Rational design of a mutation to investigate the role of the brain protein TRIP8b in limiting the cAMP response of HCN channels in neurons.

TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) is the neuronal regulatory subunit of HCN channels, a family of voltage-dependent cation channels also modulated by direct cAMP binding. TRIP8b interacts with the C-terminal region of HCN channels and controls both channel trafficking and gating. The association of HCN channels with TRIP8b is required for the correct expression and subcellular targeting of the channel protein in vivo. TRIP8b controls HCN gating by interacting with the cyclic nucleotide-binding domain (CNBD) and competing for cAMP binding. Detailed structural knowledge of the complex between TRIP8b and CNBD was used as a starting point to engineer a mutant channel, whose gating is controlled by cAMP, but not by TRIP8b, while leaving TRIP8b-dependent regulation of channel trafficking unaltered. We found two-point mutations (N/A and C/D) in the loop connecting the CNBD to the C-linker (N-bundle loop) that, when combined, strongly reduce the binding of TRIP8b to CNBD, leaving cAMP affinity unaltered both in isolated CNBD and in the full-length protein. Proof-of-principle experiments performed in cultured cortical neurons confirm that the mutant channel provides a genetic tool for dissecting the two effects of TRIP8b (gating versus trafficking). This will allow the study of the functional role of the TRIP8b antagonism of cAMP binding, a thus far poorly investigated aspect of HCN physiology in neurons.