Kv4.2 phosphorylation by cyclic AMP-dependent protein kinase

Recent evidence suggests that K+ channels composed of Kv4.2 alpha-subunits underlie a transient current in hippocampal CA1 neurons and ventricular myo cytes, and activation of the cAMP second messenger cascade has been shown t o modulate this transient current. We determined if Kv4.2 alpha-subunits we re directly phosphorylated by cAMP-dependent protein kinase (PKA). The intr acellular domains of the amino and carboxyl termini of Kv4.2 were expressed as glutathione S-transferase fusion protein constructs; we observed that b oth of these fusion proteins were substrates for PKA in vitro. By using pho sphopeptide mapping and amino acid sequencing, we identified PHA phosphoryl ation sites on the amino- and carboxyl-terminal fusion proteins correspondi ng to Thr(38) and Ser(552), respectively, within the Kv4.2 sequence. Kineti c characterization of the PKA sites demonstrated phosphorylation kinetics c omparable to Kemptide. To evaluate PKA site phosphorylation in situ, phosph o-selective antisera for each of the sites were generated. By using COS-7 c ells expressing an EGFP-Kv4.2 fusion protein, we observed that stimulation of the endogenous PKA cascade resulted in an increase in phosphorylation of Thr(38) and Ser(552) within Kv4.2 in the intact cell. We also observed mod ulation of PKA phosphorylation at these sites within Kv4.2 in hippocampal a rea CA1. These results provide insight into likely sites of regulation of K v4.2 by PKA.