N⁶-modified cAMP derivatives that activate protein kinase A also act as full agonists of murine HCN2 channels

cAMP acts as a second messenger in many cellular processes. Three protein types mainly mediate cAMP-induced effects: PKA, exchange protein directly activated by cAMP (Epac), and cyclic nucleotide-modulated channels (cyclic nucleotide-gated or hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels). Discrimination among these cAMP signaling pathways requires specific targeting of only one protein. Previously, cAMP modifications at position N⁶ of the adenine ring (PKA) and position 2'-OH of the ribose (Epac) have been used to produce target-selective compounds. However, cyclic nucleotide-modulated ion channels were usually outside of the scope of these previous studies. These channels are widely distributed, so possible channel cross-activation by PKA- or Epac-selective agonists warrants serious consideration. Here we demonstrate the agonistic effects of three PKA-selective cAMP derivatives, N⁶-phenyladenosine-3',5'-cyclic monophosphate (N⁶-Phe-cAMP), N⁶-benzyladenosine-3',5'-cyclic monophosphate (N⁶-Bn-cAMP), and N⁶-benzoyl-adenosine-3',5'-cyclic monophosphate (N⁶-Bnz-cAMP), on murine HCN2 pacemaker channels. Electrophysiological characterization in Xenopus oocytes revealed that these derivatives differ in apparent affinities depending on the modification type but that their efficacy and effects on HCN2 activation kinetics are similar to those of cAMP. Docking experiments suggested a pivotal role of Arg-635 at the entrance of the binding pocket in HCN2, either causing stabilizing cation-π interactions with the aromatic ring in N⁶-Phe-cAMP or N⁶-Bn-cAMP or a steric clash with the aromatic ring in N⁶-Bnz-cAMP. A reduced apparent affinity of N⁶-Phe-cAMP toward the variants R635A and R635E strengthened that notion. We conclude that some PKA activators also effectively activate HCN2 channels. Hence, when studying PKA-mediated cAMP signaling with cAMP derivatives in a native environment, activation of HCN channels should be considered.