Disruption of K(2P)6.1 produces vascular dysfunction and hypertension in mice

Abstract

K(2P)6.1, a member of the 2-pore domain K channel family, is highly expressed in the vascular system; however, its function is unknown. We tested the following hypotheses. K(2P)6.1 regulates the following: (1) systemic blood pressure; (2) the contractile state of arteries; (3) vascular smooth muscle cell migration; (4) proliferation; and/or (5) volume regulation. Mice lacking K(2P)6.1 (KO) were generated by deleting exon 1 of Kcnk6. Mean arterial blood pressure in both anesthetized and awake KO mice was increased by 17±2 and 26±3 mm Hg, respectively (P<0.05). The resting membrane potential in freshly dispersed vascular smooth muscle cells was depolarized by 17±2 mV in the KO compared with wild-type littermates (P<0.05). The contractile responses to KCl (P<0.05) and BAY K 8644 (P<0.01), an activator of L-type calcium channels, were enhanced in isolated segments of aorta from KO mice. However, there was no difference in the current density of L-type calcium channels. Responses to U46619, an agent that activates rho kinase, showed an enhanced contraction in aorta from KO mice (P<0.001). The BAY K 8644-mediated increase in contraction was decreased to wild-type levels when treated with Y27632, a rho kinase inhibitor, (P<0.05). K(2P)6.1 does not appear to be involved with migration, proliferation, or volume regulation in cultured vascular smooth muscle cells. We conclude that K(2P)6.1 deficiency induces vascular dysfunction and hypertension through a mechanism that may involve smooth muscle cell depolarization and enhanced rho kinase activity.

Figure 1

(A) Thoracic aorta and (B) whole heart RNA expressed as a percent of the reference mRNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The left panel shows expression of six K_2P family gene members; the right panel shows expression levels of three non-K_2P, but vascular related targets. (C) Expression of K_2P6.1 in vascular smooth muscle and endothelial cells of Tie2-GFP aorta. GFP⁺ indicates Tie2-expressing cells as the endothelial fraction (RNA profile eNOS⁺/sm22α⁻). GFP⁻ indicates non-Tie2 expressing cells as the vascular smooth muscle fraction (RNA profile eNOS⁻/sm22α⁺). Data in C are normalized to GADPH and expressed as the % of the gene with greatest expression for each individual fraction. *p<0.05, n=4-10 for each experimental group.

Figure 2

(A) Blood pressure in awake mice (tail plethysmography). (B) Blood pressure and peripheral vascular resistance (ratio of mean arterial pressure and mean aortic velocity) in Nembutal-anesthetized mice. All mice were between 8 and 12 weeks of age. n=6 animals per experimental group. *p≤ 0.05 **p<0.01 ***p<0.001.

Figure 3

(A) Force generated by addition of 60 mmol/L KCl at different resting tensions in thoracic aortas. (B) Concentration response curves for KCl at a resting force of 15 mN. (C) Raw traces of membrane potential, as measured by current clamp, in response to KCl in aortic VSMC from KO (upper trace) versus WT (lower trace). (D) Summary data from membrane potential studies. n=5-8 for each experimental group. ^#p<0.05 using 2-way RM-ANOVA, *p<0.05 and **p<0.01 using the Holm-Sidak method for multiple comparison.

Figure 4

Concentration response curves for (A) phenylephrine, an α-adrenergic agonist, (B) U46619, a thromboxane mimetic, and (C) BAY K 8644, an L-type calcium channel activator in aortic rings from WT and KO mice. n=6-9 per experimental group. ^#p<0.01 using 2-way RM-ANOVA, *p<0.05, **p<0.01, and ***p<0.001 using the Holm-Sidak method for multiple comparison.

Figure 5

(A) Contraction to BAY K 8644, an activator of L-type calcium channels, in control and after pre-incubation of aortic rings with Y27632, a rho kinase inhibitor. (B) Summary data from maximum contractions obtained at 10^-4 mol/L BAY K 8644. n=6 per experimental group. ^#p<0.05 using 2-way RM-ANOVA, *p<0.05, **p<0.01, and ***p<0.001 using the Holm-Sidak method for multiple comparison.

Figure 6

Proposed model for activation of rho kinase in VSM with K_2P6.1 dysfunction. ROCK2 (rho-associated, coiled-coil containing protein kinase 2), MYPT1 (myosin phosphatase target subunit 1), MLC20-P [phosphorylated (active) state of myosin regulatory light chain 20 kD subunit], MLCK (myosin light chain kinase).