Regulation of HCN channel surface expression by a novel C-terminal protein-protein interaction.
Hyperpolarization-activated cation currents (I(h)) are carried by channels encoded by a family of four genes (HCN1-4) that are differentially expressed within the brain in specific cellular and subcellular compartments. HCN1 shows a high level of expression in apical dendrites of cortical pyramidal neurons and in presynaptic terminals of cerebellar basket cells, structures with a high density of I(h). Expression of I(h) is also regulated by neuronal activity. To isolate proteins that may control HCN channel expression or function, we performed yeast two-hybrid screens using the C-terminal cytoplasmic tails of the HCN proteins as bait. We identified a brain-specific protein, which has been previously termed TRIP8b (for TPR-containing Rab8b interacting protein) and PEX5Rp (for Pex5p-related protein), that specifically interacts with all four HCN channels through a conserved sequence in their C-terminal tails. In situ hybridization and immunohistochemistry show that TRIP8b and HCN1 are colocalized, particularly within dendritic arbors of hippocampal CA1 and neocortical layer V pyramidal neurons. The dendritic expression of TRIP8b in layer V pyramidal neurons is disrupted after deletion of HCN1 through homologous recombination, demonstrating a key in vivo interaction between HCN1 and TRIP8b. TRIP8b dramatically alters the trafficking of HCN channels heterologously expressed in Xenopus oocytes and human embryonic kidney 293 cells, causing a specific decrease in surface expression of HCN protein and I(h) density, with a pronounced intracellular accumulation of HCN protein that is colocalized in discrete cytoplasmic clusters with TRIP8b. Finally, TRIP8b expression in cultured pyramidal neurons markedly decreases native I(h) density. These data suggest a possible role for TRIP8b in regulating HCN channel density in the plasma membrane.
Santoro
Bina
B
Center for Neurobiology and Behavior, Columbia University, New York, New York 10032, USA. bs73@columbia.edu
Wainger
Brian J
BJ
Siegelbaum
Steven A
SA
D000200:Action Potentials / Q000502:physiology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D017124:Conserved Sequence; D006224:Cricetinae; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007473:Ion Channels / Q000096:biosynthesis* / Q000737:chemistry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D000074401:Peroxins; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms; D021381:Protein Transport / Q000502:physiology; D017966:Pyramidal Cells / Q000378:metabolism / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012845:Sindbis Virus; D014982:Xenopus laevis
D007473:Ion Channels; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D000074401:Peroxins; C501732:Pex5l protein, mouse; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins