"HCN1 and TRIP8b interact in the retina as confirmed by immunoprecipitation from retinal membranes ( xref )."
"In contrast to TRIP8b (1a), binding of TRIP8b (1a-4) to HCN1 did not seem to markedly affect subcellular distribution of the channels, suggesting a more general role of TRIP8b (1a-4) in promoting surface expression and membrane insertion."
"We subsequently developed a high throughput assay based on the FP screen to identify compounds that are capable of disrupting the tripeptide interaction between 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."
"As expected, a full length TRIP8b construct, IsoA4, bound to both HCN1 (386-591) (hereafter referred to as HCN1 CNBD) and HCN1 (778-910) (hereafter referred to as HCN1 C-term)."
"In addition, reduced dendritic transport of HCN1 channels or reduced association of HCN1 with the adaptor protein TRIP8b in the chronic period (resulting in diminished transport of HCN1 channels to CA1 pyramidal cell dendrites) may also contribute to epileptogenesis ( xref )."
"We next confirmed this dual interaction between HCN1 and TRIP8b in mammalian HEK293T cells by coimmunoprecipitation."
"Together, these studies show that TRIP8b interacts with HCN1 at two distinct sites requiring two specific regions of the TRIP8b molecule."
"Moreover the binding of TRIP8b to the HCN1 DeltaSNL truncation mutant is abolished when a highly conserved arginine residue in the CNBD, which interacts with the cyclized phosphate of cAMP, is mutated to glutamate (R538E in HCN1 and R591E in HCN2)."
"Taken together, these results indicated a mechanism of dual binding of TRIP8b with HCN1 whereby the combination of both sets of TPR domains is required to bind to the C-terminal tripeptide of HCN1 (–SNL), while the first TPR set with 58 amino acid residues N-terminal to the TPR domains was required to bind to HCN1 CNBD ."
"We first investigated the interaction between HCN1 and TRIP8b by yeast two-hybrid analysis of different domains of either protein."
"As expected, we found that the interaction of TRIP8b with the final 133 amino acids of HCN1 was interrupted by removal of the –SNL ending of HCN1 ( xref )."
"Such results support the view that TRIP8b does indeed antagonize the ability of cAMP to shift HCN1 channel opening to more positive potentials and that the interaction of TRIP8b with HCN1 may be weakened following patch excision."
"Interaction with the last three amino acids of the HCN1 C terminus governed the effect of TRIP8b on channel trafficking, whereas TRIP8b interaction with the HCN1 cyclic nucleotide binding domain (CNBD) affected trafficking and gating."
"This TRIP8b (1b-2) N-terminal deletion mutant (TRIP8b DeltaNX) still strongly binds to HCN1, indicating that the extreme N-terminus of TRIP8b is not necessary for channel binding."
"Interaction of HCN1 and TRIP8b in the Retina."
"We now report that 1) interaction between TRIP8b and HCN1 was markedly reduced at 28 d but not 1 d after SE and 2) TRIP8b remained enriched along with HCN2 in distal dendrites, whereas HCN1 was mislocalized to the soma at 28–30 d after SE."
"Here, we address the structural bases of the regulatory interactions between mouse TRIP8b and HCN1."
"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."
"Using co-immunoprecipitation assays from Xenopus oocytes, we further determined that, in native conditions, both interaction sites contribute to the stability of the TRIP8b and HCN1 complex."
"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."
"Now that the structural basis for Trip8b interaction with HCN1 has been resolved, a targeted approach using the corresponding regions on HCN4 as bait may prove fruitful and perhaps more direct than the original efforts."
"We also developed an FP-based high throughput screening assay to identify small molecules capable of disrupting the TRIP8b-HCN1 interaction, and we employed the assay to screen a library of 20,000 small molecules."
"These results, together with the robust biochemical association between HCN1 and TRIP8b in vivo (XREF_FIG) and the tight co-localization of the two proteins in the distal dendrites of cortical pyramidal neurons, strongly suggest that TRIP8b plays an important role in the regulation of I h in the brain."
"The interactions between wild-type HCN1 and TRIP8b are summarized in schematic form in XREF_FIG, which also provides a more speculative model to explain certain phenotypes of HCN1 and TRIP8b mutants."
"In contrast, the downstream interaction site stabilizes the C-terminal domain of TRIP8b, allowing for optimal interaction between HCN1 and TRIP8b."
