TRIP8b Regulates HCN1 Channel Trafficking and Gating through Two Distinct C-Terminal Interaction Sites

Abstract

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in the brain associate with their auxiliary subunit TRIP8b (also known as PEX5R), a cytoplasmic protein expressed as a family of alternatively spliced isoforms. Recent in vitro and in vivo studies have shown that association of TRIP8b with HCN subunits both inhibits channel opening and alters channel membrane trafficking, with some splice variants increasing and others decreasing channel surface expression. Here, we address the structural bases of the regulatory interactions between mouse TRIP8b and HCN1. We find that HCN1 and TRIP8b interact at two distinct sites: an upstream site where the C-linker/cyclic nucleotide-binding domain of HCN1 interacts with an 80 aa domain in the conserved central core of TRIP8b; and a downstream site where the C-terminal SNL (Ser-Asn-Leu) tripeptide of the channel interacts with the tetratricopeptide repeat domain of TRIP8b. These two interaction sites play distinct functional roles in the effects of TRIP8b on HCN1 trafficking and gating. Binding at the upstream site is both necessary and sufficient for TRIP8b to inhibit channel opening. It is also sufficient to mediate the trafficking effects of those TRIP8b isoforms that downregulate channel surface expression, in combination with the trafficking motifs present in the N-terminal region of TRIP8b. In contrast, binding at the downstream interaction site serves to stabilize the C-terminal domain of TRIP8b, allowing for optimal interaction between HCN1 and TRIP8b as well as for proper assembly of the molecular complexes that mediate the effects of TRIP8b on HCN1 channel trafficking.

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

“…We have previously shown that TRIP8b core , an 80-aa sequence located in the TRIP8b protein core that directly interacts with the C-linker/CNBD region of HCN channels, is necessary and sufficient to prevent all of the effects of cAMP on the channel (10,11). TRIP8b core decreases both the sensitivity of the channel to cAMP [half maximal concentration (k 1/2 )] and the efficacy of cAMP in inducing channel opening [half activation voltage (V 1/2 )]; conversely, cAMP binding inhibits these actions of TRIP8b.…”

Distinct Amino Acids in the C-Linker Domain of the Arabidopsis K+ Channel KAT2 Determine Its Subcellular Localization and Activity at the Plasma Membrane

“…This zone has been described in HCN channels to be flexible and able to absorb conformational changes (Taraska et al, 2009). Furthermore, evidence is available in HCN channels that the C-linker E9 and F9 helices play a role in proper folding of the CNBD (Santoro et al, 2011). It is also relevant that, in HCN channels expressed in mouse brain, the C-linker E9 and F9 helices and the CNBD form a site that binds auxiliary subunits, named TRIP8b or PEX5R, which play a role in the surface expression (and activity) of the channel (Santoro et al, 2011;Han et al, 2011;Bankston et al, 2012).…”

“…Furthermore, evidence is available in HCN channels that the C-linker E9 and F9 helices play a role in proper folding of the CNBD (Santoro et al, 2011). It is also relevant that, in HCN channels expressed in mouse brain, the C-linker E9 and F9 helices and the CNBD form a site that binds auxiliary subunits, named TRIP8b or PEX5R, which play a role in the surface expression (and activity) of the channel (Santoro et al, 2011;Han et al, 2011;Bankston et al, 2012). We thus hypothesize that, in KAT2, the short sequence that connects the C-linker F9 helix to the CNBD and comprises the Val-381 and Ser-382 residues plays a role in the folding of the structure formed by the C-linker and CNBD (as in HCN channels) and that substituting Val-381 by a Phe or Ser-382 by a Pro compromises the proper folding of this structure.…”

Dynamic measurements for funny channels

“…1A) HCN in a 1:1 stoichiometry and has profound effects on channel trafficking and gating (8)(9)(10). The C-terminal tetratricopeptide repeat (TPR) domain of TRIP8b interacts with the final three amino acids of HCN (SNL in HCN1, -2, and -4; ANM in HCN3) and this interaction affects channel trafficking (9,11,12). A core domain of TRIP8b upstream of the TPR domain interacts with the CNBD of HCN, affecting trafficking, but also interfering with the ability of cAMP to activate the channel (11)(12)(13).…”

“…The C-terminal tetratricopeptide repeat (TPR) domain of TRIP8b interacts with the final three amino acids of HCN (SNL in HCN1, -2, and -4; ANM in HCN3) and this interaction affects channel trafficking (9,11,12). A core domain of TRIP8b upstream of the TPR domain interacts with the CNBD of HCN, affecting trafficking, but also interfering with the ability of cAMP to activate the channel (11)(12)(13). Although the details of the TPR interaction with the distal C terminus of HCN are known at the atomic level, the nature of the interaction between the TRIP8b core domain and the CNBD has not been determined previously (8).…”