Molecular and functional characterization of the endothelial ATP-sensitive potassium channel

Abstract

ATP-sensitive potassium (K_ATP) channels are widely expressed in the cardiovascular system, where they regulate a range of biological activities by linking cellular metabolism with membrane excitability. K_ATP channels in vascular smooth muscle have a well-defined role in regulating vascular tone. K_ATP channels are also thought to be expressed in vascular endothelial cells, but their presence and function in this context are less clear. As a result, we aimed to investigate the molecular composition and physiological role of endothelial K_ATP channels. We first generated mice with an endothelial specific deletion of the channel subunit Kir6.1 (eKO) using cre-loxP technology. Data from qRT-PCR, patch clamp, ex vivo coronary perfusion Langendorff heart experiments, and endothelial cell Ca²⁺ imaging comparing eKO and wild-type mice show that Kir6.1-containing K_ATP channels are indeed present in vascular endothelium. An increase in intracellular [Ca²⁺], which is central to changes in endothelial function such as mediator release, at least partly contributes to the endothelium-dependent vasorelaxation induced by the K_ATP channel opener pinacidil. The absence of Kir6.1 did not elevate basal coronary perfusion pressure in eKO mice. However, vasorelaxation was impaired during hypoxia in the coronary circulation, and this resulted in greater cardiac injury during ischemia-reperfusion. The response to adenosine receptor stimulation was impaired in eKO mice in single cells in patch clamp recordings and in the intact coronary circulation. Our data support the existence of an endothelial K_ATP channel that contains Kir6.1, is involved in vascular reactivity in the coronary circulation, and has a protective role in ischemia reperfusion.

Keywords: endothelium; hypoxia; ion channel; potassium channel; vascular biology.

Figure 1.

Generation and characterization of the endothelium-specific knock-out mouse (eKO). A, cre-loxP targeting strategy for the deletion of exon 2 of the Kcnj8 gene. A targeting vector construct for Kcnj8 with a 5′ LoxP site together with a FRT-flanked neomycin (Neo) selection cassette within intron 1 upstream of exon 2 and the second loxP site in intron 2 and diptheria toxin A (DTA) negative selection marker downstream. Mice with the recombined Kcnj8 locus were crossed with global flp-deleter mice to allow Flp-mediated excision of the neomycin selection cassette and generate Kir6.1 (+/flx) offspring. Kir6.1 (flx/flx) mice were then crossed with mice in which the Cre recombinase expression is driven by an endothelium-specific promoter associated with the Tie2 receptor to produce tie2cre+ Kir6.1(flx/flx) (eKO) mice. B, genotyping of DNA isolated from tail for the floxed alleles (top panel) and Cre recombinase (bottom panel). C and D, relative levels of expression of Kir6.1, Kir6.2 and SUR2 in the aorta (C) and mesenteric arteries (D) with intact endothelium (+E) and with endothelium denuded (−E) from WT and eKO mice. No CT values were detectable for SUR1. The gene expression level in each case was normalized to the WT + endothelium (WT+E). The data are shown as means ± S.E. (n = 6 mice). **, p < 0.01; ***, p < 0.001 compared with WT + endothelium.

Figure 2.

Endothelial-specific Kir6.1 deletion results in an attenuated K_ATP current in aortic endothelial cells. A and B, representative time-course traces at +40 mV (right panels) and whole-cell current density-voltage traces (left panels) taken from ECs isolated from WT (A) and eKO (B) mice showing the effects of pinacidil (Pin) and glibenclamide (Glib). The control (Con) trace is shown in gray. Current-voltage relationships were recorded using a 1-s ramp protocol (−150 to +50 mV) from a holding potential of −80 mV. C, summary of the mean current densities at +40 mV from ECs isolated from WT (left panel) and eKO (right panel) mice. D, mean glibenclamide-sensitive current in ECs from WT and eKO mice (n = 8–20 cells from 4–9 mice). E–H, representative (E and F) and mean data (G and H) of whole-cell currents recorded from SMCs isolated from WT and eKO mice (n = 10–20 cells from 4–9 mice). The data are shown as means ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with control/WT.

Figure 3.

