Separate, but interacting PKA phosphorylation and cAMP-binding domains? (A) Model of HCN4 channel (only three out of the four subunits are shown for clarity). The six transmembrane domains (S1–S6) are shown in green, with the positively charged S4 functioning as the voltage sensor. The cAMP-binding domain is based on the crystal structure of the C terminus of HCN2 (Zagotta et al., 2003), showing the cAMP-binding pocket. The >400–amino acid long C-terminal region after the CNBD is shown as unstructured, and the region with PKA sites that modulates HCN channel function is indicated with arrows. How phosphorylation of these distal C-terminal sites affect the voltage dependence is not clear. This region could interact with other cytosolic domains, such as the CNBD or the transmembrane domains, and the voltage sensor directly (arrows with question marks). (B) Schematic drawings showing the effects of cAMP and phosphatase inhibition on the voltage dependence of activation of HCN channels. Direct application of cAMP in the absence of ATP reversibly shifts the voltage dependence of activation of I_f in excised patches (DiFrancesco and Tortora, 1991). The phosphatase inhibitor calyculin A increased the ISO-induced shift in the voltage dependence of activation of I_f in SA node, suggesting some synergy between the effect of phosphatase activity and cAMP on HCN channels (Accili et al., 1997).