Sorbs2 Deficiency and Vascular BK Channelopathy in Diabetes Mellitus

Abstract

Background:

Vascular BK channel, composed of the BK-α and BK-β1 subunits, is a key determinant of coronary vasorelaxation and its function is impaired in diabetic vessels. However, our knowledge of diabetic BK channel dysregulation is incomplete. The Sorbin homology and Src homology 3 domain-containing protein 2 (Sorbs2) is ubiquitously expressed in arteries, but its role in vascular pathophysiology is unknown.

Methods:

The role of Sorbs2 in regulating vascular BK channel activity was determined using patch clamp recordings, molecular biological techniques, and in silico analysis.

Results:

Sorbs2 is not only a cytoskeletal protein but also an RNA-binding protein that binds to BK channel proteins and the Kcnma1 (encoding BK-α) mRNA, regulating BK channel expression and function in coronary smooth muscle cells (SMCs). Molecular biological studies reveal that the SH3 domain of Sorbs2 is necessary for Sorbs2 interaction with BK-α, while both the SH3 and SoHo domains of Sorbs2 interact with BK-β1. Deletion of the SH3 or SoHo domains abolishes the Sorbs2 effect on BK-α/BK-β1 current density. Additionally, Sorbs2 is a target gene of the nuclear factor erythroid-2-related factor 2 (Nrf2), which binds to the promoter of Sorbs2 and regulates Sorbs2 expression in coronary SMCs. In vivo studies demonstrate that Sorbs2 KO mice at 4 months of age display a significant decrease in BK channel expression and function, accompanied by impaired BK channel Ca²⁺-sensitivity and BK channel-mediated vasodilation in coronary arteries, without altering their body weights and blood glucose levels. Importantly, Sorbs2 expression is significantly downregulated in the coronary arteries of db/db type 2 diabetic mice.

Conclusions:

Sorbs2, a downstream target of Nrf2, plays an important role in regulating BK channel expression and function in vascular SMCs. Vascular Sorbs2 is downregulated in diabetes. Genetic knockout of Sorbs2 manifests coronary BK channelopathy and vasculopathy observed in diabetic mice, independent of obesity and glucotoxicity.

Graphical Abstract

INTRODUCTION

The incidence of diabetes mellitus has rapidly increased in recent decades. According to the WHO report in 2021, more than 422 million people worldwide suffered from diabetes with a global prevalence of 8.6%. Diabetes is associated with a significant increased risk of cardiovascular diseases in men and women, causing 1.6 million deaths annually in the world.¹ Although the morbidity and mortality of cardiovascular diseases have declined by 35% to 40% over recent decades in non-diabetic patients, such decline was not observed in patients with diabetes.^2–4 The cardiovascular complications in diabetes have been attributed to compromised coronary physiology. In coronary smooth muscle cells (SMCs), the large conductance Ca²⁺-activated K⁺ (BK) channels, composed of the pore-forming BK-α subunits (encoded by the Kcnma1 gene) and the regulatory BK-β1 subunits (encoded by the Kcnmb1 gene) in 4:4 stoichiometry, are major determinants of coronary vascular tone and myocardial perfusion.^5,6 BK-α subunit is comprised of seven transmembrane domains (S0-S6) in which the S0-S4 form the voltage sensor and the S5-S6 constitute the ion permeating pore region, conducting large amounts of potassium ions (K⁺) across cell membrane. BK-α has a huge C-terminus containing two regulators of K⁺ conductance (RCK1 and RCK2) and these RCKs have two distinct high-affinity Ca²⁺ binding sites. The first high-affinity Ca²⁺ binding site is located in (892)-DQDDDDDPD-(900) of the RCK2, known as the Ca²⁺ bowl, and the second is in the RCK1 that encompasses residues D362, D367, and E535.⁷ Moreover, the RCK1 and RCK2 of four BK-α subunits form an octameric gating-ring that connects to the voltage sensor and plays a pivotal role in BK channel activation upon voltage and Ca²⁺ stimulation.⁷ In addition, the presence of BK-β1 subunit greatly enhances BK-α sensitivity to Ca²⁺ and voltage stimulations, allowing the channel to open under physiological conditions.⁷ We have reported that BK-β1 expression is downregulated in diabetic vessels, contributing to vascular dysfunction in both type 1 and type 2 diabetes.^1,8 Moreover, the nuclear factor erythroid-2-related factor 2 (Nrf2) signaling, which regulates the Kcnma1 transcription, is significantly downregulated, leading to vascular BK channel dysregulation in diabetes.^9,10 However, the mechanisms that underlie vascular BK channel pathophysiology in diabetes remain largely unknown.

The Sorbin homology (SoHo) and Src homology 3 (SH3) domain-containing protein 2 (Sorbs2), containing one SoHo domain in the N-terminus and three SH3 domains in the C-terminus, is an important cardiac cytoskeletal protein that interacts with other cellular proteins. It has been reported that Sorbs2 is not only a cytoskeletal protein but also an RNA-binding protein (RBP) that interacts with a wide variety of cardiac ion channel mRNAs and proteins in the heart and plays a critical role in regulating cardiac contractility and electrical excitability.^11,12 Genetic deletion of the Sorbs2 gene results in cardiomyopathy,^11,13 cardiac ion channelopathy, and life-threatening arrhythmias in mice.¹² The clinical importance of Sorbs2 is underscored by the findings that Sorbs2 C-terminal deletions are linked to congenital heart diseases in human^14,15 and loss-of-function splicing variants of Sorbs2 are implicated in patients with arrhythmogenic cardiomyopathy.¹¹ However, our understanding on the role of Sorbs2 in vascular pathophysiology is primitive. In this study, we investigated the role of Sorbs2 in the regulation of vascular BK-α and BK-β1 expression and function and further determine the effects of Sorbs2 deficiency on BK channel activity and BK channel-mediated vasodilation in coronary arteries using Sorbs2 knockout (KO) mice. Results from our study indicate that Sorbs2 deficiency manifests vascular BK channel pathophysiology similar to that observed in diabetes.

