THE AUXILIARY SUBUNIT TRIP8B ANTAGONIZES 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, which controls several cognitive processes. Unique among the voltage-gated ion channel superfamily, HCN channels are modulated by the direct binding of cAMP to their cytoplasmic cyclic nucleotide binding domain (CNBD). Thus, cyclic nucleotide-dependent conformational changes of CNBD are determinant in the regulation of HCN channel opening. The rearrangements induced by cAMP in HCN CNBD are not yet elucidated, since for this protein is known only the cAMP-bound form. HCN channels are further regulated by their association with the auxiliary protein TRIP8b, which preferentially binds to the cAMP-unbound CNBD and opposes cAMP regulation. Recently, we proposed a cyclic allosteric model to explain the mutual antagonistic effect of TRIP8b and cAMP. Here, to validate this model, we first determined the model structure of the human HCN2 CNBD in the cAMP-unbound form using NMR methodologies. By comparing the cAMP-unbound and cAMP-bound structures we highlighted all the conformational changes allosterically coupled to the channel opening transition. Subsequently, we mapped the TRIP8b binding site onto the cAMP-unbound CNBD. Our results show that cAMP and TRIP8b do not compete for the same binding region, and support our allosteric antagonistic model for the dual regulation of HCN channels by cAMP and TRIP8b.