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PEX5L binds HCN1. 94 / 95
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"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."
20305382
sparser
"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 )."
25201528
sparser
"TRIP8b binds to both the extreme C-terminal Ser-Asn-Leu tripeptide of HCN1 and the cyclic nucleotide binding domain, spanning the intrinsically disordered region containing the 14-3-3 binding sites [ xref ]."
35679272
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"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."
25201528
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"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."
26045196
sparser
"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 ]."
23219411
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"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."
28057841
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"Consistent with this hypothesis, we find that HCN1 and TRIP8b are indeed tightly associated in the brain."
19555649
sparser
"We speculated that the interaction of TRIP8b with HCN1 might account for certain discrepancies between the properties of native and heterologously expressed I h ."
19555649
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"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."
22363812
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"Here, we address the structural bases of the regulatory interactions between mouse TRIP8b and HCN1."
21411649
sparser
"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 )."
19439603
sparser
"HCN1 CNBD interacted with TRIP8b IsoA4(1–451) but not TRIP8b IsoA2(1–374)."
19439603
sparser
"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 ."
19439603
sparser
"We next confirmed this dual interaction between HCN1 and TRIP8b in mammalian HEK293T cells by coimmunoprecipitation."
19439603
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"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."
21411649
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"In contrast, the downstream interaction site stabilizes the C-terminal domain of TRIP8b, allowing for optimal interaction between HCN1 and TRIP8b."
21411649
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"Briefly, the interaction of SmBiT-HCN1 with LgBiT-TRIP8b in live cells promotes assembly of functional luciferase through the proximity of the SmBiT and LgBiT fusion fragments, leading to the production of a luminescent signal in proportion to the amount of HCN1–TRIP8b binding ( xref B )."
35623388
sparser
"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."
19555649
sparser
"Furthermore, there is a decrease in the amount of TRIP8b associated with HCN1, but not HCN2."
19555649
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"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 )."
19555649
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"And knockout of TRIP8b may predispose mice to absence seizures, while TRIP8b interactions with HCN1 are disrupted by status epilepticus ( xref ; xref )."
35795651
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"We first investigated the interaction between HCN1 and TRIP8b by yeast two-hybrid analysis of different domains of either protein."
19439603
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"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."
21411649
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"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)."
21411649
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"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."
21411649
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"Together, these studies show that TRIP8b interacts with HCN1 at two distinct sites requiring two specific regions of the TRIP8b molecule."
19439603
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"Increasing concentrations of NUCC-0200590 led to a reduction in HCN1–TRIP8b binding as indicated by normalized luminescence, with an IC 50 of 5.62 ± 0.49 μM. To ensure the inhibition was specific, we also examined the interaction of the positive control pair protein kinase A catalytic (PRKACA) with type 2A regulatory subunit (PRKAR2A)."
35623388
sparser
"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."
26045196
sparser
"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."
19439603
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"Thus, TRIP8b binding to HCN1 at the upstream CNBD interaction site must be sufficient to inhibit gating."
21411649
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"This reduction of functional h channels in epileptic animals was associated with reduced interaction between HCN1 and TRIP8b ( xref )."
19439603
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"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."
26045196
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"The loss of HCN1 in distal dendrites minimizes the interaction between TRIP8b and HCN1 channels in animal models of temporal lobe epilepsy, which implies that TRIP8b interaction with HCN1 is important for appropriate HCN1 channel function in CA1 pyramidal neuron dendrites (Shin et al., 2008)."
35663267
sparser
"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."
26045196
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"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)."
21411649
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"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."
21411649
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"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)."
21411649
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"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."
21411649
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"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."
21411649
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"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."
21411649
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"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."
21411649
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"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."
21411649
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"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."
21411649
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"One such phosphorylation site, located in the loop between TPR3 and TPR4, is poised to regulate the downstream interaction between TRIP8b and HCN1."
21411649
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"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 )."
18834920
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"HCN1 and TRIP8b interact in the retina as confirmed by immunoprecipitation from retinal membranes ( xref )."
24409334
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"Next we examined whether AP trafficking proteins are, in fact, associated with the HCN1 and TRIP8b complexes."
19555649
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"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."
19555649
reach
"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]."
18346465
sparser
"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."
22363812
reach
"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."
21411649
sparser
"In the KA-induced rat model of epilepsy, the levels of TRIP8b Ser237 phosphorylation and CaMKIIα activity are reduced after SE induction, which leads to reduced TRIP8b-HCN1 binding, resulting in a disrupted HCN1 gradient from soma to dendrite, and this may be involved in epileptogenesis [ xref ]."
37366350
sparser
"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 )."
19753094
sparser
"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."
20305382
sparser
"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 ."
22363812
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"Interaction of HCN1 and TRIP8b in the Retina."
24409334
sparser
"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 )."
21555075
sparser
"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 )."
21555075
sparser
"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)."
21555075
sparser
"The loss of HCN1 in distal dendrites minimizes the interaction between TRIP8b and HCN1 channels in animal models of temporal lobe epilepsy, which implies that TRIP8b interaction with HCN1 is important for appropriate HCN1 channel function in CA1 pyramidal neuron dendrites (Shin et al., xref )."
35663267
sparser
"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."
21555075
sparser
"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 )."
21555075
sparser
"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."
15564593
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"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."
24277603
sparser
"To our knowledge, this compound represents the first described inhibitor of the TRIP8b-HCN1 interaction."
26045196
reach
"Under these conditions the binding between TRIP8b and HCN1 is reduced by cAMP in a concentration dependent manner."
24277603
reach
"We first investigated the interaction between HCN1 and TRIP8b by yeast two-hybrid analysis of different domains of either protein."
19439603
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"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)."
19439603
reach
"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."
19439603
reach
"We next confirmed this dual interaction between HCN1 and TRIP8b in mammalian HEK293T cells by coimmunoprecipitation."
19439603
sparser
"Thus, loss of the interaction between HCN1 and TRIP8b was not due to altered protein expression level of TRIP8b."
18657617
sparser
"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."
18657617
sparser
"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."
18657617
sparser
"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."
18657617
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"Together, these studies show that TRIP8b interacts with HCN1 at two distinct sites requiring two specific regions of the TRIP8b molecule."
19439603
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"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)."
24277603
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"And knockout of TRIP8b may predispose mice to absence seizures, while TRIP8b interactions with HCN1 are disrupted by status epilepticus (Shin et al., 2008; Heuermann et al., 2016)."
35795651
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"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)."
24277603
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"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."
19439603
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"This reduction of functional h channels in epileptic animals was associated with reduced interaction between HCN1 and TRIP8b."
19439603
reach
"Thus, loss of the interaction between HCN1 and TRIP8b was not due to altered protein expression level of TRIP8b."
18657617
reach
"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."
18657617
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"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."
18657617
sparser
"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."
21504900
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"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."
18657617
sparser
"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."
21504900
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"We found that isoform-wide disruption of the TRIP8b and HCN1 interaction caused HCN1 to be mistargeted throughout CA1 somatodendritic compartments."
21555075
reach
"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."
21555075
sparser
"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] ."
18346465
sparser
"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."
21593326
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"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."
15564593
sparser
"We noted that less TRIP8b bound to HCN1 in the hippocampi of mice receiving CNO in their drinking water compared with mice receiving saline in their drinking water."
34818058
sparser
"The most likely explanation for the intact gradient of HCN2 in the Hcn2 R/R mice is that there are relatively few HCN2 homomers and that TRIP8b binding to the HCN1 subunit of HCN1/HCN2(R591E) heterotetramers is sufficient to maintain HCN2 dendritic trafficking ( xref )."
34818058