Valproic Acid Stimulates Release of Ca²⁺ from InsP₃-Sensitive Ca²⁺ Stores

Reviewer 1. Consider editing the title to more accurately describe the findings of the paper. Possibilities include but are not limited to: 1) Valproic acid stimulates release of Ca2+ from InsP3-sensitive Ca2+ stores or 2) Valproic acid is a functional agonist of ER InsP3-sensitive Ca2+ receptors: A novel mechanism

Response 1:

We thank the reviewer for this helpful suggestion. We agree that the title should accurately reflect the experimental evidence and avoid potentially overinterpretative terminology. Accordingly, we have chosen the reviewer’s first suggestion and revised the title to:

“Valproic acid stimulates release of Ca²⁺ from InsP₃-sensitive Ca²⁺ stores.”

We selected this option because it precisely describes our main experimental finding—namely, the stimulation of Ca²⁺ release from InsP₃-sensitive intracellular stores—without explicitly defining valproic acid as a receptor agonist. This choice is also consistent with the recommendation of another reviewer, who advised caution in using the term agonist to describe the mechanism of action. We believe the revised title provides a more conservative and accurate description of our results while remaining fully supported by the data.

The manuscript has been updated accordingly.

Comments 2:

Figure legends should all have titles for consistency. In this draft, Figures 1 and 4 do not have titles; please edit consistency. Additionally, please edit the title for Figure 5 to read: “VPA stimulates release of Ca²⁺ from the ER in PC12 cells”.

Response 2:
We thank the reviewer for this helpful observation. We agree that figure legends should be consistent and include descriptive titles. Accordingly, we have revised the legends to ensure that all figures include a title.

Specifically, we have added titles to Figures 1 and 4, and we have revised the title of Figure 5 as suggested by the reviewer. These changes improve clarity, consistency, and alignment with the main findings of the study. The manuscript has been updated accordingly.

Figure 1

Title:
Valproic acid induces Ca²⁺ release from the endoplasmic reticulum in HeLa cells

Figure 4

Title:
Inhibition of InsP₃ receptors abolishes VPA-induced Ca²⁺ release from the ER

Figure 5:

VPA stimulates release of Ca²⁺ from the ER in PC12 cells

Reviewer comment 3:

Discussion section line 1: “data presented in this paper indicate that VPA is a functional agonist (please add the word functional to the sentence).”

Response 3:

We thank the reviewer for this suggestion. However, in order to maintain a consistent and conservative interpretation throughout the manuscript, we have chosen to emphasize that VPA induces Ca²⁺ release from InsP₃-sensitive intracellular stores, rather than defining it explicitly as an agonist. This wording is fully supported by the experimental data and is consistent with the terminology used in the revised title and figure legends.

Accordingly, the opening sentence of the Discussion has been edited to reflect this approach, focusing on the ability of VPA to stimulate Ca²⁺ release from InsP₃-sensitive stores.

Reviewer comment 4:

We thank the reviewer for helping us refine the clarity and consistency of the manuscript.

Reviewer comment:
For the methods section, provide justification for reagent concentrations (or concentration ranges used). Minimally provide context/references relative to published literature for the methods.

Response 4:
We thank the reviewer for this important comment. We agree that providing context for the concentrations used strengthens the methodological rigor of the study. Accordingly, we have revised the Materials and Methods section to include justification for the selected concentrations and ranges, supported by previously published literature.

Briefly, all reagent concentrations were chosen based on established protocols widely used for intracellular Ca²⁺ measurements, ER-targeted aequorin experiments, and functional studies of InsP₃-sensitive Ca²⁺ stores, as detailed below.

Valproic acid (VPA; 3–100 µM)

The concentration range of VPA (3–100 µM) was selected to encompass low micromolar concentrations known to exert intracellular effects while remaining within or close to the therapeutic plasma range reported for VPA. Similar micromolar concentrations have been widely used to study VPA-induced modulation of Ca²⁺ signaling and secretion in neuronal and neuroendocrine cells.

Importantly, the lower concentrations (3–30 µM) allow detection of direct intracellular actions without confounding effects related to membrane depolarization or ion channel blockade, as previously reported in chromaffin and PC12 cells.

References:

• Löscher, 2002, CNS Drugs
• Kurita et al., 2007, Prog Neuropsychopharmacol Biol Psychiatry
• Yamamoto et al., 1997, J Neurochem

InsP₃ (5 µM)

The concentration of InsP₃ (5 µM) was chosen because it is commonly used in permeabilized-cell preparations to elicit robust but non-saturating ER Ca²⁺ release through InsP₃ receptors. This concentration allows reliable kinetic comparisons between InsP₃-induced and drug-induced Ca²⁺ release.

References:

• Montero et al., 1997, J Cell Biol
• Pizzo et al., 1997, J Cell Biol
• Alvarez & Montero, 1999, Cell Calcium

2-APB (10 µM) and Heparin (200 µg/mL)

The concentrations of the InsP₃ receptor inhibitors 2-APB (10 µM) and heparin (200 µg/mL) were selected based on their well-established efficacy in selectively inhibiting InsP₃R-mediated Ca²⁺ release without affecting ER Ca²⁺ loading or RyR-dependent pathways.

