Elucidation of the mode of action of TMEM16A modulators and their effects on vascular tone.

The TMEM16A chloride channel represents a fundamental depolarising mechanism in arterial smooth muscle cells (SMCs) and cerebral contractile pericytes. The channel is a proposed target for diseases of impaired vascular tone including stroke, vascular dementia, and hypertension; however, the mechanisms of modulation of the channel by synthetic inhibitors and activators are incompletely understood (Al-Hosni, 2022, 2024). Here, we provide a functional characterization of a recently disclosed (Patent WO2021/014168, compound 1) (Mass, 2021) positive allosteric modulator (PAM_16A) of the TMEM16A channel. In the presence of sub-maximal (300 nM) intracellular free Ca²⁺ concentration [Ca²⁺]i, PAM_16A activated
the heterologously expressed TMEM16A channels at positive and negative potentials (EC₅₀≈4 nM), while being almost ineffective on the closely related TMEM16B channel. PAM_16A did not activate the TMEM16A currents in either the absence of intracellular Ca²⁺ or in the presence of saturating [Ca²⁺]i (12 uM). Mutant TMEM16A channels with the intracellular gate constitutively open were much less sensitive to PAM_16A, suggesting that PAM_16A may act as a modifier of TMEM16A channel gating. Consistent with the effects observed in heterologously expressed TMEM16A channels, PAM_16A activated endogenous TMEM16A currents in isolated rat aortic SMCs, promoted contraction of isolated aortic rings and enhanced capillary (pericyte) constriction evoked by endothelin-1 or oxygen-glucose deprivation (OGD) to stimulate cerebral ischemia. Conversely, inhibiting TMEM16A pharmacologically facilitated arterial and pericyte relaxation, and protected against OGD-mediated pericyte cell death. In summary, this work (i) enhances our understanding of fundamental mechanisms of biophysical modulation of the TMEM16A channel and (ii) supports the view that TMEM16A is a key determinant of SMCs and pericytes tone.