METHODS

DATA AVAILABILITY

Details of research designs, major resources, sporting data, and statistical methods used for each experiment are available in Supplemental Materials. The next-generation sequencing data have been deposited in the National Library of Medicine BioSample Database (https://www.ncbi.nlm.nih.gov/biosample).

Data were presented as means±standard error (S.E.M.) All representative images/figures were chosen according to the mean value of each group results. Statistically significant difference was defined as p<0.05.

RESULTS

Interaction of Sorbs2 with BK channel proteins in coronary arterial SMCs

Sorbs2 is ubiquitously expressed in mouse arteries including aortas, carotid arteries, coronary arteries, basal arteries, and mesentery arteries (Figure S1A and S1B). In human coronary arteries, the protein levels of Sorbs2 are 1.5-fold higher in human primary coronary SMCs than in human coronary endothelial cells (ECs) (Figure 1A). Using co-immunoprecipitation (co-IP), immunofluorescence staining, and in situ proximity ligation assay (PLA), we confirmed that Sorbs2 is physically associated and colocalized with BK-α and BK-β1 proteins in primary human coronary SMCs (Figure 1B and 1C). Negative controls for the immunofluorescence staining and PLA assays without the primary antibodies are shown in Figure S2 and S3 in Supplemental Materials. Moreover, a 68.3% silencing of Sorbs2 by shRNA reduced the protein levels of BK-α and BK-β1 by 54.5% and 57.3% respectively, as well as interrupted the protein interaction of Sorbs2 with BK-α and BK-β1 in freshly isolated coronary SMCs from WT mice (Figure 1D and 1E). These results suggest that Sorbs2 physically interacts with BK-α and BK-β1 and regulates BK-α and BK-β1 expression in coronary SMCs.

Figure 1: Sorbs2 interaction with BK-α and BK-β1 in human coronary SMCs.

A: Sorbs2 protein is strongly expressed in primary human coronary endothelial cells (ECs) and primary human coronary smooth muscle cells (SMCs). The primary human coronary SMCs have 52.7% higher levels of Sorbs2 expression compared to the primary human coronary ECs (n=4, the Wilcoxon test). B: BK-α and BK-β1 proteins are detected in the immunoprecipitates pulled down by anti-Sorbs2 antibodies from the lysates of primary human coronary SMCs. C: Immunofluorescence staining show the colocalization of Sorbs2 with BK-α and BK-β1 in the primary human coronary SMCs with the Pearson’s correlation coefficient (R) of 0.719 and 0.754 respectively. Note that R values between 0.7 and 1.0 indicate a strong positive association. Red lines represent the linear relationship between the colocalization Sorbs2 with BK-α and BK-β1. D: In situ proximity ligation assay (PLA) was performed in freshly isolated coronary SMCs of WT mice after a 100 min-amplification reaction at 37 °C with labelled complementary oligonucleotide probes and incubated with mouse anti-Sorbs2 and rabbit anti-BK-α or anti-BK-β1 antibodies respectively. There is a marked decrease in the fluorescent signals of ligation (red dots) in cells with Sorbs2 shRNA knockdown, compared to cells with scramble RNAi control as shown in box plot. The nuclei (blue) were counterstained with DAPI. Average fluorescent signals per cell are summarized in box plot (n=7 cells, the Shapiro-Wilk test and two-tailed unpaired t-test with Holm correction). E: Western blot analysis confirms a 68.3% knockdown of Sorbs2 by shRNA reduced BK-α and BK-β1 protein levels by 54.5% (n=6 biological replicates, the Shapiro-Wilk test and two-tailed unpaired t-test with the Holm correction) and 57.3% (n=6 biological replicates, the Shapiro-Wilk test and two-tailed unpaired t-test with the Holm correction) respectively in freshly isolated mouse coronary SMCs as shown in box plot.

Downregulation of BK channel expression and function in the arteries of Sorbs2 KO mice

To determine the role of Sorbs2 in the regulation of vascular BK channel physiology in vivo, we measured vascular BK channel expression and function in the arteries of Sorbs2 knockout (KO) mice and age-matched wild type (WT) mice at 4 months of age (Figure 2A). The cytosolic and surface expression of BK-α and BK-β1 was determined by surface biotinylation assay in the carotid arteries of Sorbs2 KO mice and WT control mice. There is a significant reduction in the cytosolic and surface expression of BK-α by 70.89±2.82% and 49.16±8.43% respectively, and that of BK-β1 by 57.42±6.13% and 40.81±10.51% respectively in Sorbs2 KO mice (Figure 2B). Moreover, voltage-dependent BK channel activation in freshly isolated coronary SMCs (Figure 2C) and BK channel-mediated coronary vasodilation were diminished in Sorbs2 KO mice, compared to those of WT mice (n=5 mice for both groups, Pr(>F)=1.1e-12, and 1.4e-18 respectively, a linear mixed effect model with a false discovery rate (FDR) correction) (Figure 2D). There results indicate that Sorbs2 KO mice have downregulated BK channel expression and impaired BK channel function in arteries. Additionally, these animals maintain normal body weights (25.9±1.3 g of WT mice vs. 23.3±1.5 g of Sorbs2 KO mice, n=14, p=0.207, the Shapiro-Wilk test and two-tailed unpaired t-test) and blood glucose levels (166.4±7.7 mg/dL of WT mice vs. 152.2±12.2 mg/dL of Sorbs2 KO mice, n=14, p=0.341, the Shapiro-Wilk test and two-tailed unpaired t-test).

