Posttranslational modifications of cyclic AMP-dependent protein kinase a affect its binding to the CFTR anion channel.

Abstract

CFTR is the only ion channel member in the ABC protein superfamily. Mutations of the protein cause malfunction of the pore or retention of the protein in the endoplasmic reticulum leading to the systemic disease cystic fibrosis, the most common lethal genetic disorder in the Caucasian population. CFTR activity relies on an irreversible gating mechanism which involves a cycle of ATP binding and hydrolysis at its cytosolic nucleotide binding domains (NBDs). Upon binding of two molecules of ATP the NBDs form a tight NBD dimer which triggers opening of the channel pore, while ATP hydrolysis closes the channel. ATP-dependent opening of the pore strongly depends on phosphorylation of the channel's regulatory (R) domain by cAMP-dependent protein kinase (PKA). At physiological ATP concentrations the NBDs of the channel are saturated by ligand, hence PKA phosphorylation plays the primary regulatory role. Our group has previously shown (PNAS 117:21740) that direct physical binding of PKA activates CFTR channels independently of, and additively to, phosphorylation. We hypothesized that PKA binding prevents its substrate, the R domain, from returning into an inhibitory position wedged in between the two NBDs, thereby shifting the closed-open equilibrium towards open. In contrast to the protein isolated from bovine heart, heterologously expressed PKA lacks such a binding effect. We therefore studied posttranslational modifications of PKA, and investigated their effects on CFTR activation by comparing the recombinant and tissue-isolated PKA isoforms. Results of the ongoing research will be presented at the meeting.