PONE-D-24-52981Complex regulation of Cav2.2 N-type Ca2+ channels by Ca2+ and G-proteinsPLOS ONE

Dear Dr. Lee,

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Academic Editor

PLOS ONE

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Additional Editor Comments:

Dear Dr. Lee,

These 2 reviews took roughly the normal time for completion.

I believe you will be able to respond effectively to both referee's concerns, which fall under Minor Revisions in my opinion.

Your emphasis would be best spent on the comments from Reviewer #2.

I look forward to your responses.

Best,

Steve Barnes

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study investigates how G-protein coupled receptors (GPCRs) inhibit Cav2.2 N-type Ca²⁺ channels through two distinct pathways: a fast, voltage-dependent pathway mediated by Gai/Gbg and a slow, voltage-independent pathway involving Gaq-dependent reductions in phosphatidylinositol 4,5-bisphosphate (PIP2) or increases in arachidonic acid. Unlike previous studies that primarily used Ba²⁺, this research compares the tonic G-protein inhibition of currents carried by Ba²⁺ (IBa) and Ca²⁺ (ICa). These results propose that when G-proteins are inhibited, Ca²⁺ influx through Cav2.2 promotes a form of VDF involving PIP2. The findings highlight the complexity of how Cav2.2 channels integrate G-protein signaling pathways, potentially enriching the information encoding capabilities of chemical synapses in the nervous system.

1. In Figure 3, why is the voltage-dependent facilitation (VDF) of ICa so strong, similar to that of IBa, while it appears much weaker than IBa in Figures 1 and 2? The differences should be clearly demonstrated.

2. To gain a deeper understanding of the molecular mechanism, it would be beneficial to compare the results of VDF experiments in channels with membrane-tethered β2a or cytosolic CaV β subunits, such as β1a or β3. I recommend testing the key findings in channels with cytosolic β subunits.

3. Please correct "β2A" to "β2a" in the first paragraph of the Materials and Methods and Results sections.

4. Kindly check the spacing between words, numbers, and units throughout the text.

Reviewer #2: I found the findings in this manuscript useful for advancing understanding of how lipids affect N-VGCC gating and modulation. The findings are timely considering there are now structural studies showing at least one PIP2 binding site to CaV2.2 (Gao et al, 2021; Dong et al 2021) as well as detailed biophysical data of channel movements from Nilsson et al. (2024).

File = Dropbox(personal)\\2024 Amy Lee Plos One ReviewF

Thomas et al have examined how tonic modulation of recombinant N-type calcium current by G-proteins varies depending on whether Ba2+ or Ca2+ is the permeant ion. Relatively few studies of N-current modulation have used physiological conditions where Ca2+ serves as the charge carrier. The authors raise the possibility that important Ca2+-dependent effects on channel gating might be lost when using Ba+ as the charge carrier. To test for differences with Ca2+ vs Ba2+ as the charge carrier, whole-cell currents were recorded from channels made up of CaV2.2-37b, α2δ-1 and �2a expressed in HEK293T cells. The authors used conditions known inhibit G-protein activity, by including in the pipette soluction GDP-�-S which blocks G-protein activity or transfection of HEK cells with the C-terminal construct of GPCR kinase (GRK), which has no kinase activity but acts to sequester Gβγ. They applied a series of classic voltage protocols to examine the voltage-dependent facilitation (VDF) of currents following a prepulse. The method is standard for looking at tonic inhibition of CaV2.2 by Gβγ.

Their results indicate that tonic current inhibition by Gβγ is context dependent. They found that tonic inhibition by Gβγ is stronger for IBa than for Ica as measured by differences in VDF. Moreover, their findings implicate PIP2 in VDF of ICa but not IBa when Gβγ-mediated inhibition is suppressed. While insensitive to high intracellular Ca2+ buffering, VDF of ICa that remained in cells dialyzed with GDP-β-S but was blunted by reductions in PIP2 when activating a voltage dependent phosphatase that acts on PI substrates. The authors concluded that when G-proteins are inhibited, Ca2+ influx through Cav2.2 promotes a form of VDF that involves PIP2. VDF of IBa2+ is minimally affected by VSP mediated metabolism of PIP2 levels. The findings reveal a complicated interplay of permeant ion, tonic G-protein activity and PIP2 levels that in part depend on the permeant ion.

The findings serve a useful purpose of pulling together previous observations about the complicated influences calcium, BAPTA, PIP2 and G-proteins exert on tonic CaV2.2 channel activity. The manuscript could contribute substantially to the calcium channel field if critical context in data interpretation on several issues is added by expanding discussion of several previous studies.

