Abstract
Proteins of the anoctamine family (ANO) form calcium-activated chloride channels (CaCC) and phospholipid scramblases. The ANO6 (TMEM16F) protein, which combines the functions of a calcium-dependent scramblase and those of an ion channel, is considered as a molecular target for the treatment of blood clotting disorders, COVID-19-associated pneumonia, neurodegenerative diseases, and other pathologies. CaCCinh-A01, which is a channel blocker of the ANO family, is studied as a potential pharmacological drug. Previously, the effect of this inhibitor was studied using methods representing the integral ion current through the membrane, which does not allow the properties of single channels to be distinguished. Therefore, it remains unknown which characteristics of single channels are sensitive to the blocker: the channel open probability, the current amplitude, or the dwelling time of the channel open state. By registration of single ANO6 channels in HEK293 cells, we showed that the action of the inhibitor is due to a decrease in both the current amplitude and the dwelling time of the single ANO6 channels open state, which, in turn, leads to a decrease in their open state probability. Thus, we have characterized the mechanism of current reduction through ANO6 channels by the inhibitor CaCCinh A01.
REFERENCES
Kalienkova V., Clerico Mosina V., Paulino C. 2021. The groovy TMEM16 family: Molecular mechanisms of lipid scrambling and ion conduction. J. Mol. Biol. 433, 166941.
Yang H., Kim A., David T., Palmer D., Jin T., Tien J., Huang F., Cheng T., Coughlin S.R., Jan Y.N., Jan L.Y. 2012. TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation. Cell. 151, 111–122.
Kostritskii A.Y., Machtens J.P. 2021. Molecular mechanisms of ion conduction and ion selectivity in TMEM16 lipid scramblases. Nat. Commun. 12, 2826.
Zhang Y., Le T., Grabau R., Mohseni Z., Kim H., Natale D.R., Feng L., Pan H., Yang H. 2020. TMEM16F phospholipid scramblase mediates trophoblast fusion and placental development. Sci. Adv. 6, 19.
Ousingsawat J., Wanitchakool P., Kmit A., Romao A.M., Jantarajit W., Schreiber R., Kunzelmann K. 2015. Anoctamin 6 mediates effects essential for innate immunity downstream of P2X7 receptors in macrophages. Nat. Commun. 6, 6245.
Ousingsawat J., Wanitchakool P., Schreiber R., Wuelling M., Vortkamp A., Kunzelmann K. 2015. Anoctamin-6 controls bone mineralization by activating the calcium transporter NCX1. J. Biol. Chem. 290, 6270–6280.
Cabrita I., Benedetto R., Schreiber R., Kunzelmann K. 2019. Niclosamide repurposed for the treatment of inflammatory airway disease. JCI Insight. 4, e128414.
Braga L., Ali H., Secco I., Chiavacci E., Neves G., Goldhill D., Penn R., Jimenez-Guardeño J. M., Ortega-Prieto A. M., Bussani R., Cannatà A., Rizzari G., Collesi C., Schneider E., Arosio D., Shah A.M., Barclay W.S., Malim M.H., Burrone J., Giacca M. 2021. Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia. Nature. 594, 88–93.
Zhang Y., Li H., Li X., Wu J., Xue T., Wu J., Shen H., Li X., Shen M., Chen G. 2020. TMEM16F aggravates neuronal loss by mediating microglial phagocytosis of neurons in a rat experimental cerebral ischemia and reperfusion model. Front. Immunol. 11, 1144.
De La Fuente R., Namkung W., Mills A., Verkman, A. S. 2008. Small-molecule screen identifies inhibitors of a human intestinal calcium-activated chloride channel. Mol. Pharmacol. 73, 758–768.
Shi S., Guo S., Chen Y., Sun F., Pang C., Ma B., Qu C., An H. 2020. Molecular mechanism of CaCCinh-A01 inhibiting TMEM16A channel. Arch. Biochem. Biophys. 695, 108650.
Taylor K.A., Mahaut-Smith M.P. 2019. A major interspecies difference in the ionic selectivity of megakaryocyte Ca2+-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01. Platelets. 30, 962–966.
Martins J. R., Faria D., Kongsuphol P., Reisch B., Schr-eiber R., Kunzelmann K. 2011. Anoctamin 6 is an essential component of the outwardly rectifying chloride channel. Proc. Natl. Acad. Sci. USA. 108, 18168–18172.
Kolesnikov D., Perevoznikova A., Gusev K., Glushankova L., Kaznacheyeva E., Shalygin A. 2021. Electrophysiological properties of endogenous single Ca2+ activated Cl-channels induced by local Ca2+ entry in HEK293. Int. J. Mol. Sci. 22, 4767.
Kolesnikov D.O., Nomerovskaya M.A., Grigorieva E.R., Reshetin D.S., Skobeleva K.V., Gusev K.O., Shalygin A.V., Kaznacheyeva E.V. 2024. Calcium chelation independent effects of BAPTA on endogenous ANO6 channels in HEK293T cells. Biochem. Biophys. Res. Comm. 693, 149378.
Tian X.Q., Ma K.T., Wang X.W., Wang Y., Guo Z.K., Si J.Q. 2018. Effects of the calcium-activated chloride channel inhibitors T16Ainh-A01 and CaCCinh-A01 on cardiac fibroblast function. Cell Physiol. Biochem. 49, 706–716.
Funding
The work was supported by Russian science foundation (project no. 22-24-00761).
Author information
Authors and Affiliations
Contributions
Authors D.O.K. and E.R.G. contributed equally.
Corresponding authors
Ethics declarations
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
This article does not contain any studies involving animals or human participants performed by any of the authors.
CONFLICT OF INTEREST
The authors declare that they have no obvious or potential conflicts of interest.
Additional information
Translated by D. Kolesnikov
Publisher’s Note.
Pleiades Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Kolesnikov, D.O., Grigorieva, E.R., Nomerovskaya, M.A. et al. The Mechanism of Calcium-Activated Chloride ANO6 Channel Inhibition by CaCCinh-A01. Biochem. Moscow Suppl. Ser. A 18, 31–35 (2024). https://doi.org/10.1134/S1990747824700041
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S1990747824700041