The auxiliary subunit TRIP8b inhibits the binding of cAMP to HCN2 channels through an allosteric mechanism

In neurons, hyperpolarization-activated cyclic nucleotide-regulated (HCN1-4) channels are the molecular determinants of the Ih current that controls cell excitability and rhythmicity [1]. HCN channels are activated by membrane hyperpolarization and directly regulated by cAMP binding to the cyclic nucleotide binding domain (CNBD) [2]. HCN channels are further regulated by their association with an auxiliary protein, TRIP8b, which binds to the CNBD and opposes cAMP regulation [3]. We asked if the effect of TRIP8b could be attributed to a direct competition with cAMP. To this end, using NMR methodologies, we determined the 3D structure of the human HCN2 CNBD in the cAMP-unbound form and subsequently mapped onto it the TRIP8b binding site. Our results show that cAMP and TRIP8b do not directly compete for binding to the same residues, and support an allosteric inhibition model for the dual regulation of HCN channels by cAMP and TRIP8b.