"Immunohistochemistry for TRIP8b and HCN1 demonstrated exquisite costaining in distal dendrites of CA1 and layer V neocortical pyramidal neurons, implying that TRIP8b’s association with at least HCN1 may play a functional role in vivo, an implication strengthened by the finding that TRIP8b enrichment in the distal dendrites of layer V neurons in the cortex was abolished in the HCN1 −/− mouse."
"Alterations in TRIP8b-HCN1 interactions may also contribute to the maladaptive changes in HCN1 expression associated with seizures that is thought to contribute to the development of epilepsy ( xref ; xref ; xref ; xref ; xref ; xref ), an effect that is, in part, due to a redistribution of HCN1 from the distal dendrites to the soma of CA1 neurons ( xref )."
"We now report that 1) interaction between TRIP8b and HCN1 was markedly reduced at 28 d but not 1 d after SE and 2) TRIP8b remained enriched along with HCN2 in distal dendrites, whereas HCN1 was mislocalized to the soma at 28-30 d after SE."
"Given the association between TRIP8b and HCN1 in regions of the mouse brain known to possess high levels of h channels and I h, XREF_BIBR, XREF_BIBR, XREF_BIBR, XREF_BIBR it was surprising that Santoro et al. found coexpression of HCN1 or HCN2 with TRIP8b led to dramatic downregulation of I h and surface associated h channel protein in oocytes as well as in cultured hippocampal neurons, with the decrease in I h dependent upon the presence of the h channel C-terminal tripeptide."
"When the interaction between HCN1 and TRIP8b is prevented by deletion of the HCN1 conserved C-terminal tripeptide (Ser-Asn-Leu), the channels are no longer targeted to the distal dendrites and are uniformly expressed throughout the CA1 somatodendritic compartment ( xref )."
"Thus, TRIP8b binding to HCN1 at the upstream CNBD interaction site must be sufficient to inhibit gating."
"Interestingly, a disruption of the interaction between the HCN1 subunits and TRIP8b has been reported as a mechanism underlying the channelopathic mislocation of h-channels in the kainate model of temporal lobe epilepsy (Shin et al., xref )."
"We subsequently developed a high throughput assay based on the FP screen to identify compounds that are capable of disrupting the tripeptide interaction between TRIP8b and HCN1."
"One explanation is that dendritic targeting and channel surface expression depend on distinct interactions of HCN1 with TRIP8b(1a-4) that are differentially sensitive to the loss of the SNL binding site."
"Furthermore, because h channel mislocalization was associated with disruption of interaction between HCN1 and TRIP8b, these data suggest abnormal h channel trafficking could contribute to increased hippocampal excitability and seizure propensity in TLE."
"To tease out the molecular foundations for these different effects of TRIP8b on HCN channel gating and surface expression, we, along with our collaborators (as well as the work of an independent group) have recently demonstrated that the interaction between TRIP8b and HCN1 channels involves two separate molecular interfaces that differentially influence channel gating and surface expression [ xref , xref ]."
"We next confirmed this dual interaction between HCN1 and TRIP8b in mammalian HEK293T cells by coimmunoprecipitation."
"Because different isoforms of TRIP8b elicited different effects on HCN1 mediated current and surface expression in HEK293T cells, yet all TRIP8b isoforms bind to HCN1, we tested if the differential structure of the isoforms might govern their effects on h channel trafficking and subcellular localization in neurons."
"This is consistent with recent reports that HCN1 and TRIP8b interact at two distinct sites ( xref ) and that the weakened binding between TRIP8b and HCN1 ΔSNL is sufficient to allow certain TRIP8b isoforms to enhance surface expression of the mutant channel (see Discussion)."
"Consistent with this hypothesis, we find that HCN1 and TRIP8b are indeed tightly associated in the brain."
"To our knowledge, this compound represents the first described inhibitor of the TRIP8b-HCN1 interaction."
"Next we examined whether AP trafficking proteins are, in fact, associated with the HCN1 and TRIP8b complexes."
"This observation is consistent with the fact that the TRIP8b DeltaTPR truncation mutant can efficiently bind to HCN1 at its C-helix and CNBD interaction site, and exert a normal inhibitory effect on channel gating (XREF_FIG, XREF_FIG)."
