Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels
10.1073/pnas.1201997109
Crossref journal-article
Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences (341)
Abstract

Ion channels operate in intact tissues as part of large macromolecular complexes that can include cytoskeletal proteins, scaffolding proteins, signaling molecules, and a litany of other molecules. The proteins that make up these complexes can influence the trafficking, localization, and biophysical properties of the channel. TRIP8b (tetratricopetide repeat-containing Rab8b-interacting protein) is a recently discovered accessory subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that contributes to the substantial dendritic localization of HCN channels in many types of neurons. TRIP8b interacts with the carboxyl-terminal region of HCN channels and regulates their cell-surface expression level and cyclic nucleotide dependence. Here we examine the molecular determinants of TRIP8b binding to HCN2 channels. Using a single-molecule fluorescence bleaching method, we found that TRIP8b and HCN2 form an obligate 4:4 complex in intact channels. Fluorescence-detection size-exclusion chromatography and fluorescence anisotropy allowed us to confirm that two different domains in the carboxyl-terminal portion of TRIP8b—the tetratricopepide repeat region and the TRIP8b conserved region—interact with two different regions of the HCN carboxyl-terminal region: the carboxyl-terminal three amino acids (SNL) and the cyclic nucleotide-binding domain, respectively. And finally, using X-ray crystallography, we determined the atomic structure of the tetratricopepide region of TRIP8b in complex with a peptide of the carboxy-terminus of HCN2. Together, these experiments begin to uncover the mechanism for TRIP8b binding and regulation of HCN channels.

Bibliography

Bankston, J. R., Camp, S. S., DiMaio, F., Lewis, A. S., Chetkovich, D. M., & Zagotta, W. N. (2012). Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels. Proceedings of the National Academy of Sciences, 109(20), 7899–7904.

Authors 6
  1. John R. Bankston (first)
  2. Stacey S. Camp (additional)
  3. Frank DiMaio (additional)
  4. Alan S. Lewis (additional)
  5. Dane M. Chetkovich (additional)
  6. William N. Zagotta (additional)
References 34 Referenced 45
  1. Z Buraei, J Yang, The ß subunit of voltage-gated Ca2+ channels. Physiol Rev 90, 1461–1506 (2010). (10.1152/physrev.00057.2009) / Physiol Rev / The ß subunit of voltage-gated Ca2+ channels by Buraei Z (2010)
  2. O Pongs, JR Schwarz, Ancillary subunits associated with voltage-dependent K+ channels. Physiol Rev 90, 755–796 (2010). (10.1152/physrev.00020.2009) / Physiol Rev / Ancillary subunits associated with voltage-dependent K+ channels by Pongs O (2010)
  3. MC Trudeau, WN Zagotta, Calcium/calmodulin modulation of olfactory and rod cyclic nucleotide-gated ion channels. J Biol Chem 278, 18705–18708 (2003). (10.1074/jbc.R300001200) / J Biol Chem / Calcium/calmodulin modulation of olfactory and rod cyclic nucleotide-gated ion channels by Trudeau MC (2003)
  4. KB Craven, WN Zagotta, CNG and HCN channels: Two peas, one pod. Annu Rev Physiol 68, 375–401 (2006). (10.1146/annurev.physiol.68.040104.134728) / Annu Rev Physiol / CNG and HCN channels: Two peas, one pod by Craven KB (2006)
  5. RB Robinson, SA Siegelbaum, Hyperpolarization-activated cation currents: From molecules to physiological function. Annu Rev Physiol 65, 453–480 (2003). (10.1146/annurev.physiol.65.092101.142734) / Annu Rev Physiol / Hyperpolarization-activated cation currents: From molecules to physiological function by Robinson RB (2003)
  6. BJ Wainger, M DeGennaro, B Santoro, SA Siegelbaum, GR Tibbs, Molecular mechanism of cAMP modulation of HCN pacemaker channels. Nature 411, 805–810 (2001). (10.1038/35081088) / Nature / Molecular mechanism of cAMP modulation of HCN pacemaker channels by Wainger BJ (2001)