Pinacidil-induced elevation of cytosolic [Ca²⁺] is attenuated in eKO mouse aortic valves. A, confocal images showing a pinacidil-evoked concentration-dependent elevation in cytosolic [Ca²⁺] in WT mice. 100 μm CPA (sarcoplasmic reticulum uptake inhibitor) was used as a control to induce an increase in cytosolic [Ca²⁺] at baseline. B, confocal images showing the effect of CPA and pinacidil on cytosolic [Ca²⁺] in valves from eKO mice. Scale bar represents 25 μm. C, summary time-course traces (S.E. in the dashed lines) illustrating the effects of 100 μm CPA, 10 and 100 μm pinacidil (Pin), and 10 μm glibenclamide (Glib) on cytosolic [Ca²⁺] in aortic valve cells from WT (black) and eKO (gray) mice. Changes in cytosolic [Ca²⁺] are expressed as a relative fluorescence ratio, F/F₀. D, bar graph showing the mean data from traces in C. The data are shown as means ± S.E. (n = 6 mice). *, p < 0.05; ***, p < 0.001 compared with WT; ^+++, p < 0.001 compared with WT 10 μm pinacidil.

Figure 4.

Kir6.1 containing K_ATP channels are present in both endothelium and smooth muscle of the coronary circulation. A, mean basal CPP in eKO and smKO mice and their littermate controls (n = 9–23). CPP was measured using the Langendorff set-up under constant flow. B, representative CPP traces from Langendorff hearts from eKO, smKO mice, and their littermate controls (WT, top panels; KO, lower panels) in response to 10 μm pinacidil (Pin) and 10 μm glibenclamide (Glib). C, summary of the mean change in CPP (relative to baseline) of WT and eKO mouse hearts in response to 10 μm pinacidil and 10 μm glibenclamide. The data are shown as means ± S.E. (n = 5–10 mice). *, p < 0.05; **, p < 0.01 compared with WT.

Figure 5.

Endothelial K_ATP channels containing Kir6.1 may be protective during metabolic challenge. A, mean CPP of eKO and smKO mouse hearts and hearts from their respective littermate controls in response to hypoxia (Krebs solution gassed with 95% N₂/5% CO₂) (n = 6–10). CPP was measured using the Langendorff set-up under constant flow. B and C, representative sections (left panel) and mean infarct size (right panel) following 30 min of global ischemia and 60 min of reperfusion of eKO (B) and smKO (C) mouse hearts stained with 1% TTC; pale tissue signifies infarction. The data are shown as means ± S.E. (n = 5–16 mice). *, p < 0.05; **, p < 0.01 compared with WT.

Figure 6.

Ablation of Kir6.2 does not attenuate K_ATP current in aortic ECs and is not protective in IR injury. A, representative time-course traces at +40 mV taken from ECs isolated from WT (left panel) and Kir6.2 eKO (right panel) mice showing the effects of pinacidil (Pin) and glibenclamide (Glib). Currents were elicited using a 1-s ramp protocol (−150 mV to +50 mV) from a holding potential of −80 mV. B, summary of the mean current-densities at +40 mV from ECs isolated from WT (left panel) and Kir6.2 eKO (right panel) mice. C, mean glibenclamide-sensitive current in ECs from WT and Kir6.2 eKO mice (n = 9–14 cells from three mice). D, mean basal CPP of WT and Kir6.2 eKO mouse hearts (n = 10–13). CPP was measured using the Langendorff set-up under constant flow. E, mean change in CPP (relative to baseline) in the presence of pinacidil, glibenclamide, and hypoxia of WT and Kir6.2 eKO hearts (n = 5–7). F, representative sections (left panel) and mean infarct size (right panel) following 30 min of global ischemia and 60 min of reperfusion of WT and Kir6.2 eKO mouse hearts stained with 1% TTC, pale tissue signifies infarction (n = 11). The data are shown as means ± S.E. ***, p < 0.001 compared with control.

Figure 7.

Effect of the adenosine agonist NECA is reduced in aortic ECs and the coronary circulation of eKO mice. A, representative time-course traces at +40 mV taken from ECs isolated from WT (left panel) and eKO (right panel) mice showing the effects of NECA. B, representative current-voltage traces from a WT (left panel) and eKO (right panel) cell in the presence and absence of NECA. Currents were elicited using a 1-s ramp protocol (−150 to +50 mV) from a holding potential of −80 mV. C, summary of the mean current-densities at +40 mV from ECs isolated from WT (left panel) and eKO (right panel) mice (n = 8–9 cells from three mice). D, representative CPP traces from Langendorff hearts from WT and eKO mice challenged with NECA and glibenclamide. E, mean change in CPP (relative to baseline) of WT and eKO mouse hearts in the presence of NECA (n = 6–10). CPP was measured using the Langendorff set-up under constant flow. The data are shown as means ± S.E. *, p < 0.05; **, p < 0.01 compared with control/WT.