Heteromeric complexes of aldo-keto reductase auxiliary KVβ subunits (AKR6A) regulate sarcolemmal localization of KV1.5 in coronary arterial myocytes

Abstract

Redox-sensitive potassium channels consisting of the voltage-gated K⁺ (K_V) channel pore subunit K_V1.5 regulate resting membrane potential and thereby contractility of vascular smooth muscle cells. Members of the K_V1 family associate with cytosolic auxiliary β subunits, which are members of the aldo-keto reductase (AKR) superfamily (AKR6A subfamily). The Kvβ subunits have been proposed to regulate Kv1 gating via pyridine nucleotide cofactor binding. However, the molecular identity of K_Vβ subunits that associate with native K_V1.5 channels in the vasculature is unknown. Here, we examined mRNA and protein expression of K_Vβ subunits and tested whether K_Vβ isoforms interact with K_V1.5 channels in murine coronary arteries. We detected K_Vβ1 (AKR6A3), K_Vβ2 (AKR6A5) and K_Vβ3 (AKR6A9) transcripts and K_Vβ1 and K_Vβ2 protein in left anterior descending coronary arteries by real time quantitative PCR and Western blot, respectively. In situ proximity ligation assays indicated abundant protein-protein interactions between K_V1.5/K_Vβ1, K_V1.5/K_Vβ2 and K_Vβ1/β2 in coronary arterial myocytes. Confocal microscopy and membrane fractionation analyses suggest that arterial myocytes from K_Vβ2-null mice have reduced abundance of sarcolemmal K_V1.5. Together, data suggest that in coronary arterial myocytes, K_V1.5 channels predominantly associate with K_Vβ1 and K_Vβ2 proteins and that K_Vβ2 performs a chaperone function for K_V1.5 channels in arterial myocytes, thereby facilitating Kv1α trafficking and membrane localization.

Keywords: Myocardial blood flow; Nicotinamide adenine dinucleotide; Oxidoreductase; Potassium channels; Vascular smooth muscle.

Figure 1. mRNA expression and protein abundance of K_V1, K_V2 and K_Vβ subunits in murine coronary arteries

A. RT-PCR gel image demonstrating amplification products for K_V channel subunits as indicated in freshly isolated coronary arteries. B. Summary of transcript expression, expressed as a percent of GAPDH, for K_V1.2, K_V1.5, K_V2.1, K_Vβ1, K_Vβ2, K_Vβ3 in coronary arteries. Mean ± SEM threshold cycle values are shown for each in table inset. (n = 3) C. Images of Western blots for K_Vβ1, K_Vβ2 and K_Vβ3 in murine coronary arteries (CA) and brain (B) lysates. Blots are representative of three independent experiments.

Figure 2. Proximity ligation detection of K_V1.5 and K_Vβ proteins in native coronary arterial myocytes

A. Representative transmitted light (i.), dapi (nuclear stain; ii.) and fluorescent PLA signal (iii.) images (left) and summary bar plot of PLA signal normalized to cell footprint (μm²; right) for isolated coronary arterial myocytes from wild type mice co-labelled with primary antibodies against K_V1.5 and K_Vβ1 (n = 7), K_Vβ2 (n = 9) or K_Vβ3 (n = 5). *P < 0.05 compared to −1° ab group. Data is also shown from negative control experiments in which primary antibodies were omitted from the PLA procedure (−1° ab). Scale bars in transmitted light images represent 10 μm. B–C. Representative images and summary plots showing PLA puncta/μm², as in A, for isolated coronary arterial myocytes from K_Vβ1.1^−/− (B; n = 6–7) and K_Vβ2^−/− (C; n = 5–6) animals co-labelled with primary antibodies against Kv1.5 and K_Vβ1 or K_Vβ2. *P < 0.05

Figure 3. Proximity ligation detection of K_Vβ1 and K_Vβ2 proteins in native coronary arterial myocytes

A. Transmitted light (i.), dapi (ii.) and fluorescent PLA (iii.) images of freshly isolated coronary arterial myocytes labeled with antibodies against K_Vβ1 and K_Vβ2 in cells from wild type (wt), K_Vβ1.1^−/−, and K_Vβ2^−/− animals. Scale bars represent 10 μm. B. Summary bar plot of PLA fluorescent signals, normalized to cell footprint (μm²) for wt (n = 12), K_Vβ1.1^−/−(n = 6), and K_Vβ2^−/− (n = 10) coronary arterial myocytes labeled for K_Vβ1 + K_Vβ2. *P < 0.05

Figure 4. Surface expression of K_V1.5 channels in wild type and K_Vβ2^−/− coronary arterial myocytes

A. Representative blot images (top) showing immunoreactive bands for K_V1.5 and cadherin (pan-cdh; membrane loading control) and corresponding densitometric summary data (bottom) for K_V1.5 subunits in membrane fractions of mesenteric arteries isolated from wild type and K_Vβ2^−/− animals (n = 3 each). B. Differential interference contrast (DIC) and confocal fluorescence images showing wt and K_Vβ2^−/− coronary arterial myocytes stained with alexa-555-conjugated wheat germ agglutinin (WGA) membrane stain and an antibody against an extracellular epitope of K_V1.5 in non-permeabilized myocytes. C. Summary bar plot showing mean intensity (arbitrary units; A.U.) of K_V1.5-associated fluorescence in coronary arterial myocytes from wild type and K_Vβ2^−/− animals (n = 5 cells each). *P < 0.05.