Ca²⁺-activated Cl^– channels (CaCC) encoded by the Anoctamin-1 (ANO1) or Tmem16a gene, Cav1.2 voltage-dependent Ca²⁺ channels and inositol-tris-phosphate receptors (IP₃R) were recently shown to be colocalized in super clusters in the plasma membrane of mouse pulmonary artery smooth muscle cells (PASMC), and suggested to play an important role in triggering Ca²⁺ waves mediated by Ca²⁺ release from IP₃R.¹  Because of the similarity of these super clusters with clusters formed by the voltage-dependent K⁺ channel Kv2.1 in both mammalian neurons^2,3 and vascular smooth muscle cells,⁴ we tested the hypothesis that surface expression of Kv2.1, which is known to be expressed in PASMCs, is confined to the same microdomains as those formed by ANO1/Cav1.2/IP₃R in mouse PASMCs. Immunolabeling and confocal imaging using specific antibodies to Kv2.1 and ANO1 showed that the two ion channels displayed both punctate staining and larger super clusters; interestingly, the two channels only colocalized in the larger microdomain structures.  Numerous studies have shown that the membrane clustering ability of Kv2.1 could be recapitulated by expression of this K⁺ channel in mammalian cell lines. GFP-tagged mouse Kv2.1 expressed in HEK-293 cells exhibited prominent clustering at the plasma membrane in agreement with previous findings by other groups.  Whole-cell patch clamp experiments were next carried out to determine if co-expression of ANO1-mCherry alters the properties of Kv2.1.  These experiments were carried out in the presence of normal solutions containing K⁺, with 5 mM EGTA in the pipette solution to minimize the activity of ANO1. While the expression of ANO1-ac had no effect on the I-V relationship of peak outward current, steady-state activation curve, or voltage-dependence of activation kinetics of Kv2.1, it decelerated deactivation kinetics as denoted by a -26 mV hyperpolarizing shift in the voltage-dependence of deactivation (Kv2.1: V_0.5 = -32.4 ± 0.7, n=7; Kv2.1 + ANO1: -55.5 ± 3.1 mV, n=8, P < 0.05).  Moreover, block of Kv2.1 at +80 mV by the highly specific Kv2.1 blocker and gating modifier Guanxitoxin-1E (GxTx; 20 nM) was significantly attenuated by co-expression of ANO1-ac (Kv2.1: 69 ± 8.6%, n=6; Kv2.1 + ANO1: 37 ± 10.5%, n=4, P < 0.05). Finally, in Cs- and TEA-based solutions, and 500 nM free internal Ca²⁺ to activate ANO1-a current, 100 nM GxTx surprisingly enhanced ~ 30% (+29 ± 13%, n=5; P < 0.001) when ANO1 was co-expressed with Kv2.1, while it inhibited the current when ANO1 was expressed alone (-52.2 ± 7.7%, n=3; P < 0.001). Although we have to remain cautious about interpreting these preliminary results, our findings suggest that when co-expressed together, ANO1 and Kv2.1 may physically and functionally interact, an observation that may bear important implications for compartmentalized Ca²⁺signaling and contractility in PASMCs, and perhaps other cell types expressing these two ion channels.  All procedures pertaining to housing conditions and animal handling were approved by the University of Nevada IACUC (protocol #20-06-1016-1) in accordance with the Guide for the Care and Use of Laboratory Animals by the National Research Council of the National Academies (8^th edition, 2011).