Trafficking and surface expression of hyperpolarization-activated cyclic nucleotide-gated channels in hippocampal neurons

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated current I(h) and thus play important roles in the regulation of brain excitability. The subcellular distribution pattern of the HCN channels influences the effects that they exert on the properties and activity of neurons. However, little is known about the mechanisms that control HCN channel trafficking to subcellular compartments or that regulate their surface expression. Here we studied the dynamics of HCN channel trafficking in hippocampal neurons using dissociated cultures coupled with time lapse imaging of fluorophore-fused HCN channels. HCN1-green fluorescence protein (HCN1-GFP) channels resided in vesicle-like organelles that moved in distinct patterns along neuronal dendrites, and these properties were isoform-specific. HCN1 trafficking required intact actin and tubulin and was rapidly inhibited by activation of either NMDA or AMPA-type ionotropic glutamate receptors in a calcium-dependent manner. Glutamate-induced inhibition of the movement of HCN1-GFP-expressing puncta was associated with increased surface expression of both native and transfected HCN1 channels, and this surface expression was accompanied by augmented I(h). Taken together, the results reveal the highly dynamic nature of HCN1 channel trafficking in hippocampal neurons and provide a novel potential mechanism for rapid regulation of I(h), and hence of neuronal properties, via alterations of HCN1 trafficking and surface expression

Trafficking and gating of hyperpolarization-activated cyclic nucleotide-gated channels are regulated by interaction with tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) and cyclic AMP at distinct sites

Ion channel trafficking and gating are often influenced by interactions with auxiliary subunits. Tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) is an auxiliary subunit for neuronal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. TRIP8b interacts directly with two distinct sites of HCN channel pore-forming subunits to control channel trafficking and gating. Here we use mutagenesis combined with electrophysiological studies to define and distinguish the functional importance of the HCN/TRIP8b interaction sites. 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. 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. In accord, increasing cAMP levels in cells antagonized the up-regulation of HCN1 channels mediated by a TRIP8b construct binding the CNBD exclusively. These data illustrate the distinct roles of the two TRIP8b-HCN interaction domains and suggest that TRIP8b and cAMP may directly compete for binding the HCN CNBD to control HCN channel gating, kinetics, and trafficking

Dysregulation of hyperpolarization-activated inward cation current (Ih) affects thalamocortical oscillations: The role of the auxiliary subunit TRIP8b on HCN channel function in thalamic and cortical neurons

19th biennial IPEG Meeting: Nijmegen, The Netherlands. 26-30 October 2016. Background: The family of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels consisiting four different isoforms (HCN1-4) have a major role in controlling neuronal excitability and generation of rhythmic oscillatory activity in individual neurons and neuronal networks. These channels activate in response to hyperpolarizing potentials negative to -50 to -60 mV and depolarize the resting membrane potential. HCN channels are regulated by small molecules like cyclic nucleotides and different accessory proteins. TRIP8b is a brain-specific accessory subunit of HCN channels which controls the gating, surface expression and trafficking of different HCN channels subunits in many regions of brain. The role of this protein for Ih characteristics in thalamic and cortical neurons and the functional consequences of TRIP8b dysregulation for thalamocortical oscillations however is not yet fully understood. The present study aimed at providing a better understanding of the functional role of TRIP8b in the thalamocortical system and shedding some light on possible dysfunctional aspects by combining in vitro and in vivo electrophysiological approaches. In this study, Ih was measured in whole cell patch clamp recordings from thalamocortical (TC) neurons of different thalamic nuclei, as well as pyramidal neurons in layer V and VI of the somatosensory cortex of TRIP8b-deficient (TRIP8b-/-) and control (C57Bl/6 J) mice (p15 - p90). Effects of TRIP8b-dependent dysregulation of Ih on thalamocortical oscillations was monitored by local field potential (LFP) recordings from the ventral-posterior medial complex of the thalamus (VPM) and somatosensory cortex (p 90 - p120), regions which are known to be involved in generation of normal and also pathological thalamocortical oscillations. Results: Characterization of Ih in the thalamocortical system in the absence of the auxiliary subunit TRIP8b showed a significant decrease in Ih density and changes in intrinsic properties and firing patterns of TC and cortical pyramidal neurons. These changes were accompanied by an increase in cAMP sensitivity in TC neurons. Dysregulation of Ih in the thalamocortical system of TRIP8b-/- mice was associated with altered thalamocortical oscillations revealing a significant increase in slow oscillations in the delta frequency range (0.5-4 Hz) during episodes of active-wakefulness. Conclusion: The results of our study point to the importance of TRIP8b, as a brain-specific auxiliary subunit of HCN channels, in regulation of cell and network oscillations. It was demonstrated here that the presence of TRIP8b is necessary for modulation of thalamocortical delta oscillations due to its direct effect on HCN channels protein expression in the thalamocortical system