  7. A Lörincz, T Notomi, G Tamás, R Shigemoto, Z Nusser, Polarized and compartment-dependent distribution of HCN1 in pyramidal cell dendrites. Nat Neurosci 5, 1185–1193 (2002). (10.1038/nn962) / Nat Neurosci / Polarized and compartment-dependent distribution of HCN1 in pyramidal cell dendrites by Lörincz A (2002)
  8. JC Magee, Dendritic lh normalizes temporal summation in hippocampal CA1 neurons. Nat Neurosci 2, 508–514 (1999). (10.1038/9158) / Nat Neurosci / Dendritic lh normalizes temporal summation in hippocampal CA1 neurons by Magee JC (1999)
  9. SR Williams, GJ Stuart, Site independence of EPSP time course is mediated by dendritic I(h) in neocortical pyramidal neurons. J Neurophysiol 83, 3177–3182 (2000). (10.1152/jn.2000.83.5.3177) / J Neurophysiol / Site independence of EPSP time course is mediated by dendritic I(h) in neocortical pyramidal neurons by Williams SR (2000)
  10. MF Nolan, et al., A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons. Cell 119, 719–732 (2004). / Cell / A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons by Nolan MF (2004)
  11. B Santoro, BJ Wainger, SA Siegelbaum, Regulation of HCN channel surface expression by a novel C-terminal protein-protein interaction. J Neurosci 24, 10750–10762 (2004). (10.1523/JNEUROSCI.3300-04.2004) / J Neurosci / Regulation of HCN channel surface expression by a novel C-terminal protein-protein interaction by Santoro B (2004)
  12. R Piskorowski, B Santoro, SA Siegelbaum, TRIP8b splice forms act in concert to regulate the localization and expression of HCN1 channels in CA1 pyramidal neurons. Neuron 70, 495–509 (2011). (10.1016/j.neuron.2011.03.023) / Neuron / TRIP8b splice forms act in concert to regulate the localization and expression of HCN1 channels in CA1 pyramidal neurons by Piskorowski R (2011)
  13. AS Lewis, et al., 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, 7424–7440 (2011). (10.1523/JNEUROSCI.0936-11.2011) / J Neurosci / Deletion of the hyperpolarization-activated cyclic nucleotide-gated channel auxiliary subunit TRIP8b impairs hippocampal Ih localization and function and promotes antidepressant behavior in mice by Lewis AS (2011)
  14. B Santoro, et al., TRIP8b splice variants form a family of auxiliary subunits that regulate gating and trafficking of HCN channels in the brain. Neuron 62, 802–813 (2009). (10.1016/j.neuron.2009.05.009) / Neuron / TRIP8b splice variants form a family of auxiliary subunits that regulate gating and trafficking of HCN channels in the brain by Santoro B (2009)
  15. AS Lewis, et al., Alternatively spliced isoforms of TRIP8b differentially control h channel trafficking and function. J Neurosci 29, 6250–6265 (2009). (10.1523/JNEUROSCI.0856-09.2009) / J Neurosci / Alternatively spliced isoforms of TRIP8b differentially control h channel trafficking and function by Lewis AS (2009)
  16. B Santoro, et al., TRIP8b regulates HCN1 channel trafficking and gating through two distinct C-terminal interaction sites. J Neurosci 31, 4074–4086 (2011). (10.1523/JNEUROSCI.5707-10.2011) / J Neurosci / TRIP8b regulates HCN1 channel trafficking and gating through two distinct C-terminal interaction sites by Santoro B (2011)
  17. Y Han, et al., 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, 20823–20834 (2011). (10.1074/jbc.M111.236125) / J Biol Chem / 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 by Han Y (2011)
  18. G Zolles, et al., Association with the auxiliary subunit PEX5R/Trip8b controls responsiveness of HCN channels to cAMP and adrenergic stimulation. Neuron 62, 814–825 (2009). (10.1016/j.neuron.2009.05.008) / Neuron / Association with the auxiliary subunit PEX5R/Trip8b controls responsiveness of HCN channels to cAMP and adrenergic stimulation by Zolles G (2009)
  19. MH Ulbrich, EY Isacoff, Subunit counting in membrane-bound proteins. Nat Methods 4, 319–321 (2007). (10.1038/nmeth1024) / Nat Methods / Subunit counting in membrane-bound proteins by Ulbrich MH (2007)
  20. W Ji, et al., Functional stoichiometry of the unitary calcium-release-activated calcium channel. Proc Natl Acad Sci USA 105, 13668–13673 (2008). (10.1073/pnas.0806499105) / Proc Natl Acad Sci USA / Functional stoichiometry of the unitary calcium-release-activated calcium channel by Ji W (2008)
