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Publications (53)

  • Y Han · R J Heuermann · K A Lyman · [...] · D M Chetkovich
    [Show abstract] [Hide abstract] ABSTRACT: Major depressive disorder (MDD) is a prevalent psychiatric condition with limited therapeutic options beyond monoaminergic therapies. Although effective in some individuals, many patients fail to respond adequately to existing treatments, and new pharmacologic targets are needed. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate excitability in neurons, and blocking HCN channel function has been proposed as a novel antidepressant strategy. However, systemic blockade of HCN channels produces cardiac effects that limit this approach. Knockout (KO) of the brain-specific HCN-channel auxiliary subunit tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) also produces antidepressant-like behavioral effects and suggests that inhibiting TRIP8b function could produce antidepressant-like effects without affecting the heart. We examined the structural basis of TRIP8b-mediated HCN-channel trafficking and its relationship with antidepressant-like behavior using a viral rescue approach in TRIP8b KO mice. We found that restoring TRIP8b to the hippocampus was sufficient to reverse the impaired HCN-channel trafficking and antidepressant-like behavioral effects caused by TRIP8b KO. Moreover, we found that hippocampal expression of a mutated version of TRIP8b further impaired HCN-channel trafficking and increased the antidepressant-like behavioral phenotype of TRIP8b KO mice. Thus, modulating the TRIP8b-HCN interaction bidirectionally influences channel trafficking and antidepressant-like behavior. Overall, our work suggests that small-molecule inhibitors of the interaction between TRIP8b and HCN should produce antidepressant-like behaviors and could represent a new paradigm for the treatment of MDD.Molecular Psychiatry advance online publication, 12 July 2016; doi:10.1038/mp.2016.99.
    Article · Jul 2016 · Molecular Psychiatry
  • Robert J. Heuermann · Dane Chetkovich
    Article · May 2016 · Epilepsy Currents
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    Robert J Heuermann · Thomas C Jaramillo · Shui-Wang Ying · [...] · Dane M Chetkovich
    [Show abstract] [Hide abstract] ABSTRACT: Absence seizures occur in several types of human epilepsy and result from widespread, synchronous feedback between the cortex and thalamus that produces brief episodes of loss of consciousness. Genetic rodent models have been invaluable for investigating the pathophysiological basis of these seizures. Here, we identify tetratricopeptide-containing Rab8b-interacting protein (TRIP8b) knockout mice as a new model of absence epilepsy, featuring spontaneous spike-wave discharges on electroencephalography (EEG) that are the electrographic hallmark of absence seizures. TRIP8b is an auxiliary subunit of the hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, which have previously been implicated in the pathogenesis of absence seizures. In contrast to mice lacking the pore-forming HCN channel subunit HCN2, TRIP8b knockout mice exhibited normal cardiac and motor function and a less severe seizure phenotype. Evaluating the circuit that underlies absence seizures, we found that TRIP8b knockout mice had significantly reduced HCN channel expression and function in thalamic-projecting cortical layer 5b neurons and thalamic relay neurons, but preserved function in inhibitory neurons of the reticular thalamic nucleus. Our results expand the known roles of TRIP8b and provide new insight into the region-specific functions of TRIP8b and HCN channels in constraining cortico-thalamo-cortical excitability.
    Full-text Article · Oct 2015 · Neurobiology of Disease
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    Ye Han · Kyle Lyman · Matt Clutter · [...] · Dane M Chetkovich
    [Show abstract] [Hide abstract] ABSTRACT: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels function in the brain to limit neuronal excitability. Limiting the activity of these channels has been proposed as a therapy for major depressive disorder, but the critical role of HCN channels in cardiac pacemaking has limited efforts to develop therapies directed at the channel. Previous studies indicated that the function of HCN is tightly regulated by its auxiliary subunit, tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), which is not expressed in the heart. To target the function of the HCN channel in the brain without affecting the channel's function in the heart, we propose disrupting the interaction between HCN and TRIP8b. We developed a high-throughput fluorescence polarization (FP) assay to identify small molecules capable of disrupting this interaction. We used this FP assay to screen a 20,000-compound library and identified a number of active compounds. The active compounds were validated using an orthogonal AlphaScreen assay to identify one compound (0.005%) as the first confirmed hit for inhibiting the HCN-TRIP8b interaction. Identifying small molecules capable of disrupting the interaction between HCN and TRIP8b should enable the development of new research tools and small-molecule therapies that could benefit patients with depression. © 2015 Society for Laboratory Automation and Screening.