Figure 2: Abnormal BK channel expression and function in Sorbs2 KO mouse arteries.

A: Sorbs2 protein expression in the arteries of Sorbs2 KO mice are undetectable using mouse monoclonal anti-Sorbs2 antibody (n=6 mice for both groups, the Shapiro-Wilk test and two-tailed unpaired t-test with the Holm correction). B: Cell surface and cytoplasmic distribution of BK-α and BK-β1 proteins in mouse carotid arteries was determined by surface biotinylation assay. The cytoplasmic and the surface protein levels of BK-α and BK-β1 were normalized to those of WT mice. Purify of membrane protein preparation was confirmed by lack of GAPDH. There is a significant reduction in the cytosolic and surface expression of BK-α by 70.89±2.82% and 49.16±8.43% respectively (the Wilcoxon test with the Holm correction) and that of BK-β1 by 57.42±6.13% and 40.81±10.51% respectively (the Wilcoxon test with the Holm correction) in Sorbs2 KO mice, compared to those of WT mice (n=4 biological replates from 8 mice for both groups). C: Whole-cell BK channel currents (defined as 0.1 μM IBTX-sensitive K⁺ components) were recorded from freshly isolated coronary SMCs. The current-voltage relationship (I-V curve) of BK current channels is significantly downregulated in Sorbs2 KO mice (n=5 for both groups, Pr(>F) =1.1e-12, a linear mixed effect model with an FDR correction). The adjusted p-values at all given voltages are available in the Statistical Analysis Table of Supplemental Materials. D: Sorbs2 KO mice have reduced coronary vasodilation to NS-1619, compared to that in WT mice (n=5 for each group, Pr(>F)=1.4e-18, a linear mixed effect model with an FDR correction).

To further examine Ca²⁺-dependent BK channel activation, we measured BK channel Ca²⁺ sensitivity in inside-out membrane patches excised from freshly isolated coronary SMCs of WT and Sorbs2 KO mice. Coronary BK channel maximal open probability (nPo) was robust in WT mice in response to gradual increase in free Ca²⁺ concentrations in the bath solution. However, the BK channel nPo in response to Ca²⁺ was blunted in Sorbs2 KO mice. There is a statistical difference in the Ca²⁺-dependent BK channel activation between WT and Sorbs2 KO mice (n=13 cells for both, Pr(>F)=3.9e-15, the Shapiro-Wilk test and a linear mixed effect model with an FDR correction), particularly at Ca²⁺ concentrations higher than 0.5 μM (Figure 3, left panel). For instance, the maximal nPo in the presence of 100 μM cytoplasmic free Ca²⁺ was 4.09±0.58 in WT mice (n=13 cells from 5 mice) but 2.31±0.45 in Sorbs2 KO mice (n=13 cells from 5 mice, p=1.8×10⁻⁷). The Ca²⁺ EC₅₀ was 0.48±0.07 μM with a Hill coefficient (nH) of 2.72 in WT mice, but it was 2.78±0.68 μM with a nH of 1.26 in Sorbs2 KO mice (Figure 3, right panel), indicating that Sorbs2 KO mice have reduced efficacy, efficiency, and cooperativity to Ca²⁺ in coronary BK channel activation.

Figure 3: Reduced BK channel Ca²⁺-sensitivity in the coronary arteriolar SMCs of Sorbs2 KO mice.

A: Representative single BK channel currents were continuously recored at +60 mV from an excised inside-out patch in the presence of various free Ca²⁺ concentrations exposed to the cytoplasmic membrane surface of freshly isolated coronary arteriolar SMCs from WT and Sorbs2 KO mice. BK channel maximal open probabilities (nPo) in response to increase of Ca²⁺ concentrations are significantly reduced in Sorbs2 KO mice (right panel) compared to WT mice (left panel). Dashed lines indicate the channel closed state (c) and multiple levels of single channel openings (o₁-o₃). B: The BK channel nPo (left panel) and Ca²⁺ concentration-dependent relationships (nPo-log[Ca²⁺] curves) (right panel) were fitted using the Hill equation. Sorbs2 KO mice have decreased nPo at given Ca²⁺ concentrations higher than 0.5 μM (left panel), rightwardly shifted Ca²⁺ concentration-dependent curve, and a smaller Hill coefficient (right panel) (n=13 cells from 5 mice for both groups, Pr(>F)=3.5e-19, the Shapiro-Wilk test and a linear mixed effect model with an FDR correction).

