Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (h channels) mediate the hyperpolarization-activated current (Ih) and are important for regulating excitability in CA1 pyramidal neurons of the hippocampus. Because localization governs h channel function in the homeostasis of neuronal excitability, and h channel dysfunction has been implicated in temporal lobe epilepsy, our longterm goal is to understand the unknown trafficking and targeting mechanism of h channels in the hippocampus, h channels of CA1 pyramidal neurons as well as neocortical layer V neurons exhibit a striking distal dendritic enrichment (DDE), shown to be critical for control of cellular excitability and synchrony. One molecular candidate implicated in h channel trafficking is the tetratricopeptide repeat-containing (TPR) Rab8b interacting protein (TRIPSb). TRIPSb is the only known interactor of h channels to demonstrate pyramidal neuron DDE in both the hippocampus and cortex mimicking that of HCN1 and 2. Additionally, TRIPSb has been shown to regulate Ih density in oocytes. TRIPSb interacts with h channels via its conserved C-terminal TPR domains in a manner homologous to the peroxisomal import receptor proteins. However, the N-terminal region of TRIPSb shares no homology with any known protein and its function is unknown. Our preliminary data indicate the N-terminus is highly alternatively spliced in a developmentally regulated manner, whereby specific isoforms are upregulated concurrent with DDE onset. Notably, alternative splicing alters the presence of cellular sorting signals thought to be important in trafficking. Because of this considerable but indirect evidence associating TRIPSb with h channel trafficking, we reason that TRIPSb plays an important role in the establishment and/or maintenance of h channel DDE in the hippocampus. To determine TRIPSb's role in hippocampal DDE, we propose the following specific aims: 1) Determine whether TRIPSb in general, and the N-terminus of TRIPSb in particular, are required for h channel DDE in CA1 pyramidal neurons. We will use lentiviral delivery of shRNA and dominant negative TRIPSb constructs in both slice culture and the living animal to determine the effect on h channel DDE in the hippocampus in vivo. 2) Determine the role of TRIPSb N-terminal alternative splicing in h channel trafficking and DDE. We will use single-cell RT-PCR, biotinylation, and viral delivery of TRIPSb splice isoforms to determine how TRIPSb splicing impacts h channel surface trafficking and DDE.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30NS064757-04
Application #
8204713
Study Section
Special Emphasis Panel (ZNS1-SRB-M (64))
Program Officer
Stewart, Randall R
Project Start
2009-01-05
Project End
2012-05-31
Budget Start
2012-01-05
Budget End
2012-05-31
Support Year
4
Fiscal Year
2012
Total Cost
$11,035
Indirect Cost
Name
Northwestern University at Chicago
Department
Pathology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Marcelin, Beatrice; Liu, Zhiqiang; Chen, Yuncai et al. (2012) Dorsoventral differences in intrinsic properties in developing CA1 pyramidal cells. J Neurosci 32:3736-47
Wilkars, Wiebke; Liu, Zhiqiang; Lewis, Alan S et al. (2012) Regulation of axonal HCN1 trafficking in perforant path involves expression of specific TRIP8b isoforms. PLoS One 7:e32181
Bankston, John R; Camp, Stacey S; DiMaio, Frank et al. (2012) Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels. Proc Natl Acad Sci U S A 109:7899-904
Chan, C Savio; Glajch, Kelly E; Gertler, Tracy S et al. (2011) HCN channelopathy in external globus pallidus neurons in models of Parkinson's disease. Nat Neurosci 14:85-92
Lewis, Alan S; Vaidya, Sachin P; Blaiss, Cory A et al. (2011) Deletion of the hyperpolarization-activated cyclic nucleotide-gated channel auxiliary subunit TRIP8b impairs hippocampal Ih localization and function and promotes antidepressant behavior in mice. J Neurosci 31:7424-40
Lewis, Alan S; Chetkovich, Dane M (2011) HCN channels in behavior and neurological disease: too hyper or not active enough? Mol Cell Neurosci 46:357-67
Han, Ye; Noam, Yoav; Lewis, Alan S et al. (2011) 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. J Biol Chem 286:20823-34
Lewis, Alan S; Schwartz, Emily; Chan, C Savio et al. (2009) Alternatively spliced isoforms of TRIP8b differentially control h channel trafficking and function. J Neurosci 29:6250-65