    Full-text Article · Jun 2015 · Journal of Biomolecular Screening
  • Kyle A Lyman · Dane M Chetkovich
    Article · Feb 2015 · Epilepsy Currents
  • [Show abstract] [Hide abstract] ABSTRACT: HCN channels are important regulators of neuronal excitability. The proper function of these channels is governed by various mechanisms, including post-translational modifications of channel subunits. Here, we provide evidence that ubiquitination via a ubiquitin ligase, neuronal precursor cell expressed developmentally downregulated (Nedd)-4-2, is involved in the regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We identified a PY motif (L/PPxY), the characteristic binding motif for Nedd4-2 in the C terminus of the HCN1 subunit, and showed that HCN1 and Nedd4-2 interacted both in vivo (rat hippocampus, neocortex, and cerebellum) and in vitro [human embryonic kidney 293 (HEK293) cells], resulting in increased HCN1 ubiquitination. Elimination of the PY motif reduced, but did not abolish, Nedd4-2 binding, which further involved a stretch of ∼100 aa downstream in the HCN1 C terminus. Coexpression of Nedd4-2 and HCN1 drastically reduced the HCN1-mediated h-current amplitude (85-92%) in Xenopus laevis oocytes and reduced surface expression (34%) of HCN1 channels in HEK293 cells, thereby opposing effects of tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b)-(1a-4), an auxiliary subunit that promotes HCN1 surface expression. Regulation may further include N-glycosylation of HCN1 channels, which is significantly enhanced by TRIP8b(1a-4), but may be reduced by Nedd4-2.Taken together, our data indicate that Nedd4-2 plays an important role in the regulation of HCN1 trafficking and may compete with TRIP8b(1a-4) in this process.-Wilkars, W., Wollberg, J., Mohr, E., Han, M., Chetkovich, D. M., Bähring, R., Bender, R. A. Nedd4-2 regulates surface expression and may affect N-glycosylation of hyperpolarization-activated cyclic nucleotide-gated (HCN)-1 channels.
    Article · Jan 2014 · The FASEB Journal
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    [Show abstract] [Hide abstract] ABSTRACT: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are cation-selective channels present in retina, brain and heart. The activity of HCN channels contributes to signal integration, cell excitability and pacemaker activity. HCN1 channels expressed in photoreceptors participate in keeping light responses transient and are required for normal mesopic vision. The subcellular localization of HCN1 varies among cell types. In photoreceptors HCN1 is concentrated in the inner segments while in other retinal neurons, HCN1 is evenly distributed though the cell. This is in contrast to hippocampal neurons where HCN1 is concentrated in a subset of dendrites. A key regulator of HCN1 trafficking and activity is tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b). Multiple splice isoforms of TRIP8b are expressed throughout the brain and can differentially regulate the surface expression and activity of HCN1. The purpose of the present study was to determine which isoforms of TRIP8b are expressed in the retina and to test if loss of TRIP8b alters HCN1 expression or trafficking. We found that TRIP8b colocalizes with HCN1 in multiple retina neurons and all major splice isoforms of TRIP8b are expressed in the retina. Photoreceptors express three different isoforms. In TRIP8b knockout mice, the ability of HCN1 to traffic to the surface of retinal neurons is unaffected. However, there is a large decrease in the total amount of HCN1. We conclude that TRIP8b in the retina is needed to achieve maximal expression of HCN1.
    Full-text Article · Jan 2014 · PLoS ONE
  • Darrin H Brager · Alan S Lewis · Dane M Chetkovich · Daniel Johnston
    [Show abstract] [Hide abstract] ABSTRACT: Hyperpolarization-activated cyclic nucleotide gated non-selective cation channels (HCN or h channels) are important regulators of neuronal physiology contributing to passive membrane properties, such as resting membrane potential and input resistance, and to intrinsic oscillatory activity and synaptic integration. The correct membrane targeting of h-channels is regulated in part by the auxiliary h-channel protein TRIP8b. The genetic deletion of TRIP8b results in a loss of functional h channels, which affects the postsynaptic integrative properties of neurons. We investigated the impact of TRIP8b deletion on long-term potentiation at the two major excitatory inputs to CA1 pyramidal neurons: Schaffer collateral (SC) and perforant path (PP). We found that SC LTP was not significantly different between neurons from wildtype and TRIP8b knockout mice. There was however, significantly more short-term potentiation in knockout neurons. We also found that the persistent increase in h current (Ih) that normally occurs following LTP induction was absent in knockout neurons. The lack of Ih plasticity was not restricted to activity-dependent induction, because the depletion of intracellular calcium stores also failed to produce the expected increase in Ih. Interestingly, pairing of SC and PP inputs resulted in a form of LTP in knockout neurons that did not occur in wildtype neurons. These results suggest that physiological impact of TRIP8b deletion are not restricted to the integrative properties of neurons but also include both synaptic and intrinsic plasticity.