Alternatively spliced isoforms of TRIP8b differentially control h channel trafficking and function

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (h channels) are the molecular basis for the current, I-h, which contributes crucially to intrinsic neuronal excitability. The subcellular localization and biophysical properties of h channels govern their function, but the mechanisms controlling these characteristics, and especially the potential role of auxiliary subunits or other binding proteins, remain unclear. We focused on TRIP8b, an h channel-interacting protein that colocalizes with HCN1 in cortical and hippocampal pyramidal neuron dendrites, and found that it exists in multiple alternative splice variants with distinct effects on h channel trafficking and function. The developmentally regulated splice variants of TRIP8b all shared dual, C terminus-located interaction sites with HCN1. When coexpressed with HCN1 in heterologous cells individual TRIP8b isoforms similarly modulated gating of I-h, causing a hyperpolarizing shift in voltage dependence of channel activation, but differentially upregulated or downregulated I-h current density and HCN1 surface expression. In hippocampal neurons, coexpression of TRIP8b isoforms with HCN1 produced isoform-specific changes of HCN1 localization. Interestingly, the TRIP8b isoforms most abundant in the brain are those predicted to enhance h channel surface expression. Indeed, shRNA knockdown of TRIP8b in hippocampal neurons significantly reduced native I-h. Thus, although TRIP8b exists in multiple splice isoforms, our data suggest that the predominant role of this protein in brain is to promote h channel surface expression and enhance I-h. Because I-h expression is altered in models of several diseases, including temporal lobe epilepsy, TRIP8b may play a role in both normal neuronal function and in aberrant neuronal excitability associated with neurological disease

Modulation of thalamocortical oscillations by TRIP8b, an auxiliary subunit for HCN channels

Contains fulltext : 189740.pdf (publisher's version ) (Open Access)Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels have important functions in controlling neuronal excitability and generating rhythmic oscillatory activity. The role of tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) in regulation of hyperpolarization-activated inward current, I h, in the thalamocortical system and its functional relevance for the physiological thalamocortical oscillations were investigated. A significant decrease in I h current density, in both thalamocortical relay (TC) and cortical pyramidal neurons was found in TRIP8b-deficient mice (TRIP8b-/-). In addition basal cAMP levels in the brain were found to be decreased while the availability of the fast transient A-type K+ current, I A, in TC neurons was increased. These changes were associated with alterations in intrinsic properties and firing patterns of TC neurons, as well as intrathalamic and thalamocortical network oscillations, revealing a significant increase in slow oscillations in the delta frequency range (0.5-4 Hz) during episodes of active-wakefulness. In addition, absence of TRIP8b suppresses the normal desynchronization response of the EEG during the switch from slow-wave sleep to wakefulness. It is concluded that TRIP8b is necessary for the modulation of physiological thalamocortical oscillations due to its direct effect on HCN channel expression in thalamus and cortex and that mechanisms related to reduced cAMP signaling may contribute to the present findings.28 p