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.
|Number of pages||6|
|Journal||Biochemical and Biophysical Research Communications|
|State||Published - 4 May 2012|
- L-type calcium channel
- PKA phosphorylation
- Protein-protein interaction