    Article · Aug 2013 · Journal of Neurophysiology
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    [Show abstract] [Hide abstract] ABSTRACT: The rodent hippocampus can be divided into dorsal (DHC) and ventral domains (VHC) based on behavioral, anatomical, and biochemical differences. Recently, we reported that CA1 pyramidal neurons from the VHC were intrinsically more excitable than DHC neurons, but the specific ionic conductances contributing to this difference were not determined. Here, we investigated the hyperpolarization-activated current (I(h)) and the expression of HCN1 and HCN2 channel subunits in CA1 pyramidal neurons from the DHC and VHC. Measurement of I(h) using cell-attached patches revealed a significant depolarizing shift in the V(1/2) of activation for dendritic h-channels in VHC neurons (but not DHC neurons), and ultrastructural immunolocalization of HCN1 and HCN2 channels revealed a significantly larger HCN1/HCN2 ratio for VHC neurons (but not DHC neurons). These observations suggest that a shift in the expression of HCN1 and HCN2 channels drives functional changes in Ih for VHC neurons (but not DHC neurons), and could thereby significantly alter the capacity for dendritic integration of these neurons.
    Full-text Article · Jan 2013 · Journal of Neurophysiology
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    [Show abstract] [Hide abstract] ABSTRACT: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are subthreshold activated voltage-gated ion channels. In the cortex, these channels are predominantly expressed in dendrites where they significantly modify dendritic intrinsic excitability as well synaptic potential shapes and integration. HCN channel trafficking to dendrites is regulated by the protein, TRIP8b. Additionally, altered TRIP8b expression may be one mechanism underlying seizure-induced dendritic HCN channel plasticity. HCN channels, though, are also located in certain mature cortical synaptic terminals, where they play a vital role in modulating synaptic transmission. In this study, using electrophysiological recordings as well as electron microscopy we show that presynaptic, but not dendritic, cortical HCN channel expression and function is comparable in adult TRIP8b-null mice and wild-type littermates. We further investigated whether presynaptic HCN channels undergo seizure-dependent plasticity. We found that, like dendritic channels, wild-type presynaptic HCN channel function was persistently decreased following induction of kainic acid-induced seizures. Since TRIP8b does not affect presynaptic HCN subunit trafficking, seizure-dependent plasticity of these cortical HCN channels is not conditional upon TRIP8b. Our results, thus, suggest that the molecular mechanisms underlying HCN subunit targeting, expression and plasticity in adult neurons is compartment selective, providing a means by which pre- and postsynaptic processes that are critically dependent upon HCN channel function may be distinctly influenced.
    Full-text Article · Oct 2012 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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    [Show abstract] [Hide abstract] ABSTRACT: Ion channels operate in intact tissues as part of large macromolecular complexes that can include cytoskeletal proteins, scaffolding proteins, signaling molecules, and a litany of other molecules. The proteins that make up these complexes can influence the trafficking, localization, and biophysical properties of the channel. TRIP8b (tetratricopetide repeat-containing Rab8b-interacting protein) is a recently discovered accessory subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that contributes to the substantial dendritic localization of HCN channels in many types of neurons. TRIP8b interacts with the carboxyl-terminal region of HCN channels and regulates their cell-surface expression level and cyclic nucleotide dependence. Here we examine the molecular determinants of TRIP8b binding to HCN2 channels. Using a single-molecule fluorescence bleaching method, we found that TRIP8b and HCN2 form an obligate 4:4 complex in intact channels. Fluorescence-detection size-exclusion chromatography and fluorescence anisotropy allowed us to confirm that two different domains in the carboxyl-terminal portion of TRIP8b--the tetratricopepide repeat region and the TRIP8b conserved region--interact with two different regions of the HCN carboxyl-terminal region: the carboxyl-terminal three amino acids (SNL) and the cyclic nucleotide-binding domain, respectively. And finally, using X-ray crystallography, we determined the atomic structure of the tetratricopepide region of TRIP8b in complex with a peptide of the carboxy-terminus of HCN2. Together, these experiments begin to uncover the mechanism for TRIP8b binding and regulation of HCN channels.
