Protein kinase A activation phosphorylates the rat ClC-2Cl(-) channel but does not change activity

Phosphorylation-dependent events have been shown to modulate the activity o f several members of the mammalian CLC Cl- channel gene family, including t he inward rectifier ClC-2. In the present study we investigated the regulat ion of rat ClC-2 expressed in the TSA-201 cell line (a transformed HEK293 c ell line that stably expresses the SV40 T-antigen) by protein kinases. Prot ein kinase A activation phosphorylated ClC-2 in vivo. whereas stimulation o f protein kinase C with phorbol 12-myristate 13-acetate did not. In vitro l abeling studies confirmed that protein kinase A could directly phosphorylat e ClC-2, and that protein kinase C and Ca2+/calmodulin-dependent protein ki nase II did not. Nevertheless, protein kinase A-dependent phosphorylation o f CLC-2 failed to regulate either the magnitude or the kinetics of the hype rpolarization-activated Cl- currents. Considered together, we demonstrate t hat protein kinase A activation results in the phosphorylation of rat ClC-2 in vivo, but this event is independent of Cl- channel activity.