"To overcome the limitations of the siRNA approach, we expressed an EGFP tagged HCN1 truncation mutant (EGFP-HCN1 DeltaSNL) that lacks the HCN1 C-terminal SNL tripeptide required for high affinity binding of HCN1 to TRIP8b."
"Taken together, these results indicated a mechanism of dual binding of TRIP8b with HCN1 whereby the combination of both sets of TPR domains is required to bind to the C-terminal tripeptide of HCN1 (-SNL), while the first TPR set with 58 amino acid residues N-terminal to the TPR domains was required to bind to HCN1 CNBD."
"Furthermore, there is a decrease in the amount of TRIP8b associated with HCN1, but not HCN2."
"In contrast to TRIP8b(1a), binding of TRIP8b(1a-4) to HCN1 did not seem to markedly affect subcellular distribution of the channels, suggesting a more general role of TRIP8b(1a-4) in promoting surface expression and membrane insertion."
"To confirm that our active compound, NUCC-5953, disrupts the TRIP8b-HCN1 interaction (i.e., its activity in the FP assay is not due to fluorescence interference), we developed an orthogonal assay utilizing AlphaScreen technology."
"This implies that the normal interaction of HCN1 and TRIP8b at the downstream binding site may induce a conformational change in the TPR domain that allows for assembly of the trafficking complex."
"We first investigated the interaction between HCN1 and TRIP8b by yeast two-hybrid analysis of different domains of either protein."
"This reduction of functional h channels in epileptic animals was associated with reduced interaction between HCN1 and TRIP8b."
"We first sought to selectively disrupt the interaction between HCN1 and TRIP8b (1b-2) by introducing a point mutation in a conserved TPR residue, N501K, which greatly diminishes the binding of the PEX5 TPR domain to the C-terminal " SKL " tripeptide in PEX5 target proteins."
"Thus, altered expression of HCN channels or I h is a phenomenon regularly observed in models of experimental epilepsy xref , xref , xref , xref , xref , xref , xref , xref , and even in human epilepsy xref , and altered interaction of HCN1 with TRIP8b could be a critical mechanism contributing to this phenomen xref ."
"Thus, loss of the interaction between HCN1 and TRIP8b was not due to altered protein expression level of TRIP8b."
"The dendritic expression of TRIP8b in layer V pyramidal neurons is disrupted after deletion of HCN1 through homologous recombination, demonstrating a key in vivo interaction between HCN1 and TRIP8b."
"One such phosphorylation site, located in the loop between TPR3 and TPR4, is poised to regulate the downstream interaction between TRIP8b and HCN1."
"Together, these studies show that TRIP8b interacts with HCN1 at two distinct sites requiring two specific regions of the TRIP8b molecule."
"Thus, disruption of TRIP8b and HCN1 binding at either the upstream or downstream interaction sites greatly reduces the ability of TRIP8b isoforms to downregulate HCN1 surface expression, but has a limited effect on the ability of TRIP8b isoforms to enhance surface expression."
"Finally, the interaction of TRIP8b with HCN1 in distal dendrites may be extremely stable and persist even when the pool of available TRIP8b is decreased ( xref )."
"Related to the HCN1 and TRIP8b interaction, it is interesting to note that this binding is dependent on SNL, the C-terminal tripeptide of mouse HCN1 [20]."
"This reduction of functional h channels in epileptic animals was associated with reduced interaction between HCN1 and TRIP8b ( xref )."
"Under these conditions the binding between TRIP8b and HCN1 is reduced by cAMP in a concentration dependent manner."
"To overcome the limitations of the siRNA approach, we expressed an EGFP-tagged HCN1 truncation mutant (EGFP-HCN1 ΔSNL ) that lacks the HCN1 C-terminal SNL tripeptide required for high affinity binding of HCN1 to TRIP8b ( xref ; Santoro et al., 2011; xref )."
"The dendritic expression of TRIP8b in layer V pyramidal neurons is disrupted after deletion of HCN1 through homologous recombination, demonstrating a key in vivo interaction between HCN1 and TRIP8b."
"Second, we find that the binding of TRIP8b to HCN1 is not altered when the corresponding arginine (R538) is substituted with alanine (HCN1 R538A), although we confirmed that the R538E mutation does weaken the binding of TRIP8b to the channel (XREF_FIG)."
