TI - Ahnak1 interaction is affected by phosphorylation of Ser-296 on Cavbeta(2) . AB - Ahnak1 has been implicated in protein kinase A ( PKA ) -mediated control of cardiac L-type Ca(2+) channels (Cav1.2) through its interaction with the Cavbeta(2) regulatory channel subunit . Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cavbeta(2) in the T-tubule system . In previous studies Cavbeta(2) attachment sites which impacted the channel's PKA regulation have been located to ahnak1's proximal C-terminus ( ahnak1 ( 4889-5535 ) , ahnak1 ( 5462-5535 ) ) . In this study , we mapped the ahnak1-interacting regions in Cavbeta(2) and investigated whether Cavbeta(2) phosphorylation affects its binding behavior . In vitro binding assays with Cavbeta(2) truncation mutants and ahnak1 ( 4889-5535 ) revealed that the core region of Cavbeta(2) consisting of Src-homology 3 ( SH3 ) , HOOK , and guanylate kinase ( GK ) domains was important for ahnak1 interaction while the C- and N-terminal regions were dispensable . Furthermore , Ser-296 in the GK domain of Cavbeta(2) was identified as novel PKA phosphorylation site by mass spectrometry . Surface plasmon resonance ( SPR ) binding analysis showed that Ser-296 phosphorylation did not affect the high affinity interaction ( K(D) approximately 35 nM ) between Cavbeta(2) and the alpha ( 1C ) I-II linker , but affected ahnak1 interaction in a complex manner . SPR experiments with ahnak1 ( 5462-5535 ) revealed that PKA phosphorylation of Cavbeta(2) significantly increased the binding affinity and , in parallel , it reduced the binding capacity . Intriguingly , the phosphorylation mimic substitution Glu-296 fully reproduced both effects , increased the affinity by approximately 2.4-fold and reduced the capacity by approximately 60% . Our results are indicative for the release of a population of low affinity interaction sites following Cavbeta(2) phosphorylation on Ser-296 . We propose that this phosphorylation event is one mechanism underlying ahnak1's modulator function on Cav1.2 channel activity .