    Full-text Article · May 2012 · Proceedings of the National Academy of Sciences
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    [Show abstract] [Hide abstract] ABSTRACT: The dorsal and ventral regions of the hippocampus perform different functions. Whether the integrative properties of hippocampal cells reflect this heterogeneity is unknown. We focused on dendrites where most synaptic input integration takes place. We report enhanced backpropagation and theta resonance and decreased summation of synaptic inputs in ventral versus dorsal CA1 pyramidal cell distal dendrites. Transcriptional Kv4.2 down-regulation and post-transcriptional hyperpolarization-activated cyclic AMP-gated channel (HCN1/2) up-regulation may underlie these differences, respectively. Our results reveal differential dendritic integrative properties along the dorso-ventral axis, reflecting diverse computational needs.
    Full-text Article · Apr 2012 · Journal of Biological Chemistry
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    [Show abstract] [Hide abstract] ABSTRACT: The dorsoventral and developmental gradients of entorhinal layer II cell grid properties correlate with their resonance properties and with their hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel current characteristics. We investigated whether such correlation existed in rat hippocampal CA1 pyramidal cells, where place fields also show spatial and temporal gradients. Resonance was absent during the first postnatal week, and emerged during the second week. Resonance was stronger in dorsal than ventral cells, in accord with HCN current properties. Resonance responded to cAMP in ventral but not in dorsal cells. The dorsoventral distribution of HCN1 and HCN2 subunits and of the auxiliary protein tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) could account for these differences between dorsal and ventral cells. The analogous distribution of the intrinsic properties of entorhinal stellate and hippocampal cells suggests the existence of general rules of organization among structures that process complementary features of the environment.
    Full-text Article · Mar 2012 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
  • Marcelin B · Liu Z · Chen Y · [...] · Bernard C
    [Show abstract] [Hide abstract] ABSTRACT: The dorsoventral and developmental gradients of entorhinal layer II cell grid properties correlate with their resonance properties and with their hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel current characteristics. We investigated whether such correlation existed in rat hippocampal CA1 pyramidal cells, where place fields also show spatial and temporal gradients. Resonance was absent during the first postnatal week, and emerged during the second week. Resonance was stronger in dorsal than ventral cells, in accord with HCN current properties. Resonance responded to cAMP in ventral but not in dorsal cells. The dorsoventral distribution of HCN1 and HCN2 subunits and of the auxiliary protein tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) could account for these differences between dorsal and ventral cells. The analogous distribution of the intrinsic properties of entorhinal stellate and hippocampal cells suggests the existence of general rules of organization among structures that process complementary features of the environment.
    Article · Mar 2012
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    [Show abstract] [Hide abstract] ABSTRACT: In sensory circuits of the brain, developmental changes in the expression and modulation of voltage-gated ion channels are a common occurrence, but such changes are often difficult to assign to clear functional roles. We have explored this issue in the binaural neurons of the medial superior olive (MSO), whose temporal precision in detecting the coincidence of binaural inputs dictates the resolution of azimuthal sound localization. We show that in MSO principal neurons of gerbils during the first week of hearing, a hyperpolarization-activated current (I(h)) progressively undergoes a 13-fold increase in maximal conductance, a >10-fold acceleration of kinetics, and, most surprisingly, a 30 mV depolarizing shift in the voltage dependence of activation. This period is associated with an upregulation of the hyperpolarization-activated and cyclic nucleotide-gated (HCN) channel subunits HCN1, HCN2, and HCN4 in the MSO, but only HCN1 and HCN4 were expressed strongly in principal neurons. I(h) recorded in nucleated patches from electrophysiologically mature MSO neurons (>P18) exhibited kinetics and an activation range nearly identical to the I(h) found in whole-cell recordings before hearing onset. These results indicate that the developmental changes in I(h) in MSO neurons can be explained predominantly by modulation from diffusible intracellular factors, and not changes in channel subunit composition. The exceptionally large modulatory changes in I(h), together with refinements in synaptic properties transform the coding strategy from one of summation and integration to the submillisecond coincidence detection known to be required for transmission of sound localization cues.
