Ahnak1 modulates L-type Ca2+ channel inactivation of rodent cardiomyocytes
- First Online:
- Cite this article as:
- Alvarez, J.L., Petzhold, D., Pankonien, I. et al. Pflugers Arch - Eur J Physiol (2010) 460: 719. doi:10.1007/s00424-010-0853-x
- 160 Downloads
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 (ICaL) 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 (Kd ∼ 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 ICaL density and/or increasing ICaL inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect ICaL density, markedly slowed ICaL 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 ICaL characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of ICaL 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.