Anoctamin 1 (ANO1) or TMEM16A gene encodes a member of Ca2+ activated Cl– channels (CaCCs) that are critical for physiological functions, such as epithelial secretion, smooth muscle contraction and sensory signal transduction. The attraction and interest in ANO1/TMEM16A arise from a decade long investigations that abnormal expression or dysfunction of ANO1 is involved in many pathological phenotypes and diseases, including asthma, neuropathic pain, hypertension and cancer. However, the lack of specific modulators of ANO1 has impeded the efforts to validate ANO1 as a therapeutic target. This review focuses on the recent progress made in understanding of the pathophysiological functions of CaCC ANO1 and the current modulators used as pharmacological tools, hopefully illustrating a broad spectrum of ANO1 channelopathy and a path forward for this target validation.

The swelling-activated chloride current ( I_Cl,swell) is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of I_Cl,swell is the volume-regulated anion channel (VRAC). Leucine-rich repeat containing 8A (LRRC8A; SWELL1) was recently identified as an essential component of VRAC, but the mechanisms of VRAC activation are still largely unknown; moreover, other Cl⁻ channels, such as anoctamin 1 (ANO1), were also suggested to contribute to I_Cl,swell. In this present study, we investigated the roles of LRRC8A and ANO1 in activation of I_Cl,swell; we also explored the role of intracellular Ca²⁺ in I_Cl,swell activation. We used a CRISPR/Cas9 gene editing approach, electrophysiology, live fluorescent imaging, selective pharmacology, and other approaches to show that both LRRC8A and ANO1 can be activated by cell swelling in HEK293 cells. Yet, both channels contribute biophysically and pharmacologically distinct components to I_Cl,swell, with LRRC8A being the major component. Cell swelling induced oscillatory Ca²⁺ transients, and these Ca²⁺ signals were required to activate both the LRRC8A- and ANO1-dependent components of I_Cl,swell. Both I_Cl,swell components required localized rather than global Ca²⁺ for activation. Interestingly, while intracellular Ca²⁺ was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca²⁺ transients linked to the I_Cl,swell activation were mediated by the G protein-coupled receptor-independent PLC isoforms.

A uricosuric drug benzbromarone, widely used for treatment of gout, hyperpolarizes the membrane potential of airway smooth muscle cells, but how it works remains unknown. Here we show a novel role of benzbromarone in activation of large conductance calcium-activated K⁺ channels. Benzbromarone results in dose-dependent activation of macroscopic BK currents about 1.7 to 14.5 folds with an EC₅₀ of 111 μM and shifts the voltage-dependent channel activation to a more hyperpolarizing direction about 10 to 54 mV in whole-cell patch clamp recordings. In single-channel recordings, benzbromarone decreases single BKα channel closed dwell time and increases the channel open probability. Co-expressing β1 subunit also enhances BK activation by benzbromarone with an EC50 of 67 μM and a leftward shift of G-V curve about 44 to 138 mV. Site-directed mutagenesis reveals that a motif of three amino acids ³²⁹RKK³³¹ in the cytoplasmic linker between S6 and C-terminal RCK gating ring mediates the pharmacological activation of BK channels by benzbromarone. Further ex vivo assay shows that benzbromarone causes reduction of tracheal strip contraction. Taken together, our findings demonstrate that uricosuric benzbromarone activates BK channels through molecular mechanism of action involving the channel RKK motif of S6-RCK linker. Pharmacological activation of BK channel by benzbromarone causes reduction of tracheal strip contraction, holding a repurposing potential for asthma and pulmonary arterial hypertension or BK channelopathies. Significance Statement We describe a novel role of uricosuric agent benzbromarone in BK channel activation and relaxation of airway smooth muscle contraction. In this study, we find that benzbromarone is an activator of the large-conductance Ca²⁺- and voltage-activated K⁺ channel (BK channel) that serves numerous cellular functions including control of smooth muscle contraction. Pharmacological activation of BK channel by an FDA approved drug benzbromarone may hold repurposing potential for treatment of asthma and pulmonary arterial hypertension or BK channeopathies.

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel, predominantly expressed in a subset of peripheral sensory neurons for pain signaling. Topical application of agonist capsaicin for desensitizing TRPV1 currents has been approved for relief of chronic pain. However, the potent TRPV1 capsaicin is not ingestible and even topical capsaicin causes common side effects such as skin irritation, swelling, erythema and pruritus, suggesting that a mild TRPV1 agonist might be helpful for reducing side effects while reliving pain. In this study, we reported on a partial and selective TRPV1 agonist 4-(5-chloropyridin-2-yl)-N-(1H-indazol-6-yl)piperazine-1-carboxamide named CPIPC that was modified based on targeting the residue Arg557, important for conversion between the channel antagonism and agonism. Whole-cell patch clamp recordings indicated a concentration-dependent activation of TRPV1 currents by CPIPC with an EC₅₀ of 1.56 ± 0.13 μM. The maximum efficacy of CPIPC (30 μM) was about 60% of saturated capsaicin (10 μM). Repetitive additions of CPIPC caused TRPV1 current desensitization in both TRPV1-expressing HEK293 cells and dorsal root ganglion (DRG) sensory neurons. Oral administration of CPIPC dose-dependently alleviated inflammatory pain in mice. Further site-directed mutagenesis combined with molecular docking revealed that residue Arg557 is critical for TRPV1 activation by CPIPC. Taken together, we identified a novel partial and selective TRPV1 agonist CPIPC that exhibits antinociceptive activity in mice.