The Ca2+-activated Cl- current (ICl(Ca)) in vascular smooth muscle cells (VSMCs) has been suggested to be important for agonist-induced contraction and rhythmic contractions, vasomotion (1). The molecular background for this current is, however, elusive. Bestrophins (BEST) and TMEM16 proteins are the most prominent candidates for this role. We have previously characterized two distinct ICl(Ca) in VSMCs: the cGMP-dependent and the ‘classical’ currents (2).Experiments were approved by the Danish Animal Experiments Inspectorate. Rats were anesthetized for transfection with s.c. injection of hypnorm (1mg/100g) and midazolam (0.5mg/100g), the painkiller Temgesic (2mL/100g) was given. Best-3 or TMEM16A were downregulated in rat mesenteric small arteries in vivo using siRNA. Downregulation was confirmed 3 days after transfection. The contractile responses were tested in vitro using isometric myograph. The ICl(Ca) was measured in isolated VSMCs.Best-3-siRNA reduced Best-3 mRNA and protein to 24±4 % (n=8) and to 28±9 % (n=6) of the level in non-transfected arteries (3). Tmem16a-siRNA reduced TMEM16A mRNA and protein to 25±5 % (n=6) and 37±3 % (n=15) (4). Best-3 downregulation induced secondary reduction in Best-1 and -2, while TMEM16A expression was not affected (5). In contrast, TMEM16A knockdown reduced the expression of all bestrophins.Downregulation of Best-3 suppressed only the cGMP-dependent ICl(Ca) but TMEM16A knockdown suppressed both the cGMP-dependent and the ‘classical’ ICl(Ca) (3; 4). There was no effect on arterial contraction of BEST-3 downregulation (5). In contrast, agonist-induced membrane depolarization, increase in Ca2+ and contraction were decreased in TMEM16A downregulated arteries. Moreover, K+-induced Ca2+ influx was suppressed in the TMEM16A downregulated arteries although K+-induced depolarization was normal. Importantly, L-type Ca2+ channels were found secondary downregulated in the TMEM16A-downregulated arteries (4).Downregulation of either TMEM16A or Best-3 resulted in reduced amplitude of vasomotion, while frequency was unaffected (4; 5).Co-immunoprecipitation and proximity ligation assays suggested an interaction between BEST-3 and TMEM16A proteins. Proximity ligation assay suggested physical proximity between TMEM16A and L-type Ca2+ channels.Both TMEM16A and BEST-3 are associated with the ICl(Ca) in the VSMCs where they interact physically. We suggest that TMEM16A forms the channel pore, while BEST-3 may be a subunit modifying the properties of this CaCC. We suggest that TMEM16A may regulate the expression of BEST-3. Finally, TMEM16A is a protein having more than one function, e.g. regulation of other channel activity and gene transcription, and is also involved in protein-protein interaction.