Ahnak1 modulates L-type Ca2+ channel inactivation of rodent cardiomyocytes

Ahnak1, a giant 700 kDa protein, has been implicated in Ca²⁺ signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavβ₂ subunit plays a role in L-type Ca²⁺ current (I _CaL) regulation. Here, we performed structure–function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavβ₂ (K _d?～?60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavβ₂ affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca²⁺ influx by enhancing I _CaL density and/or increasing I _CaL inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I _CaL density, markedly slowed I _CaL inactivation resulting in a 50–70% increase in transported Ca²⁺ during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba²⁺ as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I _CaL characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I _CaL inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavβ₂ and/or scaffolds selectively different β-subunit isoforms in the heart.