Abstract
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.
Highlights
Edited by Mike Shipston cAMP acts as a second messenger in many cellular processes
It has been shown that the cyclic nucleotide– binding domain has an inhibitory effect on channel gating that is relieved by cAMP binding [13]
We performed a comparative study of three cAMP derivatives, N6-Phe-cAMP, N6-Bn-cAMP, and N6-Bnz-cAMP, earlier described as activators of PKA, to investigate their effects on HCN2 channels
Summary
Exchange protein directly activated by cAMP; CN, cyclic nucleotide; HCN, hyperpolarization-activated and cyclic nucleotide–modulated; CNBD, cyclic nucleotide– binding domain; N6-PhecAMP, N6-phenyladenosine-3Ј,5Ј-cyclic monophosphate; N6-Bn-cAMP, N6-benzyladenosine-3Ј,5Ј-cyclic monophosphate; N6-Bnz-cAMP, N6-benzoyl-adenosine-3Ј,5Ј-cyclic monophosphate; RMSD, root mean square deviation. Epac tolerates 2Ј-OH modifications, modifications at the N6 position of the purine ring are not accepted by this protein Such N6-modified derivatives are often potent agonists for PKA and can be used to exclude Epac activation [23]. Besides a systematic study performed by Ng et al [24] comparing binding and gating parameters for different cyclic nucleotides in whole HCN channels, the effect of purine ring modifications at cAMP on functional channels have so far not been tested systematically. To contribute to the urgent problem of developing cAMP analogs that can discriminate between the different cAMP-binding proteins, here we tested three N6-modified cAMP derivatives: N6-phenyladenosine-3Ј,5Ј-cyclic monophosphate (N6-Phe-cAMP), N6-benzyladenosine-3Ј,5Ј-cyclic monophosphate (N6-BncAMP), and N6-benzoyladenosine-3Ј,5Ј-cyclic monophosphate (N6-Bnz-cAMP), known to be activators of PKA, on functional HCN2 channels. Docking and mutagenesis experiments revealed the specific interactions between molecule and binding pocket that underlie those differences
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
