Ahnak1 has been implicated in protein kinase A (PKA)-mediated control of cardiac L-type Ca2+ channels (Cav1.2) through its interaction with the Cav beta(2) regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cav beta(2) in the T-tubule system. In previous studies Cav beta(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 Cav beta(2) and investigated whether Cav beta(2) phosphorylation affects its binding behavior. In vitro binding assays with Cav beta(2) truncation mutants and ahnak1(4889-5535) revealed that the core region of Cav beta(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 Cav beta(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 approximate to 35 nM) between Cav beta(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 Cav beta(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 approximate to 2.4-fold and reduced the capacity by approximate to 60%. Our results are indicative for the release of a population of low affinity interaction sites following Cav beta(2) phosphorylation on Ser-296. We propose that this phosphorylation event is one mechanism underlying ahnak1's modulator function on Cav1.2 channel activity. (C) 2012 Elsevier Inc. All rights reserved.