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 Cavβ ₂ regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cavβ ₂ in the T-tubule system. In previous studies Cavβ 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 Cavβ ₂ and investigated whether Cavβ ₂ phosphorylation affects its binding behavior. In vitro binding assays with Cavβ ₂ truncation mutants and ahnak1 ^4889-5535 revealed that the core region of Cavβ 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 Cavβ 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 Cavβ ₂ and the α _1C I-II linker, but affected ahnak1 interaction in a complex manner. SPR experiments with ahnak1 ^5462-5535 revealed that PKA phosphorylation of Cavβ 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 ahnak1́s modulator function on Cav1.2 channel activity.