Comparison of PKA-phosphorylated residues on HCN4 to predicted PKA phosphorylation sites. Amino acid sequence of mHCN4 is shown with color coding to illustrate phosphorylated residues identified by MS compared with predicted PKA sites. Red residues were phosphorylated in multiple peptide fragments, and green residues in single peptide fragments. Sequences identified as high probability PKA phosphorylation sites (score ≥ 0.5) by the pkaPS algorithm are highlighted in yellow. The cyclic nucleotide binding domain is indicated by a black outline, and transmembrane regions by gray shading. (Inset) Schematic representation of an HCN4 subunit with the approximate location of identified phosphorylated residues marked by red and green circles. The cyclic nucleotide binding domain is represented by a thick black line.