Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function

Abstract

cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.

Fig. 1.

cAMP-free structure of the human HCN2 CNBD and comparison with the bound structure. (A) Ribbon representation of the cAMP-free CNBD structure (orange). For better visualization of the structure, only one conformation of the unstructured C-terminal part of the C-helix (residues 659–663) is shown, whereas the unfolded region at the N terminus of the construct (residues 521–532) and the stretch following the C-helix (residues 664–672) are not shown. Secondary structure elements are labeled. (B) Ribbon representation of the X-ray cAMP-bound CNBD structure (gray) [PDB ID code 1Q5O (ref. 16)]. The cAMP molecule is shown in stick representation in black. (C) Superposition of the cAMP-free and cAMP-bound structures of the CNBD.

Fig. 2.

Conformational changes in the helical components following cyclic nucleotide binding to the CNBD. (A) Close-up view of the PBC. The phosphate-sugar moiety of cAMP binds to the PBC, inducing its rearrangement. In the absence of cAMP, Leu₆₁₂ of PBC occupies the space that is filled by Phe₆₃₈ of the B-helix in the cAMP-bound conformation. (B) Highlighted in red is the portion of the PBC loop that folds into αP upon cAMP binding. (C) Translational movement of the B- and C-helices moving as a rigid body toward the cAMP molecule bound to the PBC. (D) Folding of the C-terminal portion of the C-helix from Arg₆₅₉ to Ile₆₆₃ (shown in red). cAMP apolar interactions with the side chains of Arg₆₅₉ and Ile₆₆₃ are represented as dotted spheres. (E) Close-up view of the N-terminal helical bundle (αE′–turn–αA). The cAMP-induced movement of the B- and C-helices element forces the N-terminal helical bundle to adopt a new position. (F) Red-marked loop between αE′ and αA folds into αF′.

Fig. 3.

Proposed binding region for TRIP8b_core on the HCN2 CNBD. A ribbon representation of the CNBD shows in red the residues whose amide proton (^NH) signals were perturbed upon the addition of TRIP8b_core. van der Waals volumes are reported for the perturbed residues in the N-bundle loop and in the C-helix. For simplicity, only one conformation of the unstructured region of the C-helix (residues 659–663) is shown. The N- and C-terminal regions of the construct are omitted as in Fig. 1.

Fig. 4.

Contribution of the N-bundle loop and the C-helix/stretch to the TRIP8b binding site. (A, Lower) Bacterial lysates from cells coexpressing Strep-tagged TRIP8b_core (blue arrowhead) and His₆-MBP–tagged CNBD WT and mutants (green arrowhead) were loaded onto a Strep-tactin affinity column for Strep-tag purification. Eluted samples were analyzed by Coomassie Blue staining following SDS/PAGE separation. Numbers on the left indicate molecular mass markers (kDa), loaded in the first lane. Lane 1 contains CNBD_ΔN. Lane 2 contains CNBD_ΔC. Lanes 3–5 contain mutants obtained by progressive truncation of the C-helix. A stop codon was introduced after the following residues: Ala₆₅₁ (lane 3), Ile₆₅₇ (lane 4), and Ile₆₆₃ (lane 5). Pro₆₄₆ and Ile₆₆₃ correspond to the first and last amino acids of the C-helix, respectively. Lanes 6 and 7 contain mutants obtained by progressive truncation of the stretch following the C-helix. A stop codon was introduced after the following residues: Lys₆₆₆ (lane 6) and Ile₆₆₉ (lane 7). Lane 8 contains K₆₆₅E/K₆₆₆E double-CNBD point mutant. (A, Upper) Sequence and cartoon representation of the C-helix/stretch are shown. Arrows indicate the last residue of the deletion constructs. Numbering of the arrows corresponds to gel lanes. (B) Coomassie Blue staining of the bacterial lysates before Strep-tactin affinity purification, showing an equivalent expression level of all the mutant constructs tested. The green arrowhead indicates the His₆-MBP–tagged CNBD proteins. Lane numbers are as in A.