PKA phosphorylates HCN4 channel fragments in vitro. (A) Schematic representation of HCN4 GST fusion proteins. (B) In vitro phosphorylation and Western blotting of HCN4 channel fragments fused to GST. Fusion proteins were incubated with ³²P-ATP in the absence or presence of PKA (20 U/ml) or PKA plus PKI (20 µM) as indicated. Incorporated ³²P was visualized by autoradiography (top), and GST fusion proteins were visualized by Western blotting with an anti-GST antibody (1:50,000; Oncogene; bottom). (C) Average densitometric analysis of ³²P incorporation into the HCN4 GST fusion proteins for three independent experiments. Red bars show average density of ³²P normalized to the density of corresponding protein bands in the presence of PKA, blue bars, in PKA plus PKI. Asterisks indicate a significant reduction in normalized ³²P incorporation in the presence of PKI.