Background Kv7 voltage-gated potassium channels (Kv7.1-5) are expressed in rodent and human arterial beds, with Kv7.4 and Kv7.5 playing a key functional role in regulating vascular tone1. Previously we have shown Kv7.4 and Kv7.5 form predominantly heteromeric channels in cerebral vascular smooth muscle cells (VSMCs)2. Here we extend this study to investigate the molecular architecture of Kv7s in renal and mesenteric VSMCs using Proximity Ligation Assay (PLA) technology3.Methods The Duolink PLA detection kit 563 was used to visualise single molecule interactions for Kv7.4, Kv7.5 and Kv7.1 in renal and mesenteric arteries isolated from male Wistar rats. Single myocytes were enzymatically isolated and incubated with pairs of primary antibodies (rabbit Kv7.4+goat Kv7.4; rabbit Kv7.4+goat Kv7.5; goat Kv7.4+rabbit Kv7.5; rabbit Kv7.5+goat Kv7.5; goat Kv7.4+rabbit Kv7.1; 1:200). Controls omitted primary antibodies or employed only 1 primary antibody. Cells were labelled with anti-rabbit PLUS and anti-goat MINUS probes attached to synthetic oligonucleotides that hybridise when <40nm apart, producing a red spot visualised using confocal microscopy. Mid-cell xy sections were selected and puncta assessed. Statistical analysis used 1-way ANOVA, Tukey’s multiple comparisons test.Results Quantification of the mean number (±SEM) of PLA signals in mesenteric VSMCs revealed a high number of puncta per cell and of similar magnitude in the Kv7.4/Kv7.4 (52.75±12.28, n=12) and Kv7.4/Kv7.5 combinations (44.88±7.31, n=17; 33.44±4.12, n=17, P=ns). Kv7.5/Kv7.5 and Kv7.4/Kv7.1 produced a significantly lower number of PLA signals (4.50±1.22, n=13, and 6.63±1.81, n=19, respectively, P<0.0001 vs Kv7.4/7.4). In renal VSMCs, Kv7.4/Kv7.4 combination again produced a high number of puncta (63.59 ±18.16, n=15) that was not significantly different from Kv7.4/7.5 (67.04±15.70, n=12; 71.65±11.79, n=17; P=ns). However, unlike in mesenteric VSMCs, Kv7.5/Kv7.5 and Kv7.4/Kv7.1 combinations were of similar magnitude to Kv7.4/7.4 and Kv7.4/7.5 (61.74±10.24, n=19; 41.05±7.52, n=19, respectively, P=ns). In both cell types no chromatic product was visualised for Kv7.4 and Kv7.5 antibodies applied alone or no primary controls. All experiments used N=3-4 rats.Conclusions In mesenteric VSMCs, PLA technology strongly suggests Kv7.4/7.5 heterotetramers predominate, consistent with recent findings4. Kv7.5 is unlikely to exist as a homomer and Kv7.4/Kv7.1 are not in close proximity. In renal VSMCs, a different molecular architecture is proposed. Kv7.5/7.5 PLA signals were high and equivalent to Kv7.4/7.4 or Kv7.4/Kv7.5 indicating that again Kv7.4/7.5 heterotetramers may predominate but with a different stoichiometry. Kv7.4/7.1 subunits were also shown to coexist in the same cellular microdomain. The study provides new insights into tetrameric subunit assembly that could be key to regulation of vascular Kv7 channels.