To determine whether the observed in vitro affinity differences resulted in a difference in cellular compartmentalization, we examined the association of the AKB variants and the PKA regulatory subunits in cells. In this assay, the AKB domains of D-AKAP2 from mouse (Val) and human (Ile) were fused to the mitochondrial anchoring domain of D-AKAP1, respectively, and tethered to the outer mitochondrial membrane (27, 28). The binding of the AKB domain to the PKA regulatory subunits was detected as the cotransfected PKA regulatory domains colocalized to the mitochondria. As expected, both the Val and Ile AKB variants can target RII to the mitochondria effectively, in accordance with their similar affinity in vitro (Fig. 3h and k). However, for RI, there was a difference in colocalization between the variants. The Val variant effectively targeted RI to the mitochondria (Fig. 3b). The Ile variant, however, was unable to target RI, which was evenly diffused in the cytosol (Fig. 3e) demonstrating that the Ile variant of D-AKAP2 has an impaired ability to sequester RI.

Increasing evidence suggests that the RI and RII isoforms of PKA have distinct functions (30). The RI isoform has been implicated in a variety of biological functions such as cell proliferation (31), tumor suppression (32, 33), immune regulation (34), and embryonic development (35, 36). In addition, the RI isoform plays a significant role in maintaining cAMP regulation of PKA as evidenced by the embryonic lethality of mice deficient in the gene (35). Interestingly, these mice have defects in cardiac morphogenesis.

The correlation of the I646V variant with the EKG PR interval indicates that this polymorphism might be a predisposing factor for a cardiac phenotype. Individuals homozygous for the Val variant exhibit shorter atrium depolarization intervals (PR) than individuals homozygous for Ile. This phenotypic correlation combined with literature reports supporting a role for AKAP-mediated PKA signaling in normal cardiac function suggests plausible hints of how the Val variant may lead to pathogenesis.

We have identified SNPs in the D-AKAP2 gene that are associated with morbidity by using a genome-wide association study from an age-stratified healthy population and 6,500 gene-based SNPs. The combined genetic and biochemical evidence points to the Ile-Val variant as the functional polymorphism. PKA phosphorylates numerous substrates in response to adrenergic stimulation that influence the contractility of cardiac myocytes (37). Some of these substrates include the L-type Ca2+ channel, the ryanodine receptor, and phospholamban of the sarcoplasmic reticulum. AKAP-mediated targeting of PKA in cardiac myocytes has also been directly implicated in regulating cell contractility (38). Three AKAPs have been shown to interact with PKA in cardiac myocytes; muscle-selective AKAP (mAKAP), AKAP18 and Yotiao. mAKAP targets PKA to the perinuclear region of differentiated myocytes, coordinating both PKA and phosphodiesterase activity in a single complex (39). AKAP18 couples PKA to L-type Ca2+ channels, which enhances Ca2+ influx through the channel after adrenergic stimulation (40). Yotiao, an AKAP associated with N-methyl-D-aspartate (NMDA) receptors (41), has been shown to interact with the KCNQ1-KCNE1 K+ channel subunits in human hearts (42). Mutations in this channel associated with hereditary long QT syndrome abolish Yotiao interactions with the channel and attenuN. Marx, S. O., Reiken, S., Gaburjakova, M., Richardson, J. A., Marks, A. R. & Olsen, E. N. (Circ. Res.), Robinson, M.L., Wallert, M.A., Reinitz, C.A. & Shabb, J.B. (Arch. Biochem. Biophys.), Reinitz, C.A., Bianco, R.A. & Shabb, J.B. (Arch. Biochem. Biophys.), Risch, N. & Merikangas, K. (Science), Puca, A.A., Daly, M.J., Brewster, S.J., Matise, T.C., Barrett, J., Shea-Drinkwater, M., Kang, S., Joyce,E., Nicoli,J., Benson,E., Kunkel,L.M.&Perls,T.(Proc.Natl.Acad.Sci.USA), Haviland,M.B.,Lussier-Cacan,S.,Davignon,J.&Sing,C.F.(Am.J.Med.Genet.), Schachter,F.,Faure-Delanef,L.,Guenot,F.,Rouger,H.,Froguel,P.,Lesueur-Ginot,L.&Cohen,D.(Nat.Genet.), Andrew,T.,Hart,D.J.,Snieder,H.,Dange,M.,Spector,T.D.&Maregor,A.J.(Twin Res.), Buetow,K.H.,Edmonson,M.,MacDonald,R.,Clifford,R.,Yip,P.,Kelley,J.,
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