Functional interplay of CFTR and TMEM16A channels in mesenteric artery smooth muscle.

H.A.T Pritchard, H.R Askew-Page, G. Carr, A.P Albert, I.A Greenwood. St. George's, University of London, London, UK

Introduction: As vascular smooth muscle cells actively accumulate Cl- ions, activation of Cl- channels results in Cl- efflux. This leads to membrane depolarisation and activation of voltage gated Ca2+ channels, resulting in smooth muscle contraction. This is the method by which Ca2+-activated Cl- channels, encoded by TMEM16A, cause vasoconstriction (1). Vascular smooth muscle contains also express the cAMP/PKA activated Cl- channel CFTR; however the role of this protein within vascular smooth muscle is not clear. The aim of this study was to establish the effect of CFTR and TMEM16A modulators on vascular tone.

Methods & results: Male Wistar rats were killed in accordance with Schedule 1 of the United Kingdom Animals Act (1986) and 3rd order mesenteric artery segments were isolated. Using qPCR, both CFTR and TMEM16A mRNA were detected at similar levels, relative to the house keeping genes (CYC1 & ACTB). Proximity ligation assay (PLA) identified that CFTR and TMEM16A co-locate within 40 nm of each other, in freshly isolated mesenteric smooth muscle cells. To study the role of CFTR and TMEM16A in vascular physiology isometric tension recordings were carried out on mesenteric segments, with statistical differences determined using the two-way ANOVA test. Vessels were pre-contracted with 300nM U46619 and the specific TMEM16A inhibitor T16Ainh-A01 produced a concentration-dependent relaxation. At low concentrations the CFTR inhibitor (0.3-1 μM), oxo-CFTR-172, produced a small contraction, on top of the U46619 induced contraction, whereas higher concentrations resulted in a full relaxation. The increase in contraction was hypothesised to be due to increased TMEM16A activity, which was confirmed with the application of a low concentration of T16Ainh-A01 (300nM), prior to the increasing concentrations of oxo-CFTR-172. This resulted in an increased efficacy of oxo-CFTR-172, and the absence of the small contraction at low concentrations (N=6, p<0.05). Pre-application of a low concentration of oxo-CFTR-172 increased the potency of T16Ainh-A01 significantly (N=6, p<0.01). Increasing cAMP levels with either 1 nM Forskolin or 1 μM 8-Br-cAMP, which theoretically increased CFTR activity, decreased the potency of T16Ainh-A01 (N=7-8, p<0.01 & p<0.05 respectively). To confirm this effect, vessels were exposed to 1 μM Rp-8-Br-cAMP or 1 μM H89, to inhibit PKA/cAMP activity and decrease CFTR activity, which resulted in an increase in T16Ainh-A01 potency (N=7, p<0.01 & p<0.05 respectively).

Conclusions: We have shown that TMEM16A is involved in vascular smooth muscle contraction whereas a CFTR has more complex role including a negative interaction with TMEM16A.

References: 1. Davis et al. (2013) Br J Pharmacol 171: 4311-21