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 activation of beta-adrenergic receptors, PKA phosphorylates the α1C(Ca_V1.2)- and β2-channel subunits and ahnak, an associated 5643-amino acid (aa) protein (Haase et al. 1999). Herein, we examined the role of a naturally occurring, genetic variant I5236T-ahnak on I_CaL elicited under whole-cell patch-clamp conditions on ventricular myocytes obtained from humanely killed 3 month old Sprague Dawley rats. On the native cardiomyocytes, intracellular application of 10 μM of the synthetic ahnak peptide carrying the I5236T mutation (GGLPGTGVQGLE) increased I_CaL amplitude from 11.9±0.8 to 18.9±1.4 pA/pF (P<0.05; n=18,20) at 0 mV depolarization, slowed the inactivation together with a leftward shift in the current-voltage relationship. Importantly, the mutated I5236T-peptide prevented specifically the further upregulation of I_CaL by the beta-adrenoceptor agonist, isoprenaline. Under the same experimental conditions, the respective wild-type peptide did not mimic the isoprenaline effects on I_CaL. Equilibrium binding experiments performed with recombinant β2 subunit and the ahnak-C1 fragment (aa 4646-5288) by analytical ultracentrifugation revealed a dissociation constant for the ahnak-C1/β2 subunit complex of 155±31 nM (n=14). The binding affinity between ahnak-C1 and β2 subunit decreased by ~50% following PKA phosphorylation of both protein partners. A similar decrease in ahnak-C1/β2 subunit binding affinity was induced by the presence of 10 μM mutated peptide (but not wild-type peptide) in equilibrium binding assays. Hence, we suggest the ahnak-C1 domain serves as a physiological brake on I_CaL. Relief from this inhibition is proposed as a common pathway used by sympathetic signalling and I5236T-ahnak fragments to increase I_CaL. This genetic ahnak variant might cause individual differences in I_CaL regulation upon physiological challenges or therapeutic interventions.