"These observations suggest that TRIP8b interaction with HCN1 may be critical for h channel trafficking in CA1 pyramidal neuron dendrites, and that yet unknown echanisms associated with TLE disrupt TRIP8b interaction with HCN1 and lead to h channel mislocalization."
"Because of the importance of the CNBD in h channel gating ( xref ; xref ), we reason that the TRIP8b interaction with the HCN1 CNBD could introduce steric effects to alter h channel gating."
"Biochemical studies revealed that direct interaction between TRIP8b and the HCN1 CNBD was disrupted by cAMP and that TRIP8b binding to the CNBD required an arginine residue also necessary for cAMP binding."
"Moreover, it suggests that when the TRIP8b and HCN1 interaction at the downstream site is prevented, there is an unmasking of a latent inhibitory effect exerted by the extreme C-terminus of HCN1 and/or the TPR domain of TRIP8b (see below and XREF_FIG)."
"Furthermore, because h channel mislocalization was associated with disruption of interaction between HCN1 and TRIP8b, these data suggest abnormal h channel trafficking could contribute to increased hippocampal excitability and seizure propensity in TLE."
"Of further interest, the activation of I h in CA1 distal apical dendrites is shifted by ∼6 mV to more negative voltages than those required to activate I h in more proximal regions of the dendrite ( xref ), an effect that might be explained by a more prominent association of TRIP8b with HCN1 channels in the distal dendrites, where TRIP8b and HCN1 co-localization is tightest ( xref )."
"Our findings that mutations that perturb the downstream interaction between TRIP8b and HCN1 differentially alter the functional consequences of residual binding at the upstream site have interesting implications for the dynamic regulation of the TRIP8b and HCN1 interaction in vivo."
"We speculated that the interaction of TRIP8b with HCN1 might account for certain discrepancies between the properties of native and heterologously expressed I h ."
"Here, we address the structural bases of the regulatory interactions between mouse TRIP8b and HCN1."
"A displaced TPR domain could interfere with clathrin mediated endocytosis of the HCN1 and TRIP8b complex; deletion of the C-terminal portion of the TPR domain would remove this inhibitory effect, rescuing the ability of TRIP8b to mediate HCN channel endocytosis."
"Related to the HCN1 and TRIP8b interaction, it is interesting to note that this binding is dependent on SNL, the C-terminal tripeptide of mouse HCN1 [20] ."
"A recent biochemical study focused on the nature of the interaction between TRIP8b and HCN1 at the upstream interaction site, using a channel in which the C-terminal SNL tripeptide was deleted to prevent the interaction at the downstream site."
"When the interaction between HCN1 and TRIP8b is prevented by deletion of the HCN1 conserved C-terminal tripeptide (Ser-Asn-Leu), the channels are no longer targeted to the distal dendrites and are uniformly expressed throughout the CA1 somatodendritic compartment."
"HCN1 CNBD interacted with TRIP8b IsoA4(1–451) but not TRIP8b IsoA2(1–374)."
"On the other hand, if interaction of HCN1 with TRIP8b alone enhances HCN1 protein expression or stability, then coexpression with either TRIP8b(1a-4) or TRIP8bΔN should enhance HCN1 protein levels."
"We found that isoform-wide disruption of the TRIP8b and HCN1 interaction caused HCN1 to be mistargeted throughout CA1 somatodendritic compartments."
"These observations suggest that TRIP8b interaction with HCN1 may be critical for h channel trafficking in CA1 pyramidal neuron dendrites, and that yet unknown echanisms associated with TLE disrupt TRIP8b interaction with HCN1 and lead to h channel mislocalization."
"Indeed, we find that whereas the extreme N- and C-termini of HCN1 (outside of the SNL sequence) do not directly bind TRIP8b, these domains appear to modulate the functional association between HCN1 and TRIP8b."
"Thus, loss of the interaction between HCN1 and TRIP8b was not due to altered protein expression level of TRIP8b."
"Here we have mapped distinct upstream and downstream sites of interaction between HCN1 and TRIP8b, and defined the differential roles of these sites in the functional effects of three different TRIP8b splice variants on channel trafficking and cyclic nucleotide gating."
"Furthermore, this result confirms that an intact TPR domain is not required for an interaction between HCN1 and TRIP8b at the upstream CNBD and core binding site in native conditions (see below)."