The Role Of The ATP-Sensitive Potassium Channel Subunit, Kir6.1, In Vascular Endothelium

Introduction: ATP-sensitive potassium channels (K_ATP) are widely expressed in the cardiovascular system and they are activated by declining ATP/increasing ADP levels, thus regulating a range of biological activities by linking cellular metabolism with membrane excitability (1). In vascular smooth muscle (SM), K_ATP channels play a role in regulating vascular tone and have been shown to be composed of Kir6.1/SUR2B (2). In addition to SM, K_ATP channels are also thought to be expressed in vascular endothelial cells (ECs) and Kir6.1 is thought to be the underlying pore-forming subunit for this channel. To investigate the role of K_ATP channels in the endothelium, we have generated a murine model that allows conditional deletion of the Kir6.1 subunit in vascular ECs.

Methods: Cre\loxP technology was used to delete exon 2 of the kcnj8 gene in ECs by crossing the Kir6.1 (+, flx) mice with a cre line that expresses the recombinase in ECs (Tie2 cre). Whole tissue organ bath studies, wire myography and Ca²⁺ imaging were used to investigate Kir6.1 expression and their role in the endothelium. Data are expressed as mean ± S.E.M., Student's t test was used for statistical analysis. p<0.05 was considered significant; n=number of animals.

Results: Organ bath studies with wild type (WT) and Kir6.1 endothelial knock-out (KO) mouse aortic rings showed that relaxation of phenylephrine (1µM)-constricted rings evoked by the K_ATP opener pinacidil was; (a) not different between WT and KO in endothelium-intact (+E) rings (EC50s: WT+E,1.32±0.51µM, n=15; KO+E, 2.38±0.86µM; n=11, p>0.05); (b) significantly reduced in WT endothelium-denuded (-E) rings compared with WT+E (EC50s: WT-E, 20.53±6.86µM, n=10, p<0.01); (c) but there was no difference between KO+E and KO-E rings (KO-E, 3.68±0.62µM, n=7, p>0.05). To investigate if the pinacidil response is linked with the nitric oxide (NO) and prostaglandin (PG) pathways, rings were treated with the NO synthase inhibitor, Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 300µM) and Indomethacin, a PG synthesis inhibitor (Indo, 10µM). The relaxation evoked by pinacidil was (a) significantly reduced in WT+E+L-NAME+Indo compared with WT+E rings (EC50s: WT+E+L-NAME+Indo, 5.59±2.77µM, n=5, p<0.05); (b) not different between KO+E+L-NAME+Indo and KO+E rings (EC50s: KO+E+L-NAME+Indo, 4.40±1.91µM, n=5, p>0.05). In comparison, myograph studies with WT and KO mesenteric arteries showed that relaxation evoked by pinacidil was (a) similar in WT and KO in +E rings (EC50s: WT+E, 3.14±1.15µM, n=19; KO+E, 3.30±1.21µM; n=5, p>0.05); (b) significantly reduced in WT-E rings compared with WT+E (EC50s: WT-E, 8.61±1.56µM, n=12, p<0.01); (c) not affected in KO-E rings when compared with KO+E (KO-E, 5.00±1.31µM, n=9, p>0.05). In rings treated with L-NAME+Indo, the pinacidil-induced relaxation was (a) unaffected in WT+E+L-NAME+Indo compared with WT+E rings (EC50s: WT+E+L-NAME+Indo, 2.22±1.06µM, n=9, p>0.05); (b) not significantly different between KO+E+L-NAME+Indo and KO+E rings (EC50s: KO+E+L-NAME+Indo, 1.33±0.16µM, n=5, p>0.05). Imaging of cytosolic [Ca²⁺] in WT mice aortic valve ECs loaded with Fluo-4 demonstrated that pinacidil induced an increase in intracellular Ca²⁺ which can be inhibited by glibenclamide, in contrast, the effects of pinacidil and glibenclamide were not significant in KO mice.

Conclusion: Thus, our data support the existence of an endothelial K_ATP channel composed of Kir6.1 in both mouse aorta and mesenteric arteries. An increase in intracellular [Ca²⁺] and NO/PG release at least partly contribute to the endothelium-dependent vasorelaxation induced by the K_ATP opener pinacidil.

1. Tinker et al (2014). Br J Pharmacol 171(1): 12-23.

2. Aziz et al, (2014). Hypertension 64(3): 523-9.