Angiotensin II reduces the surface abundance of KV 1.5 channels in arterial myocytes to stimulate vasoconstriction

Abstract

Key points: Several different voltage-dependent K⁺ (K_V ) channel isoforms are expressed in arterial smooth muscle cells (myocytes). Vasoconstrictors inhibit K_V currents, but the isoform selectivity and mechanisms involved are unclear. We show that angiotensin II (Ang II), a vasoconstrictor, stimulates degradation of K_V 1.5, but not K_V 2.1, channels through a protein kinase C- and lysosome-dependent mechanism, reducing abundance at the surface of mesenteric artery myocytes. The Ang II-induced decrease in cell surface K_V 1.5 channels reduces whole-cell K_V 1.5 currents and attenuates K_V 1.5 function in pressurized arteries. We describe a mechanism by which Ang II stimulates protein kinase C-dependent K_V 1.5 channel degradation, reducing the abundance of functional channels at the myocyte surface.

Abstract: Smooth muscle cells (myocytes) of resistance-size arteries express several different voltage-dependent K⁺ (K_V ) channels, including K_V 1.5 and K_V 2.1, which regulate contractility. Myocyte K_V currents are inhibited by vasoconstrictors, including angiotensin II (Ang II), but the mechanisms involved are unclear. Here, we tested the hypothesis that Ang II inhibits K_V currents by reducing the plasma membrane abundance of K_V channels in myocytes. Angiotensin II (applied for 2 h) reduced surface and total K_V 1.5 protein in rat mesenteric arteries. In contrast, Ang II did not alter total or surface K_V 2.1, or K_V 1.5 or K_V 2.1 cellular distribution, measured as the percentage of total protein at the surface. Bisindolylmaleimide (BIM; a protein kinase C blocker), a protein kinase C inhibitory peptide or bafilomycin A (a lysosomal degradation inhibitor) each blocked the Ang II-induced decrease in total and surface K_V 1.5. Immunofluorescence also suggested that Ang II reduced surface K_V 1.5 protein in isolated myocytes; an effect inhibited by BIM. Arteries were exposed to Ang II or Ang II plus BIM (for 2 h), after which these agents were removed and contractility measurements performed or myocytes isolated for patch-clamp electrophysiology. Angiotensin II reduced both whole-cell K_V currents and currents inhibited by Psora-4, a K_V 1.5 channel blocker. Angiotensin II also reduced vasoconstriction stimulated by Psora-4 or 4-aminopyridine, another K_V channel inhibitor. These data indicate that Ang II activates protein kinase C, which stimulates K_V 1.5 channel degradation, leading to a decrease in surface K_V 1.5, a reduction in whole-cell K_V 1.5 currents and a loss of functional K_V 1.5 channels in myocytes of pressurized arteries.

Keywords: KV channel; angiotensin II; smooth muscle; vasoconstriction.

Figure 1. Angiotensin II reduces total and surface K_V1.5 channel protein through protein kinase C (PKC) activation in mesenteric arteries

A, representative Western blot images of total arterial K_V1.5, K_V2.1 and actin protein at 0 or 2 h in 6 mm K⁺ physiological saline solution (PSS), 30 mm K⁺ PSS, or 30 mm K⁺ PSS with angiotensin II (Ang II; 100 nm), bisindolylmaleimide (BIM; 10 μm), Ang II plus BIM, or a myristoylated PKC inhibitory peptide (myr‐psi; 100 μm). B, mean data. n = 6 for each. ^* P < 0.05 vs. 0 h. C, immunofluorescence of a section cut from a biotinylated mesenteric artery labelled with Alexa Fluor 546 streptavidin (red) and DAPI (blue), a nuclear stain. Scale bars represent 20 μm. D, representative Western blot images of arterial biotinylation samples illustrating surface (S) and intracellular (I) K_V1.5 and K_V2.1 following 2 h in 30 mm K⁺ alone (control) or in the presence of Ang II (100 nm), BIM (10 μm), Ang II plus BIM, or Ang II plus myr‐psi (100 μm). E, mean data for surface protein in conditions from experiments shown in C calculated as a percentage of control surface protein. n = 6 for each. ^* P < 0.05 vs. control. F, quantitative analysis of the relative cellular distribution of total K_V1.5 and K_V2.1 protein in conditions from C calculated as the percentage of total protein located at the cell surface. n = 6 for each. G, representative image of a Western blot probed with horseradish peroxidase‐conjugated avidin, illustrating that the avidin bead pull‐down procedure is 100% efficient and that no biotinylated proteins are present in the non‐biotinylated protein fraction.

Figure 2. Angiotensin II reduces surface K_V1.5 channels in isolated arterial myocytes

A, immunofluorescence images illustrating colocalization of K_V1.5 (red) and wheat germ agglutinin (WGA, green) in isolated myocytes maintained for 2 h in 30 mm K⁺ alone (control), with Ang II (100 nm) or with Ang II plus BIM (10 μm). Scale bars represent 5 μm. B, quantitative analysis for the percentage of WGA pixels that colocalize with K_V1.5. n = 8 for each. ^* P < 0.05 vs. control.

Figure 3. Angiotensin II stimulates K_V1.5 lysosomal degradation in mesenteric arteries

A, representative Western blot images illustrating surface and intracellular K_V1.5 and K_V2.1 protein after 2 h in 30 mm K⁺ (control), 30 mm K⁺ plus Ang II (100 nm) or 30 mm K⁺ plus Ang II and bafilomycin (Baf, 50 nm). B, mean data illustrating surface K_V1.5 and K_V2.1 protein normalized to control. n = 6 for each. ^* P < 0.05 vs. control. C, quantitative data showing relative cellular distribution of total K_V1.5 and K_V2.1 protein. n = 6 for each.

Figure 4. Angiotensin II reduces whole‐cell K_V and K_V1.5 currents in arterial myocytes

A, representative whole‐cell K_V current recordings in myocytes isolated from arteries that had been maintained for 2 h in 30 mm K⁺ (control), 30 mm K⁺ plus Ang II (100 nm) or 30 mm K⁺ plus Ang II and BIM (10 μm). Currents were measured in myocytes immediately following isolation. B, mean current–voltage relationships for whole‐cell (top, n = 7 for each) and Psora‐4‐sensitive currents (bottom, n = 7 for each). ^* P < 0.05 vs. control.

Figure 5. Angiotensin II reduces functional K_V1.5 channel activity in pressurized arteries

A, representative diameter traces for vasoconstriction stimulated by Psora‐4 (100 nm) or 4‐aminopyridine (4‐AP, 1 mm) at an intravascular pressure of 80 mmHg for arteries maintained for 2 h in 30 mm K⁺ alone (control) or plus Ang II (100 nm) or plus Ang II with BIM (10 μm). Initial arterial diameters before application of Psora‐4 or 4‐AP were normalized. B, mean data illustrating vasoconstriction stimulated by Psora‐4 or 4‐AP. n = 6 for each. Mean passive diameters were similar for arteries used in each data set (P > 0.05 for each, e.g. mean passive diameter for control arteries was 231.6 ± 4.7 μm, for Ang II‐treated arteries 228.0 ± 4.1 μm, and for arteries treated with Ang II and BIM 232.8 ± 4.8 μm). ^* P < 0.05 vs. control. C, mean data for vasoconstrictive response to 60 mm K⁺ PSS at 80 mmHg intravascular pressure. n = 6 for each.