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

Defective L-type Ca2+ channel (I-CaL) regulation is one major cause for contractile dysfunction in the heart. The I-CaL is enhanced by sympathetic nervous stimulation: via the activation of beta-adrenergic receptors, PKA phosphorylates the alpha 1C(Ca(V)1.2)- and beta 2-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 I-CaL. Binding experiments with ahnak fragments (wild-type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr-ahnak critically affected both beta 2 subunit interaction and I-CaL regulation. Binding affinity between ahnak-C1 (aa 4646-5288) and beta 2 subunit decreased by approximate to 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 I-CaL increasing the amplitude by approximate to 60% and slowing its inactivation together with a leftward shift of its voltage dependency. Both mutated Ile5236Thr-peptide and Ile5236Thr-fragment (aa 5215-5288) prevented specifically the further up-regulation of I-CaL by isoprenaline. Hence, we suggest the ahnak-C1 domain serves as physiological brake on I-CaL. Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr- ahnak fragments to increase I-CaL. This genetic ahnak variant might cause individual differences in I-CaL regulation upon physiological challenges or therapeutic interventions.