These concentrations have been extensively validated in aequorin-based ER Ca²⁺ measurements and permeabilized-cell systems.

References:

• Ehrlich et al., 1994, Trends Pharmacol Sci
• Zima et al., 2007, J Physiol
• Montero et al., 1997, J Cell Biol

Cyclopiazonic acid (CPA; 3–30 µM)

CPA concentrations were chosen according to classical studies demonstrating dose-dependent inhibition of SERCA activity and progressive ER Ca²⁺ depletion. The selected range allows comparison of kinetic profiles between SERCA inhibition and VPA-induced Ca²⁺ release.

References:

• Pizzo et al., 1997, J Cell Biol
• Alvarez & Montero, 1999, Cell Calcium

Digitonin (100 µM)

Digitonin was used at 100 µM to selectively permeabilize the plasma membrane while preserving ER integrity, a concentration routinely employed in aequorin-based ER Ca²⁺ studies.

References:

• Montero et al., 1997, J Cell Biol
• Alvarez & Montero, 1999, Cell Calcium

High K⁺, caffeine, dantrolene, veratridine

The concentrations of depolarizing K⁺ solutions, caffeine, dantrolene, and veratridine were selected according to previous functional studies in PC12 cells and bovine chromaffin cells, including several from our own group, where these conditions reliably activate VDCCs, RyR-mediated Ca²⁺ release, or epileptiform Ca²⁺ oscillations.

References:

• García-Sancho et al., 1999, J Cell Biol
• Cano-Abad et al., 1998, Br J Pharmacol
• Cano-Abad et al., 2001, J Chem Biol.
• Otoom & Alkadhi, 1998; 2000, Brain Res / Epilepsy Res

Overall, the concentrations used in this study are consistent with well-established protocols in the Ca²⁺ signaling field and with previous work by Pozzan, Alvarez, and our group, ensuring both physiological relevance and methodological robustness. The Methods section added a supplementary Table 1 as follow.

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1: One of the major concern, is the categorization of VPA as an ‘agonist’. While the data convincingly show that VPA-induced ER Ca2+ release requires INSP3R, the conclusion that VPA acts as a direct agonist may be overstated based only on the dose response curve, which is not fully resolved. No direct binding or competition are provided, and the bottom and max of dose-response curve are also not fully resolved. additional evidence supporting direct receptor engagement would be greatly appreciated, or change the categorization accordingly.

Response 1: We thank the reviewer for this valuable and well-founded comment. We agree that, in strict pharmacological terms, the designation of a compound as an agonist generally requires direct evidence of ligand–receptor interaction, such as binding or competition assays. Our study was not designed to assess direct binding of valproic acid (VPA) to the inositol 1,4,5-trisphosphate receptor (InsP₃R), and we therefore acknowledge that the original terminology may have been overly strong.

In response to this concern, we have revised the manuscript throughout (Abstract, Discussion, and Conclusions) to replace the term “agonist” with “InsP₃R-mediated Ca²⁺ mobilization” or “InsP₃R-dependent ER Ca²⁺ release”, which more accurately reflects the scope of our experimental evidence.

Importantly, although we do not claim direct receptor binding, our results provide robust functional evidence demonstrating that VPA induces Ca²⁺ release from the endoplasmic reticulum through InsP₃ receptors. This conclusion is supported by multiple complementary experimental approaches based on direct Ca²⁺ measurements within intracellular organelles, a technically demanding and highly specific methodology:

VPA-induced ER Ca²⁺ release persists in digitonin-permeabilized cells, where plasma membrane receptors, phospholipase C activation, and second-messenger generation are eliminated, indicating an intracellular mechanism independent of upstream signaling cascades.

The kinetics of ER Ca²⁺ release elicited by VPA are indistinguishable from those triggered by InsP₃, as assessed using ER-targeted aequorin, a well-established and widely accepted tool for quantitative Ca²⁺ measurements in the endoplasmic reticulum.

Pharmacological blockade of InsP₃R with heparin or 2-aminoethoxydiphenyl borate (2-APB) completely abolishes VPA-induced ER Ca²⁺ release. Both inhibitors are standard and widely accepted tools to define InsP₃R-dependent Ca²⁺ signaling.

Alternative ER Ca²⁺ release pathways are excluded, as neither SERCA inhibition nor ryanodine receptor blockade reproduces or interferes with the VPA effect.

Together, these data demonstrate a functional dependence on InsP₃R activity for VPA-induced ER Ca²⁺ mobilization. While indirect, this level of functional evidence—obtained using direct organellar Ca²⁺ measurements and established InsP₃R pharmacology—is consistent with accepted criteria in the field for defining InsP₃R-mediated Ca²⁺ signaling.

We believe that this revision appropriately addresses the reviewer’s concern and improves the precision and rigor of the manuscript without altering its central mechanistic conclusions.

