partition coefficient and affects a wide array of membrane proteins. Whether CBD's promiscuity is generally due to bilayer effects or protein binding is unclear. The use of gramicidin in patch clamped cells to test for bilayer effects is smart. The study appears to be straightforward and observational as promised. This study will have value to readers interested int he topic of CBD-membrane interactions.

We thank the reviewer for a positive and constructive assessment of our manuscript.

My major concern is that the data do not strongly support the conclusions. I suggest softening the conclusions. The CBD effect is weak, and I was not convinced that it is not a fluke of serial errors. The statement " CBD ... slightly increased gramicidin currents at ... 10 μM (p>0.05)." is logically flawed because the test indicates that the increase was not significant. The statistic of p<0.05 for 1 μM is not compelling. Additional statistical analyses and discussion of why the statistical test is appropriate could be helpful. Any information about how opportunities for serial errors were minimized between compound and cell prep could be important. A positive control for gA activation is lacking. I think this manuscript would better err on the side of caution and describe the weakness of the CBD effect, and discuss the possibility the effect could not even be real. I suggest changing the conclusion to "CBD does not appear to inhibit gA currents at concentrations where is active against other membrane proteins, and the statistical analyses suggest there may even be an activating effect.

We thank the reviewer for raising these points and suggestions. The statistical tests that were used in this study are outlined in the Methods section, in which each drug condition was compared with the gA (no drug) condition. The automated patch clamp system that we used takes measurements, head-to-head over the time of a given experiment, and therefore, the effect of a given drug and/or concentration on a given cell was taken at the exact same time. We agree with the reviewer that the effect of CBD is not a strong one. We have added/reflected this onto the revised manuscript. This new addition is under the Discussion section.

Reporting transparency:

What specific gramicidin & CBD were used? Report source.

This information has been added to the Methods section. CBD powder was purchased from Cayman Chemicals. Gramicidin was obtained Sigma.

Report on care taken handling of gramicidin and CBD (tubes tubing etc). Both stick to surfaces and easily depleted from solution.

In this study, we used nonbinding plates and tubes for preparing and the experiments with CBD and gramicidin. This information has been added to the Methods section.

Report p-values rather than just p<0.05.

Done.

Report if blinding and randomization were implemented and how.

This info has been added to methods: The Sophion Qube is an automated electrophysiology instrument that is blinded to cell selections and experimentation, and selection was performed in a randomized manner. All subsequent data filtering and analysis was performed in a non-biased manner, in which automated filters were applied to the entire dataset from a given Qube run.

Report the sequence and timing of compound additions to HEK cells.

Gramicidin was first perfused followed by 5 mins of waiting followed by CBD/TX100 addition.

Please provide a metric for assessing what the maximum tolerated voltage error was. Series resistance compensation was applied at 100% yet the accuracy of the compensation is dependent on the accuracy of the series resistance estimate itself, and these estimates can be noisy. As current densities of up to 10 nA/pF are reported the potential for error seems real.

The Qube uses a proprietary algorithm to perform 100% compensation, after accurately measuring series resistance, as outlined below. Information can be found here: https://sophion.com/products/rs-compensation-qube-384/

Please report whether series resistance was reassessed after every compound addition. The Sophion Qube actively measures series resistance at every experimental block. The compensation is applied at each measurement.

Reviewer #2: This manuscript by Ghovanloo Goodchild and Ruben describes the effects of CBD on gramicidin channels incorporated in HEK293 cells, using patch clamp.

Major comments

The description of the methods appears insufficient. In particular, application of gramicidin on only one side of the membrane would be difficult to compare to gramicidin added in the tiny amount used when added to both side of lipid bilayers. Did the authors thought of using a low concentration of gramicidin in the pipette to let the two sides bring a small amount of gramicidin as in the bilayer experiments of Andersen’s lab? How much was the stock in DMSO? How much DMSO was added to get to 26 μM?

We thank the reviewer for a positive and constructive assessment of our manuscript.

“We made fresh gramicidin stock from powder (5 mg/100 µL) in DMSO (26.56 mM). Gramicidin was dissolved in 100% DMSO, and the final concentration of 26 µM. CBD was purchased from Cayman Chemicals (No. 90080) and gramicidin was obtained from Sigma-Aldrich (CAS 11029-61-1).”

It would be useful to see the full temporal course of the experiment. This is because the actions of gramicidin on biological membranes tend to take long to equilibrate when gramicidin is added on only one side (although this would depend on the concentration used and the biological membrane being considered). This will demonstrate that the drugs were always added only after the current is completely stable can the drugs be added. If this is not possible, at least give the time course of increase in conductance after application of gramicidin and how long after gramicidin application were the drugs added, and how long was the waiting time before measurements in the presence of drug.

We added a supplementary figure and new section on this, as follows:

“We added the gramicidin and compound combinations after about 22 minutes, when the currents were stable, then gramicidin/compound combos were added for a 5-minute interval, and the measurements were taken at the end of the interval (Fig S1).”

I believe the large concentrations of gramicidin required to form pores in the whole cell are the reason why opposite than expected results are observed with triton. It would be good to discuss the differences between what is observed in HEK cells and what has been described in lipid bilayers and the possibility that at these massive concentrations part of the gramicidin channels are not as well behaved as in the bilayers. I think that it is good that the authors have the results in both systems. Perhaps what happens is that large part of the channels are not in the conformation normally seen at the low concentrations used in artificial lipid bilayers.

Done. We thank the reviewer for this point. We have added this to the discussion section.

The gramicidin monomer/dimer association dissociation is not a conformational change. The channel associates and dissociates and the energetic cost for this only comes from the membrane deformation, please correct that statement.

Done.

About the discussion

I don’t think this results suggest that there is a different mechanism, I think this suggests that the channels formed by gramicidin are not the well behaved channels seen in artificial bilayers. This will require reviewing literature about different structures of gramicidin channels can adopt (I won’t dare to say this is a double stranded gramicidin, see Andersen et al NSB, 1999), but clearly gramicidin channel in artificial membranes form at concentrations of 10 nM not the 26 μM used in this work or to increase permeability in biological membranes. The possibility of the channel adopting strange conformations should be considered here, even if its cation selectivity remains.

Done. We have added a section on the possibility of other conformations of gramicidin.

As presented more than indicating that CBD acts through a different mechanism than bilayer modification, the data indicates that triton may be interacting directly with the gramicidin channel. I say this, because the observation with CBD is an increase in current, as expected from the GFA. The effect of triton is not.

Done. We added this important point to the manuscript.

Minor comments.

3rd line of the discussion, please correct the statement on bilateral modification to

These findings indicate that CBD is a modifier of the bilayer physical properties at the tested concentrations of 1-30 μM ...

Done.

The room temperature appears to be very high (27C), I am just confirming this is not a typo occurring twice.

Yes, the temperature on our patch-clamp setup was 27 degrees.

8 Jul 2022

Cannabidiol increases gramicidin current in human embryonic kidney cells: An observational study

PONE-D-22-02483R1

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14 Jul 2022

PONE-D-22-02483R1

Cannabidiol increases gramicidin current in human embryonic kidney cells: An observational study

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