Coronary Large Conductance Ca2+-Activated K+ Channel Dysfunction in Diabetes Mellitus

Figures

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  Figure 1. Schematic illustration of vascular Ca2+-activated K+ (BK) channel subunits. BK-α, BK channel α-subunit; BK-β1, BK channel β1-subunit; BK-γ1, BK channel γ1-subunit; S or TM, transmembrane domain segment; VDS, voltage-sensor domain; RCK, regulator of K+ conductance; LRR, leucine-rich repeat; LRRD, leucine-rich repeat domain; LRRCT, leucine-rich repeat C-terminus; LRRNT, leucine-rich repeat N-terminus; COOH, C-terminus; and NH2, N-terminus.

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  Figure 2. Impaired vascular BK channel function in patients with T2DM. (A) Coronary arterioles of T2DM patients exhibit diminished BK channel Ca2+ sensitivity. Left panel: Representative tracings of inside-out single BK channel currents recorded at +60mV in an excised patch of freshly isolated atrial coronary arteriolar myocytes from non-diabetic (Ctrl) and T2DM patients. With an increase in free Ca2+ concentration, BK channel open probability (nPo) was robust in controls but not in T2DM patients. Dashed lines indicate the closed state (c) of channel. Right panel: The nPo plotted against logarithm Ca2+ concentrations (nPo-log[Ca2+] curve) was fitted using the Hill equation. There were significant reductions in Ca2+ log[EC50] and BK channel maximal nPo in T2DM patients (n=9) compared to those in non-diabetic controls (n=12). A rightward shift on the normalized nPo-log[Ca2+] curve of T2DM patients. Data are presented as mean±SEM. The BK channel maximal nPo and log[EC50] were significantly reduced in diabetic patients. (B) Impaired BK channel voltage sensitivity in the coronary arterioles of T2DM patients. Left panel: Representative tracings of inside-out single BK channel currents elicited…

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  Figure 3. Regulation of BK channels by AT1R signaling and caveolae compartmentalization. In DM, AT1R expression, and caveolae formation are upregulated in vascular SMCs. Upon Ang II activation, AT1R translocates to caveolae, where G-proteins, BK-α, NOX-1, and c-Src are colocalized. In caveolae, AT1R interacts with Gαq to activate PKC and NOX-1 through IP3/DAG signaling pathway, leading to an increase of ROS production. Meanwhile, the Gαi and β-arrestin complex induces c-Src activation. As a result of AT1R activation, BK-α protein oxidation, tyrosine phosphorylation, and tyrosine nitration are enhanced. In addition, AKT phosphorylates FOXO-3a, which in turn suppresses FOXO-3a nuclear translocation and reduces its transcriptional activities. With high glucose, increased ROS production inhibits AKT function, which promotes FOXO-3a nuclear translocation and facilitates Cav-1 expression. Since BK-β1 is not present in the caveolae, an increase in BK-α compartmentalization in caveolae may lead to physical uncoupling between BK-α and BK-β1 in vascular SMCs. The symbols “n,” “o,” and “p” represent protein nitration, oxidation, and phosphorylation, respectively.

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  Figure 4. Regulation of BK channel expression by ubiquitin proteasome system (UPS) and nuclear factor erythroid-2-related factor 2 (Nrf2) signaling. FBXO and MuRF1 are the E3 ligases targeting BK-β1 protein degradation via the UPS in vascular SMCs. FBXO is one of downstream targets of FOXO-3a. FOXO-3a activity is negatively controlled by AKT-dependent phosphorylation, while MuRF1 expression is controlled by NF-κB/p65. Under baseline conditions, p65 is bound to an inhibitory subunit, IκB that keeps it sequestered in an inactive state in the cytoplasm. Phosphorylation of IκB by IκB kinase promotes IκB degradation through the UPS, which in turn releases p65 and facilitates nuclear translocation. Under hyperglycemic conditions, overproduction of ROS inhibits AKT and activates NF-κB/p65, which in turn promotes FBXO and MuRF1 expression, leading to BK-β1 ubiquitination and accelerated degradation in vascular SMCs. Nrf2 is the master regulator of the antioxidant response. Under normal conditions, each molecule of Nrf2 interacts with two molecules of Keap1 resulting in UPS-mediated degradation. ROS modifies specific cysteine residues in Keap1 and releases Nrf2 from binding with Keap1. The unbound…

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  Figure 5. Regulation of BK-β1 expression by NFATc3 signaling. Calcineurin is a Ca2+/calmodulin (CaM)-activated phosphatase. In the membranes of vascular SMCs, AKAP150 proteins anchor calcineurin (CaN) with PKA and L-type Ca2+ channels (Cav1.2) to form dynamic Ca2+ signaling complexes. L-type Ca2+ channel activity is upregulated by PKA, which increases Ca2+ influx. Upon Ca2+ binding to calmodulin, calcineurin is activated, which then dephosphorylates NFATc3 and promotes NFATc3 nuclear translocation, inhibiting BK-β1 mRNA expression. In DM, the activity of the AKAP150-NFATc3 signaling pathway is upregulated, resulting in enhanced suppression of BK-β1 expression and impaired BK channel function in vascular SMCs. The symbol “p” represents protein phosphorylation.