    Full-text Article · Feb 2012 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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    Dataset: Figure S1
    [Show abstract] [Hide abstract] ABSTRACT: Strategy of TRIP8b[1b/2] knock out by NIH KOMP/Regeneron. The Pex5l gene encoding TRIP8b consists of 16 exons. An expression-selection cassette containing a lacZ reporter followed by a neomycin resistance gene driven by the human ubiquitin C gene promoter and flanked by loxP sites was inserted by homologous recombination in place of exons 1b and 2 after the 1b ATG start codon to generate the allele Pex5ltm1(KOMP)Vlcg. (TIF)
    Full-text Dataset · Feb 2012
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    [Show abstract] [Hide abstract] ABSTRACT: The functions of HCN channels in neurons depend critically on their subcellular localization, requiring fine-tuned machinery that regulates subcellular channel trafficking. Here we provide evidence that regulatory mechanisms governing axonal HCN channel trafficking involve association of the channels with specific isoforms of the auxiliary subunit TRIP8b. In the medial perforant path, which normally contains HCN1 channels in axon terminals in immature but not in adult rodents, we found axonal HCN1 significantly increased in adult mice lacking TRIP8b (TRIP8b(-/-)). Interestingly, adult mice harboring a mutation that results in expression of only the two most abundant TRIP8b isoforms (TRIP8b[1b/2](-/-)) exhibited an HCN1 expression pattern similar to wildtype mice, suggesting that presence of one or both of these isoforms (TRIP8b(1a), TRIP8b(1a-4)) prevents HCN1 from being transported to medial perforant path axons in adult mice. Concordantly, expression analyses demonstrated a strong increase of expression of both TRIP8b isoforms in rat entorhinal cortex with age. However, when overexpressed in cultured entorhinal neurons of rats, TRIP8b(1a), but not TRIP8b(1a-4), altered substantially the subcellular distribution of HCN1 by promoting somatodendritic and reducing axonal expression of the channels. Taken together, we conclude that TRIP8b isoforms are important regulators of HCN1 trafficking in entorhinal neurons and that the alternatively-spliced isoform TRIP8b(1a) could be responsible for the age-dependent redistribution of HCN channels out of perforant path axon terminals.
    Full-text Article · Feb 2012 · PLoS ONE
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    Full-text Article · Jan 2012 · Biophysical Journal
  • Marcelin B · Liu Z · Chen Y · [...] · Bernard C
    Article · Jan 2012
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    [Show abstract] [Hide abstract] ABSTRACT: Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels represent the molecular substrate of the hyperpolarization-activated inward current (I(h)). Although these channels act as pacemakers for the generation of rhythmic activity in the thalamocortical network during sleep and epilepsy, their developmental profile in the thalamus is not yet fully understood. Here we combined electrophysiological, immunohistochemical, and mathematical modeling techniques to examine HCN gene expression and I(h) properties in thalamocortical relay (TC) neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in an epileptic (WAG/Rij) compared to a non-epileptic (ACI) rat strain. Recordings of TC neurons between postnatal day (P) 7 and P90 in both rat strains revealed that I(h) was characterized by higher current density, more hyperpolarized voltage dependence, faster activation kinetics, and reduced cAMP-sensitivity in epileptic animals. All four HCN channel isoforms (HCN1-4) were detected in dLGN, and quantitative analyses revealed a developmental increase of protein expression of HCN1, HCN2, and HCN4 but a decrease of HCN3. HCN1 was expressed at higher levels in WAG/Rij rats, a finding that was correlated with increased expression of the interacting proteins filamin A (FilA) and tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Analysis of a simplified computer model of the thalamic network revealed that the alterations of I(h) found in WAG/Rij rats compensate each other in a way that leaves I(h) availability constant, an effect that ensures unaltered cellular burst activity and thalamic oscillations. These data indicate that during postnatal developmental the hyperpolarizing shift in voltage dependency (resulting in less current availability) is compensated by an increase in current density in WAG/Rij thereby possibly limiting the impact of I(h) on epileptogenesis. Because HCN3 is expressed higher in young versus older animals, HCN3 likely does not contribute to alterations in I(h) in older animals.
    Full-text Article · Sep 2011 · Neurobiology of Disease