Identification of the functional domains in Sorbs2 interacting with BK channel subunits

To determine the functional domains in Sorbs2 that participate in the interaction with BK-α and BK-β1, we constructed the Sorbs2 SH3 domain truncation (Sorbs2ΔC-EGFP) and the SoHo domain truncation (Sorbs2ΔN-EGFP) mutants. These Sorbs2 truncation mutants are properly expressed in HEK293 cells as illustrated in Figure 4A. After a 48-h transfection with Sorbs2-EGFP WT, Sorbs2ΔC-EGFP or Sorbs2ΔN-EGFP cDNAs into HEK293 cell lines stably expressing BK-α (Flag-BK-α-HEK) or BK-β1 (Flag-BK-β1-HEK), protein immunoprecipitates (IPs) against mouse anti-Flag antibody (for Flag-BK-α or Flag-BK-β1) were pulled down and then detected by rabbit anti-EGFP antibody (for Sorbs2-EGFP and its truncation mutants). As shown in Figure 4B and 4C, the EGFP band was detected in the anti-Flag IPs extracted from Flag-BK-α-HEK cells co-expressed with Sorbs2ΔN-EGFP but not in those co-expressed with Sorbs2ΔC-EGFP, indicating that the SH3 domain is needed for Sorbs2-BK-α interaction. The EGFP band was present in the anti-Flag IPs from Flag-BK-β1-HEK cells co-expressed with either Sorbs2ΔN-EGFP or Sorbs2ΔC-EGFP, suggesting that both SH3 and SoHo domains of Sorbs2 interact with BK-β1. In addition, there is no significant difference in the surface and cytosolic protein levels of BK-α and BK-β1 in HEK293 stable cell lines co-expressing Sorbs2 WT, Sorbs2ΔC or Sorbs2ΔN (Figure 4D).

Figure 4: Schematic representation of the truncation mutants of pEGFP-Sorbs2 and their effects on BK-α and BK-β1 expression.

A: Schematic representation of the full-length (WT), N-terminal (ΔN) and C-terminal (ΔC) truncation mutants of Sorbs2. There is one SoHo domain in the N-terminus and three SH3 domains in the C-terminus of Sorbs2. Western blot bands demonstrate EGFP-tag Sorbs2 protein expression in HEK293 cells 48 h after transfection with EGFP-Sorbs2 WT (157 kD), EGFP-Sorbs2ΔN (126 kD) and EGFP-Sorbs2ΔC (50 kD) cDNAs. B and C: EGFP-Sorbs2 WT, EGFP-Sorbs2ΔN and EGFP-Sorbs2ΔC cDNAs were transiently transfected into HEK cell line stably expressing Flag-BK-α (Flag-BK-α-HEK cell line) and Flag-BK-β1 (Flag-BK-β1-HEK cell line). Forty-eight hours after expression, immunoprecipitation assay was performed to determine the physical domain of Sorbs2 required for interaction with BK-α and BK-β1. In Flag-BK-α-HEK cells, EGFP-Sorbs2ΔC is not detected in the immunoprecipitates against Flag antibody, but EGFP-Sorbs2ΔN is found in the Flag antibody pull-downs (B). In Flag-BK-β1-HEK cells, both EGFP-Sorbs2ΔN and EGFP-Sorbs2ΔC are present in the immunoprecipitates of Flag antibody pulldown (C). Protein lysates (inputs) serve as positive controls. D: The cytosolic and surface proteins of Flag-BK-α and Flag-BK-β1 were measured in Flag-BK-α-HEK cell line and Flag-BK-β1-HEK cell line 48 h after transfection with EGFP-Sorbs2 WT, EGFP-Sorbs2ΔN and EGFP-Sorbs2ΔC cDNAs respectively. The cytosolic and surface protein expression of BK-α and BK-β1 in cells with EGFP-Sorbs2ΔN and EGFP-Sorbs2ΔC transfection was not statistically different from those of EGFP-Sorbs2 WT transfection (n=3 biological replicates, the Wilcoxon test with the Holm correction).

The effects of Sorbs2 WT, Sorbs2ΔC, and Sorbs2ΔN on BK-α/BK-β1 current density were examined in Flag-BK-α/Flag-BK-β1 HEK293 cells 48 h after co-transfection with Sorbs2 WT-EGFP, Sorbs2ΔN-EGFP or Sorbs2ΔC-EGFP cDNAs respectively, compared to the cells without Sorbs2 expression (empty controls). There is an overall effect on the current-voltage (I-V) relationship of BK-α/BK-β1 channels with Sorbs2 WT con-transfection (n=12) in comparison with empty controls (n=20), Sorbs2ΔN-EGFP (n=11), and Sorbs2ΔC-EGFP (n=11) co-transfection (Pr(>F)=1.7e-147, the Shapiro-Wilk test and a linear mixed effect model with an FDR correction). However, no statistical difference was observed in the I-V relationship of BK-α/BK-β1 channels without Sorbs2 expression and with Sorbs2ΔC-EGFP or Sorbs2ΔN-EGFP expression (Figure 5). Hence, our results suggest that Sorbs2 upregulates BK-α/BK-β1 channel activity and the integrity of Sorbs2 is necessary for functional regulation of BK-α/BK-β1 channels.

Figure 5: Effects of Sorbs2 truncation mutants on BK-α/BK-β1 currents in heterologous expression system.

Whole-cell BK-α/BK-β1 currents were recorded in HEK293 cell lines stably expressing Flag-BK-α and Flag-BK-β1 (Flag-BK-α/BK-β1-HEK cells) 48 h after transfection with EGFP-Sorbs2 WT, EGFP-Sorbs2ΔN and EGFP-Sorbs2ΔC cDNAs respectively. Flag-BK-α/BK-β1-HEK cells without transfection with Sorbs2 cDNAs served as controls. The I-V curves illustrate a significant increase in IBTX-sensitive K⁺ currents (defined as BK-α/BK-β1 currents) elicited from the cells with EGFP-Sorbs2 WT transfection at membrane voltages above +40 mV, compared to those of controls (Pr(>F)=1.7e-147, the Shapiro-Wilk test and a linear mixed effect model with an FDR correction). However, this effect of Sorbs2 on BK-α/BK-β1 current density was absent in cells expressing Sorbs2ΔN or Sorbs2ΔC. The difference observed in BK-α/BK-β1 current density was not statistically significant among the cells co-expressing Sorbs2ΔN, Sorbs2ΔC, and no Sorbs2 co-expression (the Shapiro-Wilk test and a linear mixed effect model with an FDR correction). The adjusted p-values between each group comparison at different voltages are listed in the Statistical Analysis Table of Supplemental Materials.

Interaction between Sorbs2 proteins and BK channel mRNAs in the cardiovascular tissue of WT mice

To determine the mechanism through which Sorbs2 directly regulates BK channel expression, we performed the next generation RNA-sequencing analysis in the ribonucleoprotein precipitates against anti-Sorbs2 antibodies in mouse hearts. A total of 48 genes encoding cardiovascular ion channels and associated receptors at a given RPKM (reads per kilobase of transcript per million reads mapped) cutoff value of 1 are listed in Table S1. Among them, the Kcnma1 mRNA was the most abundant ion channel mRNA pulled down by anti-Sorbs2 antibodies, whereas the Kcnmb1 mRNA was undetectable. Hence, Sorbs2 may function as an RBP for the regulation of BK-α expression in mouse cardiovascular tissues. Details of RNA-sequencing results are available in Supplemental Materials.

Upregulation of Sorbs2 expression by Nrf2

Nrf2 regulates downstream targets by binding to the Nrf2-binding motifs [TGA(G/C)xxxGC] in their promoters, where x represents any amino acid.¹⁶ There are at least two Nrf2-binding motifs in the promoter of the human SORBS2 and mouse Sorbs2 gene. We examined whether Sorbs2 expression is regulated by Nrf2 using the luciferase reporter assay. Our results confirmed that the presence of Nrf2 increased the luciferase signals by 3.26 folds in HEK293 cells 48 h after co-transfection of Sorbs2 luciferase reporter plasmids with Nrf2 plasmids, compared to controls without Nrf2 plasmid transfection. However, the luciferase signals were only increased by 1.62 folds after co-transfection of mutant cDNAs containing the Nrf2-binding motif deletion, compared to cells without Nrf2 co-expression, which were 50.3% lower compared to WT cDNA co-transfection with intact Sorbs2 promoter (Figure 6A). In addition, a 48-h transduction with Ad-Nrf2 (50 MOI) produced a 15.5-fold augmentation of Nrf2 expression accompanied by a 2.10-fold enhancement of Sorbs2 protein levels in primary human coronary SMCs, compared to Ad-CMV transduction (Figure 6B). In contrast, a 70.2% knockdown of Nrf2 expression by Ad-Nrf2 shRNA resulted in a 38.8% reduction in Sorsb2 protein levels in primary human coronary SMCs (Figure 6C). Hence, these results indicate that vascular Sorbs2 expression is regulated by Nrf2.

Figure 6: Regulation of Sorbs2 expression by Nrf2.

A: Nrf2-binding motifs [TGA(G/C)xxxGC] are present in the Sorbs2 promoter region of human, monkey, mouse and rat. Luciferase reporter assay showed a 3.26-fold increase of luciferase signals in HEK293 cells co-transfected with the Sorbs2 luciferase reporter plasmids (Sorbs2-pEZX-luc) and Nrf2 plasmids (Nrf2-pcDNA3.1). Co-transfection with empty pcDNA3.1 served as a plasmid control. Deletion of the Nrf2-binding motifs by replacing G/C to A [TGAAxxxGC] in the Sorbs2 luciferase reporter plasmids [Sorbs2-ΔNrf2-pEZX-luc) significantly decreased the luciferase signals by 50% (n=6 biological replicates, the Shapiro-Wilk test and two-tailed unpaired t-test with the Holm correction). B: A 48-h transduction with adenovirus carrying Nrf2 (50 MOI) increased Nrf2 protein expression by 21 folds, accompanied by a 2.71-fold increase of Sorbs2 expression in human coronary SMCs (n=6 biological replicates, the Shapiro-Wilk test and two-tailed unpaired t-test with the Holm correction). C: A 70.2% knockdown of Nrf2 by Nrf2-shRNA resulted in a 38.8% reduction of Sorbs2 expression in human coronary SMCs (n=6 biological replicates, Shapiro-Wilk test and two-tailed unpaired t-test with the Holm correction).

Molecular docking analysis of the interaction between Sorbs2 and BK-α proteins

It is known that the SH3 domain of Sorbs2 binds to partner proteins that contain the SH3 domain-binding motif (xPxxPx, where P is proline and x is any amino acid).¹⁷ There is one SH3 domain-binding motif in the BK-α gating-ring. To better understand the structural basis of Sorbs2-BK-α interaction, we downloaded the crystal structures of human Sorbs2 SH3-domain protein (PDB ID: 5VEI) and human BK-α gating-ring protein (PDB ID: 3NAF) from the RCSB protein data bank (PDB). Docking of two proteins was conducted using the ClusPro 2.0 program,¹⁸ which resulted in the generation of top 10 structures according to the highest weighted scores of the lowest energy. The docking structure selected as a putative binding model was ranked at the top 2 with a weighted score of −775, showing multiple polar interactions between the BK-α gating-ring and the Sorbs2 SH3 domain (Figure 7A). Specifically, the middle part of Sorbs2 SH3 domain interacts directly with the Ca²⁺ bowl of BK-α gating-ring (Figure 7B), and the distal end of Sorbs2 SH3 domain connects to the groove constituted by the SH3-binding motif and two nearby α-helixes in the BK-α gating-ring (Figure 7C).

Figure 7: Detailed intermolecular interaction between the Sorbs2 SH3 domain and the BK-α gating-ring proteins.

A: Structure of Sorbs2 SH3 domain ans BK-α gating-ring protein interaction. The Sorbs2 SH3 domain (cyan) forms multiple polar interactions with the BK-α gating-ring (green) with a weighted score of −775. There are two regions of the BK-α gating-ring (green) involved in the interactions with the Sorbs2 SH3 domain (cyan): the Ca²⁺ bowl [(892)-DQDDDDDPD-(900)] (magenta) and the groove constituted by the SH3-binding motif [(780)-PGTP-(783)] (yellow) and two nearby α-helixes [(786)-RADLRAV-(792)] (red) and [(760)-IEYLKREWETLH-(771)] (orange). B: Surface representation of Sorbs2 SH3 domain and BK-α gating-ring protein interaction. C: Close-up view of the interaction between the Sorbs2 SH3 domain (cyan) and the Ca²⁺ bowl (magenta) of the BK-α gating ring (green). R-894 in the Sorbs2 SH3 domain interacts with D-894 (1.7 and 1.9Å, via a salt bridge) and D-895 (2.0 Å, via a hydrogen bond) of the Ca²⁺ bowl. R-908 in the Sorbs2 SH3 domain binds to D-900 (1.9 and 2.0 Å, via a salt bridge) of the Ca²⁺ bowl. D: Close-up view of the Sorbs2 SH3 domain (cyan) interaction with the SH3 domain-binding motif (yellow) and nearby α-helixes (red and orange) of the BK-α gating-ring. R-928 in the Sorbs2 SH3 domain forms two polar interactions with T-782 (yellow, 2.4 Å, via a hydrogen bond) at the SH3 domain-binding motif and with D-788 (1.9 and 1.9 Å, via a salt bridge) at a nearby α-helix (red) of the BK-α gating-ring, while D-938 in the Sorbs2 SH3 domain connects to E-761 (2.2 Å, via a N-O covalent bond) and K-764 (1.8 Å and 1.8 Å, via a salt bridge) at another α-helix (orange) of the BK-α gating-ring.

Downregulation of Sorbs2 expression in the arteries of diabetic animals

It is well-established that impaired vascular BK channel function is a common feature in both type 1 and type 2 diabetes, which are associated with decreased BK-β1 expression in diabetic vessels.^1,8 We found that Sorbs2 protein expression was reduced by 60% in the coronary arteries of db/db diabetic mice (Figure 8A), concomitant with a significant reduction in the cytosolic and surface expression of BK-α by 76.0% and 84.6% respectively, as well as the cytosolic and surface expression of BK-β1 by 57.3% and 42.4% respectively (Figure 8B). BK channel current density in freshly isolated coronary SMCs and BK channel-mediated vasodilation in coronary arteries were diminished in db/db diabetic mice, compared to those of Lean control mice (Figure 8C and 8D). Hence, vascular Sorbs2 deficiency may contribute to BK channelopathy and vasculopathy in diabetes.

Figure 8: Decreased Sorbs2 protein levels with reduced BK channel expression and function in the coronaries of db/db type 2 diabetic mice.

A: Immunoblots showed that Sorbs2 protein levels are significantly decreased in the coronary arteries of db/db type 2 diabetic mice, compared to Lean control mice (n=6, Shapiro-Wilk test and two-tailed unpaired t-test with the Holm correction). B: The cytosolic and surface protein levels of BK-α and BK-β1 are significantly reduced in freshly dissected carotid arteries of db/db mice 6 months after developing hyperglycemia, compared to age-matched Lean control mice (n=6 mice for BK-α and BK-β1 cytosolic protein expression and n=4 biological replicates from 8 mice for BK-α and BK-β1 surface expression, Shapiro-Wilk test and two-tailed unpaired t-test with the Holm correction). C: The I-V relationships of coronary BK channels recorded from freshly isolated coronary SMCs from Lean control mice and db/db diabetic mice are shown. There is a significant reduction of the BK current densities in the coronary SMCs of db/db diabetic mice (n=7), compared to Lean control mice (n=6, Pr(>F)=1.7e-13, Shapiro-Wilk test and a linear mixed effect model with an FDR correction). The adjusted p-values at all given voltages are available in the Statistical Analysis Table of Supplemental Materials. D: Lean mice (n=6) have substantial concentration-dependent coronary vasodilation to NS-1619 (a BK channel activator), but the NS-1619 effect is significantly diminished in the coronary arteries of db/db diabetic mice (n=7, Pr(>F)=1.3e-014, Shapiro-Wilk test and a linear mixed effect model with an FDR correction).

DISCUSSION

In this study, we have made several novel findings regarding the role of Sorbs2 in regulating vascular BK channel physiology: 1) Sorbs2 is abundant in arteries where Sorbs2 physically interacts with BK-α and BK-β1 and upregulates BK-α and BK-β1 expression in vascular SMCs. 2) Molecular docking analysis demonstrates that the Sorbs2 SH3 domain interacts with the Ca²⁺ bowl and the SH3-binding motif at the BK-α gating-ring. 3) Sorbs2 is a downstream target of Nrf2 in coronary SMCs. 4) Sorbs2 KO mice manifest vascular BK channelopathy and coronary vasculopathy like those observed in diabetes, where BK-α and BK-β1 protein expression is downregulated with impaired BK channel Ca²⁺-dependent activation and BK channel-mediated vasodilation in coronary arteries, but these animals are normoglycemic with normal body weights. 5) Importantly, Sorbs2 expression is markedly reduced in the arteries of db/db diabetic mice. Hence, Sorbs2 is an important regulator for vascular BK channel physiology. Sorbs2 deficiency is an independent risk factor of vascular BK channelopathy and vasculopathy in diabetes.