Context Issues:

1) CaV2 model of 2 PIP2 binding sites: The authors do not address previous findings that CaV2 channels appear to have 2 PIP2 binding sites that exert different effects on CaV2.2 activity. It may not appear obvious that their data may arise from both or just one PIP2 binding site. Previously, Wu et al (2002) presented a strong argument for two PIP2 binding sites on CaV2 channels: one site regulates whether channels open with “willing” voltage sensitivity (S site) and the second, lower affinity PIP2 binding site (R) promotes reluctant gating that is voltage-dependent where strong voltage pulses facilitate current amplitude to “willing” kinetics and amplitude. Loss of PIP2 from the R site enhances current amplitude promoting willing gating such that a prepulse no longer facilitates current amplitude. Thus, conditions that shift equilibrium in favor of PIP2 bound at the R site would be predicted to promote VDF following a prepulse. Loss of PIP2 at this site shifts the activation to more negative voltages and the channels undergo rapid activation and no VDF. Thomas et al’s should discuss their data in context of the Wu et al model since their data are complimentary to this model.

2) Palmitoylated �2a: The Hille, Rittenhouse, & Suh labs have each published findings that when �2a is expressed in HEK cells, its 2 palmitoyl groups, located on vicinal cysteines, appear occupy the “S” PIP2 binding site and confer resistance to current inhibition by M1R/Gq signaling and PIP2 breakdown. Under Thomas et al’s recording conditions, as with the above studies, the palmitoylated �2a will block the PIP2 “S” putative binding site leaving modulation of the voltage-dependent “R” site. The authors need to point this out since it is directly relevant to their data interpretation as mentioned above.

3) Previous expts with BAPTA & Ca2+: BAPTA can interact with PIP2 protecting it from breakdown. The authors need to look at Shapiro et al 1994 in Neuron, Rousset et al 2004a (JBC) and 2004b (FEBS Letters). Each of these studies address biophysical aspects of interactions among Ca2+, BAPTA, G-proteins. The data in these papers may help further mechanistic insight in the differences in VDF with Ba2+ vs Ca2+.

4) Alternative theories: while the authors conclude that PIP2 regulates ICa2+, rather than CaM kinases or G-protein mediated signaling cascades, the authors should not rule out a role for enzymes directly activated by Ca2+ and/or PIP2 without G�� activity. Good to cover all possibilities.

Major Issues

The prepulse voltage used varies with different figures. For example, Figs 1 &2 use a prepulse to +60 mV whereas Fig 7 it is +20 mV. The I-FF figs were apparently gathered from different prepulse voltages, e.g., Figs. 3F,5D, 6D plot I-FF80, whereas Figs 1G,2G & 4G, plot I-FF60, and Fig 7 data are plotted P2/P1 vs P2-P1/P1as with the other figures. It is not clear why in Fig. 7 the Pre pulse is only to +20 mV. The authors need to explain the thinking behind the different voltages and calculations. Explain why they used different voltages since it adds an additional variable when comparing experimental data.

For Fig. 7 include 2 graphs like those in Figs. 1C & F.

Activation kinetics of current traces were not measured despite it being a hallmark of G-protein modulation. The authors have the traces. The activation kinetics, e.g. time to peak or �o measurements should support changes in current amplitudes following prepulses. The extra data would strengthen their observation that Ca2+ vs Ba2+ current modulation show different characteristics.

Minor Issues

1. Everywhere Gi is written should be changed to Go/i. Indeed, Go may mediate more VDF than Gi.

2. State where the amplitude of current traces were measured, e.g., at the end of the test pulse, the peak tail current?

3. Explain why data are expressed as median rather than the mean.

4. The figures need more detail: what is the test pulse for all figs where current amplitudes and FF were calculated, e.g, for Fig 2 the test voltage was 0 mV but it is not clear for Fig. 2 C & F.

5. In Fig. 1, the IBa individual P1 traces are so small that the currents can’t be compared directly to the P2 traces. If possible, replace the recording for one where the current amplitudes are greater.

6. The I-FF data would be clearer if in Figs 1G, 2G, #F & 4G graphs use symbols other than circles, e.g. squares or triangles to distinguish the difference in data graphed compared to C & F.

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Reviewer #1: No

Reviewer #2: No

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Complex regulation of Cav2.2 N-type Ca2+ channels by Ca2+ and G-proteins

PONE-D-24-52981R1

Dear Dr. Lee,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Steven Barnes

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors provided excellent responses and changes to the manuscript based on the 2 reviewers thorough and insightful comments and requested corrections. This manuscript is now ready for publication.

Reviewers' comments: