Abstract

Calcium-activated chloride channels (CaCCs) play critical roles in numerous physiological processes, including epithelial secretion, smooth muscle contraction, and cell volume regulation. Anoctamin 6 (ANO6), a member of the TMEM16 family, functions as a CaCC and is a key target for pharmacological modulation. This study investigates the precise mechanism by which the small-molecule inhibitor CaCCinh-A01 reduces the activity of the human ANO6 channel. Using patch-clamp electrophysiology in a heterologous expression system, we characterized the effect of CaCCinh-A01 on both macroscopic and single-channel ANO6 currents. Our results demonstrate that CaCCinh-A01 acts as a non-competitive inhibitor, significantly reducing the maximum current amplitude in a concentration-dependent manner. At the single-channel level, the inhibitor was found to decrease the open-state dwelling time and increase the closed-state duration, suggesting a mechanism of open-channel block and/or allosteric modulation that destabilizes the open conformation. These findings provide molecular insight into the inhibitory action of CaCCinh-A01, clarifying its utility as a pharmacological tool and contributing to the development of more selective therapeutic agents targeting ANO6 in diseases such as cystic fibrosis, cancer, and pain.