The Mechanism of Calcium-Activated Chloride ANO6 Channel Inhibition by CaCCinh-A01

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Abstract

Proteins of the anoctamine family (ANO) form calcium-activated chloride channels (CaCC) and phospholilpid 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: 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 open state dwelling time of single ANO6 channels, 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.

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About the authors

D. O. Kolesnikov

Institute of Cytology, Russian Academy of Sciences

Email: shalygin.alexey@gmail.com
Russian Federation, St. Petersburg, 194064

E. R. Grigorieva

Institute of Cytology, Russian Academy of Sciences

Email: shalygin.alexey@gmail.com
Russian Federation, St. Petersburg, 194064

M. A. Nomerovskaya

Institute of Cytology, Russian Academy of Sciences

Email: shalygin.alexey@gmail.com
Russian Federation, St. Petersburg, 194064

D. S. Reshetin

Institute of Cytology, Russian Academy of Sciences

Email: shalygin.alexey@gmail.com
Russian Federation, St. Petersburg, 194064

A. V. Shalygin

Institute of Cytology, Russian Academy of Sciences

Author for correspondence.
Email: shalygin.alexey@gmail.com
Russian Federation, St. Petersburg, 194064

E. V. Kaznacheyeva

Institute of Cytology, Russian Academy of Sciences

Email: evkazn@incras.ru
Russian Federation, St. Petersburg, 194064

References

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Supplementary files

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2. Fig. 1. Effect of CaCCinh-A01 on the probability of the open state of single ANO6 channels of HEK293 cells. Experiments performed in an inside-out configuration at a potential of +40 mV were used for analysis. a - Fragment of current recording through CaCC ANO6 channels after addition of 10 μM [Ca2+]i and subsequent application of 20 μM CaCCinh-A01. Extended current recording fragments and amplitude histograms are indicated below the current recording fragment. b - Volt-ampere characterization of CaCC ANO6 channels after addition of 20 μM CaCCinh-A01 to the intracellular side of the membrane. c - Open-state probability of CaCC ANO6 channels activated by addition of 10 μM [Ca2+]i at 30 s intervals immediately before (1, black squares) and after application of CaCCinh-A01 (2, gray squares) (p < 0.05).

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3. Fig. 2. Effect of CaCCinh-A01 on the open state lifetime of single ANO6 channels of HEK293 cells. Experiments performed in an inside-out configuration at a potential of +40 mV were used for analysis. a - Fragment of current recording through single CaCC ANO6 activated with 10 μM [Ca2+]i. b - Fragment of current recording through single CaCC ANO6 in the presence of 20 μM CaCCinh-A01. Corresponding histograms of channel lifetime are shown below the current recording fragments. c - Cumulative plot showing the change in lifetime of single CaCC ANO6 channels upon addition of 10 μM [Ca2+]i (1, black squares) and after subsequent application of 20 μM CaCCinh-A01 (2, gray squares) (n = 4, p < 0.05).

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4. Fig. 3. Effect of CaCCinh-A01 on the current amplitude through CaCC ANO6 channels of HEK293 cells. Experiments performed in an inside-out configuration at a potential of +40 mV were used for analysis. a - Extended fragments of current recordings of CaCC ANO6 channels activated by 10 μM [Ca2+]i before and after addition of 20 μM CaCCinh-A01. Amplitude histograms are indicated next to the current recording fragments. b - Graph of the change in current amplitude through CaCC ANO6 channels before (1, black squares) and after (2, gray squares) application of CaCCinh-A01 inhibitor (p < 0.05).

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