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

Defective L-type Ca super(2+) channel (I sub(CaL)) regulation is one major cause for contractile dysfunction in the heart. The I sub(CaL) is enhanced by sympathetic nervous stimulation: via the activation of ss-adrenergic receptors, PKA phosphorylates the alpha 1C (Ca sub(V)1.2)- and ss2-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 sub(CaL). Binding experiments with ahnak fragments (wild-type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr-ahnak critically affected both ss2 subunit interaction and I sub(CaL) regulation. Binding affinity between ahnak-C1 (aa 4646-5288) and ss2 subunit decreased by approximately 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 sub(CaL) increasing the amplitude by approximately 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 sub(CaL) by isoprenaline. Hence, we suggest the ahnak-C1 domain serves as physiological brake on I sub(CaL). Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr-ahnak fragments to increase I sub(CaL). This genetic ahnak variant might cause individual differences in I sub(CaL) regulation upon physiological challenges or therapeutic interventions.