Abstract 2332: PKA Activation Regulates L-type Cav1.3 Calcium Channel in vivo
Introduction: We have recently established that Cav1.3 L-type Ca channel is exclusively expressed in the supraventricular tissue and is regulated by protein kinase A (PKA) pathway in vitro (JBC 2009). Here, we addressed whether PKA regulates Cav1.3 in vivo and investigated the role of the C-terminus in this phosphorylation.
Methods: Mice were studied under basal conditions and after administration of norepinephrine (NE, 2mg/kg), a beta-adrenergic agonist. Cav1.3 proteins were extracted from mice atria and the phosphorylation status was assessed by Western blot using an anti-phosphoserine antibody. The presence of C-terminal fragment was assessed using a 17 amino acid peptide (1708 to 1724) of the C-terminus (anti-C-term) and a commercial antibody directed to the intracellular loop between domain II and III (anti-II-III loop) for the full length Cav1.3.
Results: Under basal condition, anti-II-III loop antibody detected a 190 kD band corresponding to a truncated Cav1.3. Cav1.3 was neither phosphorylated under basal conditions nor in the presence of NE. However, a 50 kD band corresponding to a portion of C-terminal fragment was detected with anti-phosphoserine antibody in the presence of NE. A band (above 64 kD) corresponding to the cleaved C-terminus was identified with anti-C-term antibody in the presence of NE consistent with the previously identified phosphorylation sites.
Conclusion: The data are first to show that C-terminus of Cav1.3 L-type Ca channel is truncated in vivo, and that C-terminal fragment is regulated, in vivo, by PKA. These findings have physiological and clinical significance for the regulation of heart rhythm by the autonomic nervous system in vivo.