Hormones can transmit signals through adenosine 3′,5′-monophosphate (cAMP) to precise intracellular locations. The fidelity of these responses relies on the activation of localized protein kinase A (PKA) holoenzymes. Association of PKA regulatory type II (RII) subunits with A-kinase—anchoring proteins (AKAPs) confers location, and catalytic (C) subunits phosphorylate substrates. Single-particle electron microscopy demonstrated that AKAP79 constrains RII-C subassemblies within 150 to 250 angstroms of its targets. Native mass spectrometry established that these macromolecular assemblies incorporated stoichiometric amounts of cAMP. Chemical-biology— and live cell—imaging techniques revealed that catalytically active PKA holoenzymes remained intact within the cytoplasm. These findings indicate that the parameters of anchored PKA holoenzyme action are much more restricted than originally anticipated.

Rationale Increased contractility of arterial myocytes and enhanced vascular tone during hyperglycemia and diabetes may arise from impaired large conductance Ca2+-activated K+ (BKCa) channel function. The scaffolding protein AKAP150 is a key regulator of calcineurin (CaN), a phosphatase known to modulate expression of the regulatory BKCa β1 subunit. Whether AKAP150 mediates BKCa channel suppression during hyperglycemia and diabetes is unknown. Objective To test the hypothesis that AKAP150-dependent CaN signaling mediates BKCa β1 downregulation and impaired vascular BKCa channel function during hyperglycemia and diabetes. Methods and Results We found that AKAP150 is an important determinant of BKCa channel remodeling, CaN/NFATc3 activation, and resistance artery constriction in hyperglycemic animals on high fat diet (HFD). Genetic ablation of AKAP150 protected against these alterations, including augmented vasoconstriction. D-glucose-dependent suppression of BKCa channel β1 subunits required Ca2+ influx via voltage-gated L-type Ca2+ channels and mobilization of a CaN/NFATc3 signaling pathway. Remarkably, HFD mice expressing a mutant AKAP150 unable to anchor CaN resisted activation of NFATc3 and downregulation of BKCa β1 subunits, and attenuated HFD-induced elevation in arterial blood pressure. Conclusions Our results support a model whereby subcellular anchoring of CaN by AKAP150 is a key molecular determinant of vascular BKCa channel remodeling, which contributes to vasoconstriction during diabetes.