A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) to intracellular sites where they preferentially phosphorylate target substrates. Most AKAPs exhibit nanomolar affinity for the regulatory (RII) subunit of the type II PKA holoenzyme, whereas dual-specificity anchoring proteins also bind the type I (RI) regulatory subunit of PKA with 10-100-fold lower affinity. A range of cellular, biochemical, biophysical, and genetic approaches comprehensively establish that sphingosine kinase interacting protein (SKIP) is a truly type I-specific AKAP. Mapping studies located anchoring sites between residues 925-949 and 1,140-1,175 of SKIP that bind RI with dissociation constants of 73 and 774 nM, respectively. Molecular modeling and site-directed mutagenesis approaches identify Phe 929 and Tyr 1,151 as RI-selective binding determinants in each anchoring site. SKIP complexes exist in different states of RI-occupancy as single-molecule pulldown photobleaching experiments show that 41 AE 10% of SKIP sequesters two YFP-RI dimers, whereas 59 AE 10% of the anchoring protein binds a single YFP-RI dimer. Imaging, proteomic analysis, and subcellular fractionation experiments reveal that SKIP is enriched at the inner mitochondrial membrane where it associates with a prominent PKA substrate, the coiled-coil helix protein ChChd3.

Background— KCNJ2 encodes Kir2.1, a pore-forming subunit of the cardiac inward rectifier current, I K1 . KCNJ2 mutations are associated with Andersen-Tawil syndrome and catecholaminergic polymorphic ventricular tachycardia. The aim of this study was to characterize the biophysical and cellular phenotype of a KCNJ2 missense mutation, V227F, found in a patient with catecholaminergic polymorphic ventricular tachycardia. Methods and Results— Kir2.1-wild-type (WT) and V227F channels were expressed individually and together in Cos-1 cells to measure I K1 by voltage clamp. Unlike typical Andersen-Tawil syndrome-associated KCNJ2 mutations, which show dominant negative loss of function, Kir2.1WT+V227F coexpression yielded I K1 indistinguishable from Kir2.1-WT under basal conditions. To simulate catecholamine activity, a protein kinase A (PKA)-stimulating cocktail composed of forskolin and 3-isobutyl-1-methylxanthine was used to increase PKA activity. This PKA-simulated catecholaminergic stimulation caused marked reduction of outward I K1 compared with Kir2.1-WT. PKA-induced reduction in I K1 was eliminated by mutating the phosphorylation site at serine 425 (S425N). Conclusions— Heteromeric Kir2.1-V227F and WT channels showed an unusual latent loss of function biophysical phenotype that depended on PKA-dependent Kir2.1 phosphorylation. This biophysical phenotype, distinct from typical Andersen-Tawil syndrome mutations, suggests a specific mechanism for PKA-dependent I K1 dysfunction for this KCNJ2 mutation, which correlates with adrenergic conditions underlying the clinical arrhythmia.

The fidelity of excitation-contraction (EC) coupling in ventricular myocytes is remarkable, with each action potential evoking a [Ca2+]i transient. The prevalent model is that the consistency in EC coupling in ventricular myocytes is due to the formation of fixed, tight junctions between the sarcoplasmic reticulum (SR) and the sarcolemma where Ca2+ release is activated. Here, we tested the hypothesis that the SR is a structurally inert organelle in ventricular myocytes. Our data suggest that rather than being static, the SR undergoes frequent dynamic structural changes. SR boutons expressing functional ryanodine receptors moved throughout the cell, approaching or moving away from the sarcolemma of ventricular myocytes. These changes in SR structure occurred in the absence of changes in [Ca2+]i during EC coupling. Microtubules and the molecular motors dynein and kinesin 1(Kif5b) were important regulators of SR motility. These findings support a model in which the SR is a motile organelle capable of molecular motor protein-driven structural changes.

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