Trafficking and Surface Expression of Hyperpolarization-activated Cyclic Nucleotide-gated Channels in Hippocampal Neurons

Tallie Z. Baram,Rui-Lin Wu,Wytse J. Wadman,Qinqin Zha,Yoav Noam,Dane M. Chetkovich,Lise Phan

doi:10.1074/jbc.m109.070391

Abstract

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.

Highlights

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated current Ih and play important roles in the regulation of brain excitability
The results reveal the highly dynamic nature of HCN1 channel trafficking in hippocampal neurons and provide a novel potential mechanism for rapid regulation of Ih, and of neuronal properties, via alterations of HCN1 trafficking and surface expression
The subcellular distribution of HCN channels varies in different cell types and brain regions and is important for determining the effects that these channels exert on neuronal excitability [5]

Summary

Hippocampal Cell Culture

Dissociated hippocampal primary cultures were prepared from postnatal day 0 (P0) Sprague-Dawley rats. Cultures were subsequently refreshed every 3– 4 days with the conditioned medium. On the third day in vitro (DIV), 1 ␮M cytosine-arabinoside (Sigma) was added to the culture medium to inhibit glial proliferation. Plasmid DNA Constructs cDNA constructs containing the GFP sequence in the C terminus of the mouse HCN1 cDNA (inserted between amino acid residues 885 and 886) or the hemagglutinin (HA) tag at the HCN1 extracellular domain between transmembrane domains S3 and S4 (residues 231–232) were generated as described previously [12]. MCherry-HCN1 was created by inserting the cDNA encoding mCherry from pRSET-BmCherry (generously provided by Dr Roger Tsien, University of California, San Diego) instead of the GFP in the AgeI/XhoI site of the HCN1-GFPn construct The N terminus GFP-conjugated mHCN1 (HCN1-GFPn) and mHCN2 (HCN2-GFP) constructs were a gift from Dr Santoro (Columbia University); previous studies found that the two constructs yielded functional Ih with biophysical properties typical for the respective isoform expressed in heterologous systems [13]. mCherry-HCN1 was created by inserting the cDNA encoding mCherry from pRSET-BmCherry (generously provided by Dr Roger Tsien, University of California, San Diego) instead of the GFP in the AgeI/XhoI site of the HCN1-GFPn construct

Neuronal Transfection of Plasmid DNA

Time Lapse Live Imaging and Analysis

Hippocampal Organotypic Slice Cultures and Biotinylation Assay

RESULTS

Increased Glutamatergic Input

DISCUSSION