There is growing evidence that ion channels are usually assembled not only with the same type of channels as clusters but also with different types of channels to form microdomain complexes on the membrane of excitable cells, exhibiting the “cooperative gating” and “functional coupling” properties that are important for cellular physiology and pathophysiology.¹⁹ In vascular SMCs, the voltage-gated L-type Ca²⁺ channels appear to cluster and colocalize with BK channels in the surface membrane, facilitating the cooperative generation of Ca²⁺ sparklets and activation of BK channels.^20–22 It is known that scaffold proteins provide an important platform for organizing ion channels, receptors and signaling pathways to form channel-receptor-enzyme microdomain complexes and maintain their structural stability at cell membranes, which are critical in maintaining ion channel physiology and tissue function.^23,24 As a cytoskeletal protein, Sorbs2 contains one SoHo domain in the N-terminus and three SH3 domains in the C-terminus, which are known to mediate protein-protein interactions in different tissues.²⁵ It has been reported that Sorbs2 is localized in the myocardial intercalated discs amid other important cytoskeletal proteins, including cadherin, α-actinin, actin stress fibers, and adhesion junctions, helping to maintain cytoskeletal organization and signal transduction in heart.^11,13,26 Importantly, Sorbs2 is not only a cardiac cytoskeletal protein that physically interacts with many cardiovascular ion channel proteins, such as the voltage-gated Na⁺ channels (Nav1.5), the L-type Ca²⁺ channels (Cav1.2), the voltage-gated K⁺ channels (Kv1.4 and Kv4.2) and the inward rectifier K⁺ channels (Kir2.1 and Kir6.2), but is also an RBP that binds to the mRNAs of these ion channels in the heart, with broad effects on regulating cardiovascular ion channel expression and function.¹² In this study, we found that Sorbs2 is ubiquitously expressed in arteries and physically interacted with BK-α and BK-β1 proteins, as well as with the Kcnma1 mRNAs, thereby playing an important role in regulating vascular BK channel physiology. These observations are supported by in vivo and in vitro studies, showing that silencing of Sorbs2 not only interrupts the interaction of Sorbs2 with BK-α and BK-β1, but also reduces the expression of BK-α and BK-β1 in coronary SMCs. Downregulated BK-α expression by Sorbs2 can be explained by its RBP mechanism. However, the cause of reduced BK-β1 by Sorbs2 is unclear since the Kcnmb1 mRNAs are not detectable in the ribonucleoprotein precipitates against anti-Sorbs2 antibodies. Nevertheless, loss of Sorbs2 diminishes BK channel activity and BK channel-mediated vasodilation in the coronary arteries of Sorbs2 KO mice.

Structure-function relationship studies confirmed that the SH3 domains of Sorbs2 are involved in the interaction between Sorbs2 and BK-α, and both SoHo and SH3 domains participate in the Sorbs2-BK-β1 interaction (Figure 4). These interactions are important for BK-α/BK-β1 activity as deletion of the SH3 or the SoHo domain abolishes the regulatory effect of Sorbs2 on BK-α/BK-β1 current density. We found that Sorbs2 upregulates the BK channel sensitivity to free Ca²⁺, and genetic deletion of Sorbs2 impaired the efficiency, efficacy, and cooperativity of Ca²⁺-dependent BK channel activation in coronary SMCs. Protein-protein docking studies demonstrated that the SH3 domain of Sorbs2 directly interacts with the BK-α gating-ring, especially with D984, D985 and D900 in the Ca²⁺ bowl. Based on these results, it is therefore not surprising to find that Sorbs2 functions as a BK channel modulator by stabilizing the 3D-structure of the BK channel Ca²⁺ bowl and organizing BK-α and BK-β1 with signaling pathways to regulate BK channel physiology and vascular function. Loss of Sorbs2 would lead to compromised Ca²⁺-dependent BK channel activation.

Impaired vascular BK channel function is a common feature in both type 1 and type 2 diabetes, leading to diabetic cardiovascular complications. We and other investigators have delineated that BK-β1 dysregulation occurs at the transcriptional, translational and post-translational levels in diabetic vessels.^10,27–29 BK-α function is also dysregulated in diabetes due to increased oxidative stress in vascular SMCs.^30,31 Moreover, BK channel kinetic analysis from excised membranes containing only one channel showed that the intrinsic properties on BK channel Ca²⁺-dependent activation are compromised in the coronary arteries of diabetes, indicating that abnormal Ca²⁺ responsivity can occur at BK channel protein per se.^32,33 Recent studies revealed that L-type Ca²⁺ channel clustering and cooperative gating behaviors were enhanced, giving rise to an increase in vascular myogenic tone in the vascular SMCs of type 2 diabetes.³⁴ However, it has been reported that reduced Ca²⁺ sparks and BK channel coupling occurred in the cerebral arterial SMCs of db/db diabetic mice.³⁵ Taken together, loss of BK channel negative feedback regulation in vascular tone as results of an increase of intracellular Ca²⁺ signals concomitant with a drastic decrease in BK channel Ca²⁺ sensitivity would produce diminished vascular relaxation in diabetes. Since Sorbs2 physically interacts with L-type Ca²⁺ channels and BK channels, whether Sorbs2 participates in their cooperative gating and functional coupling in vascular SMCs, particularly in diabetes, warrants to further investigation.