We updated the text in the manuscript:

ABSTRACT

Before:

“Together, these results support the conclusion that VPA acts as a novel functional agonist of InsP₃Rs and identify ER Ca²⁺ mobilization as a previously unrecognized intracellular mechanism contributing to its modulatory effects on calcium signaling and excitability in epilepsy.”

After:

“Together, these results support the conclusion that VPA induces InsP₃R-mediated Ca²⁺ mobilization from the endoplasmic reticulum and identify ER Ca²⁺ release as a previously unrecognized intracellular mechanism contributing to its modulatory effects on calcium signaling and excitability in epilepsy.”

INTRODUCTION

Before:

“…supporting the hypothesis that VPA acts as a novel InsP₃R agonist.”

After:

“…supporting the hypothesis that VPA induces InsP₃R-mediated Ca²⁺ mobilization from the endoplasmic reticulum.”

DISCUSSION

Before:

“The major finding of the present study is that the antiepileptic drug valproic acid (VPA) acts as an agonist for the inositol 1,4,5-trisphosphate receptor (InsP₃R).”

After:

“The major finding of the present study is that the antiepileptic drug valproic acid (VPA) induces InsP₃R-mediated Ca²⁺ release from the endoplasmic reticulum.”

Mechanistic interpretation

Before:

“…indicating that VPA mimics the physiological agonist of the InsP₃R.”

After:

“…indicating a functional convergence on InsP₃R-dependent Ca²⁺ release mechanisms.”

SERCA vs InsP₃R mechanism

Before:

“These data suggest that VPA does not act through SERCA inhibition, but rather directly targets the InsP₃R.”

After:

“These data indicate that VPA does not act through SERCA inhibition and that its effect depends on InsP₃R-mediated ER Ca²⁺ mobilization.”

CONCLUSION

Before:

“The drug behaves as an InsP₃R agonist, causing Ca²⁺ release from ER stores.”

After:

“The drug induces InsP₃R-dependent Ca²⁺ release from endoplasmic reticulum stores.”

New text added

“Although direct ligand–receptor binding was not assessed, the strict functional dependence on InsP₃R activity demonstrated by kinetic, pharmacological, and organellar Ca²⁺ measurements supports InsP₃R-mediated Ca²⁺ mobilization as a key intracellular effect of valproic acid.”

KEYWORDS

Before:

“InsP₃ receptor activation”

After:

“InsP₃ receptor–mediated Ca²⁺ release”

Comments 2: The manuscript would benefit from a clearer discussion of how the effective concentrations of VPA used here (3–30 µM) relate to free intracellular and brain concentrations achieved clinically, particularly given protein binding and compartmentalization.

Response 2: We thank the reviewer for this important and insightful comment. We agree that contextualizing the concentrations of valproic acid (VPA) used in our study with clinically relevant exposure levels is essential.

We have now expanded the Discussion to explicitly address this point. As added to the revised manuscript:

Regarding the translational relevance of the concentrations of valproic acid (VPA) used in this study (3–30 µM), it is important to consider that VPA is highly bound to plasma proteins, mainly albumin, such that only a small fraction circulates as free drug [16]. Consequently, total plasma concentrations commonly used for therapeutic drug monitoring do not directly reflect the pharmacologically active fraction. Previous clinical and experimental studies have shown that VPA-induced intracellular Ca²⁺ signaling and secretory effects occur at low micromolar concentrations compatible with the free fraction of the drug [2,12,19]. Moreover, available pharmacological evidence indicates that brain and cerebrospinal fluid exposure to VPA is more closely related to unbound plasma concentrations than to total serum levels [16]. In this context, the concentration range of VPA effective in our experiments (3–30 µM) falls within the lower-to-mid range of clinically relevant free VPA concentrations. Finally, given that our study focuses on an intracellular target—the endoplasmic reticulum—additional intracellular compartmentalization or accumulation cannot be excluded, further supporting the physiological relevance of the concentrations used.

We believe that the added discussion, now supported by relevant clinical and pharmacokinetic references, clarifies the translational relevance of the concentrations used and strengthens the physiological interpretation of our findings.

Comments 3: Both 2-APB and heparin have well-known off-target effects and complex actions on Ca²⁺ signaling. A brief discussion acknowledging these limitations, and why the combined pharmacological and kinetic evidence still supports InsP3R involvement, would improve rigor.

Response 3:

We acknowledge that both 2-aminoethoxydiphenyl borate (2-APB) and heparin exhibit complex pharmacological profiles and have been reported to display off-target effects on Ca²⁺ signaling [22]. However, their use at low concentrations has been extensively validated as a functional approach to assess InsP₃ receptor (InsP₃R) involvement in intracellular Ca²⁺ release pathways [8,22]. Importantly, heparin experiments were performed in digitonin-permeabilized cells, a classical strategy that minimizes indirect effects mediated by plasma membrane receptors or cytosolic signaling pathways and is widely accepted for functional interrogation of InsP₃R-dependent Ca²⁺ release [26,30]. Moreover, despite their distinct chemical nature and mechanisms of action, both heparin and 2-APB fully abolished valproic acid–induced ER Ca²⁺ release, providing convergent pharmacological evidence for InsP₃R involvement.