Introduction

KCNQ-encoded KV7 channels are key regulators of arterial reactivity. Of the five KCNQ subtypes, KCNQ4 is the most predominantly expressed in the vasculature, followed by KCNQ5 then KCNQ1, with little to no contribution from KCNQ2/3 (Ng et al., 2011; Ohya, Sergeant, Greenwood, & Horowitz, 2003; Yeung et al., 2007). In human and rodent blood vessels KV7 channels contribute to resting tone (Ohya et al. , 2003; Yeung et al. , 2007; Ng et al. , 2011, Mackie et al., 2008) whereby KV7 blockers like linopirdine or XE991 produce contractions or enhance vasoconstrictor responses. In addition, a range of compounds like retigabine or S-1 that increase the activity of KV7.2-7.5 are effective relaxants of pre-contracted arteries. KV7 channels are also functional end targets for a myriad of endogenous vasoactive responses. Channel activity is impaired during PKC-mediated vasoconstriction (L. I. Brueggemann et al., 2006) and enhanced as a result of cGMP and cAMP dependent receptor-mediated vasodilatations (e.g. Chadha et al. , 2012; Khanamiri et al., 2013; Stott, Jepps and Greenwood, 2014; Stott et al. , 2015; Mani et al. , 2016; Brueggemann et al. , 2018). To date, vascular studies on KV7 channels have focused predominantly on vascular smooth muscle cells (VSMCs), and as a result it is currently unclear whether endothelial cell (EC) KV7 channels exist and if so, what their functional role may be.
The inner layer of blood vessels is comprised of ECs, which constitute a paracrine signaling platform that lines all blood vessels. These cells regulate VSMC contractility, vascular resistance and ultimately blood flow through the release of nitric oxide (NO), prostacyclin, epoxyeicosatrienoic acid and others as well as the generation and spread of endothelium-derived hyperpolarization (EDH) (McGuire, Ding, & Triggle, 2001). Myoendothelial projections within fenestrations (holes) of the internal elastic lamina (IEL) facilitate the presence of myoendothelial gap junctions (MEGJs) at a proportion of such sites (~50% in adult rat 1st-3rd order ‘large’ mesenteric arteries; MA; (Sandow et al., 2009). Such junctions facilitate heterocellular electrochemical coupling via connexins at junction sites (Sandow, Senadheera, Bertrand, Murphy, & Tare, 2012). Ultimately, MEGJs enable EC-derived signaling pathways via the flow of both small molecules <~1 kDa and selective currents between cell types. Within rat MA EC-derived vasorelaxant microdomain signaling cascades, previous data has implicated fundamental roles for small/intermediate conductance calcium-activated potassium channels (SKCa and IKCa, respectively; (Sandow, Neylon, Chen, & Garland, 2006); Dora et al., 2008 Circ Res), transient receptor potential canonical type 3 channels (Senadheera et al., 2012), inositol-1,3,4 trisphosphate receptor/s (Sandow CEPP 2009), and inwardly rectifying potassium channels (KIR2) (Goto, Rummery, Grayson, & Hill, 2004). More recently, within mouse MA, KIR2.1 has been identified as a propagator of EC derived signals, acting as a hyperpolarization ‘booster’ (Sonkusare, Dalsgaard, Bonev, & Nelson, 2016).
Despite the contribution of KV7 channels to baseline VSMC tension and receptor-mediated responses, their expression and function within ECs remains relatively unknown. This study shows that KV7 channels are expressed in rat mesenteric ECs and contribute to both KV7 activator mediated vasorelaxation via a novel functional interaction with KIR2 channels and endothelial nitric oxide synthase (eNOS)-dependent carbachol (CCh)-mediated vasorelaxation.