Ahnak is critical for cardiac Ca(v)1.2 calcium channel function and its β-adrenergic regulation

Defective L-type Ca2+ channel (ICaL) regulation is one major cause for contractile dysfunction in the heart. The ICaL is enhanced by sympathetic nervous stimulation: via the activation of β-adrenergic receptors, PKA phosphorylates the α1C(CaV1.2)- and β2-channel subunits and ahnak, an associated 5643-amino acid (aa) protein. In this study, we examined the role of a naturally occurring, genetic variant Ile5236Thr-ahnak on ICaL. Binding experiments with ahnak fragments (wild-type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr-ahnak critically affected both β2 subunit interaction and ICaL regulation. Binding affinity between ahnak-C1 (aa 4646-5288) and β2 subunit decreased by ≈50% after PKA phosphorylation or in the presence of Ile5236Thr-ahnak peptide. On native cardiomyocytes, intracellular application of this mutated ahnak peptide mimicked the PKA-effects on ICaL increasing the amplitude by ≈60% and slowing its inactivation together with a leftward shift of its voltage dependency...