Alternatively Spliced Isoforms of TRIP8b Differentially Control h Channel Trafficking and Function

Abstract

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.

Differential Dorso-ventral Distributions of Kv4.2 and HCN Proteins Confer Distinct Integrative Properties to Hippocampal CA1 Pyramidal Cell Distal Dendrites

“…Western blotting for total HCN1, HCN2, and TRIP8b in dorsal and ventral hippocampus was performed as described previously, with sample loading differences controlled by normalizing to ␣-tubulin (32). Quantitative analysis of the density of immunoreactive bands was performed with the National Institutes of Health ImageJ or Scion Image software.…”

“…The auxiliary protein TRIP8b is a key regulator of HCN channels in physiological and pathological conditions (32,35,36). However, the expression of TRIP8b was similar in the dorsal and ventral hippocampus.…”

Role of cysteine 288 in nucleophosmin cytoplasmic mutations: sensitization to toxicity induced by arsenic trioxide and bortezomib

“…Lentiviral supernatants were prepared using 293T cells co-transfected with packaging plasmids pCMV-VSV-G and pCMVDR8.9 with Lipofectamine 2000 (Invitrogen) as previously described. 34 After an overnight incubation, the medium was replaced with fresh growth medium and at 48 h post transfection, virus-containing supernatants were collected by centrifugation at 500 g for 10 min at 4 1C and filtered through a 0.45-mm filter. K562, HL60 and OCI-AML3 cells were infected by spin inoculation (600 g, 90 min) in the presence of 2 mg/ml polybrene with lentiviral supernatants.…”

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).…”

“…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).…”