Another important finding in this study is that Sorbs2 is a downstream target of Nrf2, a master regulator of cellular redox, in vascular SMCs. We have shown that Nrf2 plays a central role in the regulation of BK-α and BK-β1 expression and function in vascular SMCs through its antioxidant response and transcriptional regulation.^1,10,36 In diabetes, Nrf2 expression and its downstream genes are slightly increased during the early stages of hyperglycemia as a compensatory response for increased reactive oxygen species (ROS) production, but then Nrf2 becomes significantly downregulated in the advanced stages of diabetes because of mechanism burnout and overwhelmed.^37–39 We have reported that Nrf2 signaling is compromised in diabetic vessels, contributing to vascular BK channelopathy as a result of accelerated BK-β1 protein degradation and reduced the Kcnma1 gene transcription in coronary SMCs.^1,9,10,36 Treatment with FDA-approved Nrf2 activators such as dimethyl fumarate and sulforaphane significantly improves the protein expression, nuclear translocation and transcriptional activity of Nrf2, which in turn reduces ROS production, increases coronary BK-α and BK-β1 protein expression, and protects coronary BK channel activity and BK channel-mediated vasodilation in diabetic mice.^9,10,36 In this study, we found that Sorbs2 expression is regulated by Nrf2 in vascular SMCs. Adenoviral delivery of Nrf2 significantly increases Sorbs2 protein levels, while knockdown of Nrf2 markedly decreases Sorbs2 expression in coronary SMCs. Using the luciferase reporter assay, we confirmed that Nrf2 binds to the Nrf2-binding motifs of the Sorbs2 promoter. Deletion of the Nrf2-binding motifs in Sorbs2 promoter reduced the luciferase signals by 50%, suggesting that Nrf2 is a key determinant of Sorbs2 expression in vascular SMCs. Thus, Nrf2 downregulation in diabetes may contribute to the decreased Sorbs2 protein levels in the coronary arteries of db/db mice. Since Nrf2 regulates Sorbs2, BK-α, and BK-β1 expression in vascular SMCs, a novel strategy of upregulating the Nrf2-Sorbs2-BK channel pathway may have therapeutic potential for reducing cardiovascular complications in patients with diabetes.

Novelty and Significance.

What is known

• The Sorbin homology and Src homology 3 domain-containing protein 2 (Sorbs2), a cytoskeletal protein, is densely expressed in heart and is essential for normal cardiac architecture and electrical conductivity.

• Loss of Sorbs2 leads to cardiac structural and electrical remodeling, causing cardiomyopathy and premature death in Sorbs2 knockout (KO) mice.

• However, the role of Sorbs2 in vascular physiology and pathophysiology is unknown.

What new information does this article contribute

• Sorbs2 is not only a cytoskeletal protein that physically interacts with vascular BK channel subunits but also an RNA-binding protein that binds to Kcnma1 (encoding BK-α subunit) mRNA, regulating BK channel expression and function in coronary smooth muscle cells.

• Sorbs2 protein interacts with the Ca²⁺ bowl of BK channel gating-ring, in turn regulating BK channel Ca²⁺ sensitivity. Loss of Sorbs2 diminishes the BK channel Ca²⁺-dependent activation in the coronary smooth muscle cells (SMCs).

• Sorbs2 expression is transcriptionally regulated by the nuclear factor erythroid-2-related factor 2 (Nrf2) and Sorbs2 protein levels are decreased in db/db diabetic mouse vessels.

• Sorbs2 knockout mice exhibit coronary BK channelopathy and vasculopathy, including reduced BK channel expression and activation and impaired BK channel-mediated vasodilation in coronary arteries similar to those observed in diabetic mice, despite having normal glucose levels and body weights.

Summary.

In this study, we reported for the first time that Sorbs2 is highly expressed in vascular SMCs and its expression is regulated by Nrf2. Vascular Sorbs2 is important for the modulation of BK channel expression and function in coronary arteries. Particularly, Sorbs2 binds to the BK channel Ca²⁺ bowl, affecting BK channel Ca²⁺-dependent activation. Knockout of Sorbs2 gene in mice manifest BK channelopathy and vasculopathy observed in the coronary arteries of diabetes without affecting their blood glucose levels and body weights. Importantly, vascular Sorbs2 protein levels are significantly downregulated in db/db diabetic mice. Hence, Sorbs2 deficiency could be an independent risk of coronary BK channelopathy and vasculopathy in diabetes.

Nonstandard Abbreviations and Acronyms:

BK channel

the large conductance Ca²⁺-activated K⁺ channel

BK-α

the large conductance Ca²⁺-activated K⁺ channel α subunit

BK-β1

the large conductance Ca²⁺-activated K⁺ channel β1 subunit

EC

endothelial cell

nH

the Hill coefficient

nPo

maximal channel open probability

Nrf2

the nuclear factor erythroid-2-related factor 2

PLA

in situ proximity ligation assay

Pr(>F)

the probability larger than the F-value

RCK

the regulator of K⁺ conductance

RBP

RNA-binding protein

RPKM

Reads Per Kilobase Million

ROS

reactive oxygen species

SMC

smooth muscle cell

Sorbs2

the Sorbin homology and Src homology 3 domain-containing protein 2

SH3

the Src homology 3

SoHo

the Sorbin homology