Ahnak1 interaction is affected by phosphorylation of Ser-296 on Cavβ(2)

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 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)≈35nM) 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 ≈2.4-fold and reduced the capacity by ≈60%. Our results are indicative for the release of a population of low affinity interaction sites following Cavβ(2) phosphorylation on Ser-296. We propose that this phosphorylation event is one mechanism underlying ahnak1's modulator function on Cav1.2 channel activity