  21. T Kawate, E Gouaux, Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006). (10.1016/j.str.2006.01.013) / Structure / Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins by Kawate T (2006)
  22. AM Rossi, CW Taylor, Analysis of protein-ligand interactions by fluorescence polarization. Nat Protoc 6, 365–387 (2011). (10.1038/nprot.2011.305) / Nat Protoc / Analysis of protein-ligand interactions by fluorescence polarization by Rossi AM (2011)
  23. GJ Gatto, BV Geisbrecht, SJ Gould, JM Berg, Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nat Struct Biol 7, 1091–1095 (2000). (10.1038/81930) / Nat Struct Biol / Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5 by Gatto GJ (2000)
  24. CP Williams, WA Stanley, Peroxin 5: A cycling receptor for protein translocation into peroxisomes. Int J Biochem Cell Biol 42, 1771–1774 (2010). (10.1016/j.biocel.2010.07.004) / Int J Biochem Cell Biol / Peroxin 5: A cycling receptor for protein translocation into peroxisomes by Williams CP (2010)
  25. SJ Gould, GA Keller, N Hosken, J Wilkinson, S Subramani, A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol 108, 1657–1664 (1989). (10.1083/jcb.108.5.1657) / J Cell Biol / A conserved tripeptide sorts proteins to peroxisomes by Gould SJ (1989)
  26. GJ Gatto, et al., Correlating structure and affinity for PEX5:PTS1 complexes. Biochemistry 42, 1660–1666 (2003). (10.1021/bi027034z) / Biochemistry / Correlating structure and affinity for PEX5:PTS1 complexes by Gatto GJ (2003)
  27. ER Liman, J Tytgat, P Hess, Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron 9, 861–871 (1992). (10.1016/0896-6273(92)90239-A) / Neuron / Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs by Liman ER (1992)
  28. WN Zagotta, T Hoshi, RW Aldrich, Gating of single Shaker potassium channels in Drosophila muscle and in Xenopus oocytes injected with Shaker mRNA. Proc Natl Acad Sci USA 86, 7243–7247 (1989). (10.1073/pnas.86.18.7243) / Proc Natl Acad Sci USA / Gating of single Shaker potassium channels in Drosophila muscle and in Xenopus oocytes injected with Shaker mRNA by Zagotta WN (1989)
  29. Collaborative Computational Project Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50: 760–763. (10.1107/S0907444994003112)
  30. AJ McCoy, Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr D Biol Crystallogr 63, 32–41 (2007). (10.1107/S0907444906045975) / Acta Crystallogr D Biol Crystallogr / Solving structures of protein complexes by molecular replacement with Phaser by McCoy AJ (2007)
  31. PD Adams, et al., PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213–221 (2010). (10.1107/S0907444909052925) / Acta Crystallogr D Biol Crystallogr / PHENIX: A comprehensive Python-based system for macromolecular structure solution by Adams PD (2010)
  32. P Emsley, K Cowtan, Coot: Model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126–2132 (2004). (10.1107/S0907444904019158) / Acta Crystallogr D Biol Crystallogr / Coot: Model-building tools for molecular graphics by Emsley P (2004)
  33. NA Baker, D Sept, S Joseph, MJ Holst, JA McCammon, Electrostatics of nanosystems: Application to microtubules and the ribosome. Proc Natl Acad Sci USA 98, 10037–10041 (2001). (10.1073/pnas.181342398) / Proc Natl Acad Sci USA / Electrostatics of nanosystems: Application to microtubules and the ribosome by Baker NA (2001)
  34. F DiMaio, et al., Improved molecular replacement by density- and energy-guided protein structure optimization. Nature 473, 540–543 (2011). (10.1038/nature09964) / Nature / Improved molecular replacement by density- and energy-guided protein structure optimization by DiMaio F (2011)
Dates
Type When
Created 13 years, 3 months ago (May 1, 2012, 10:57 p.m.)
Deposited 3 years, 2 months ago (June 7, 2022, 4:26 a.m.)
Indexed 1 month, 2 weeks ago (July 14, 2025, 11:36 p.m.)
Issued 13 years, 3 months ago (May 1, 2012)
Published 13 years, 3 months ago (May 1, 2012)
Published Online 13 years, 3 months ago (May 1, 2012)
Published Print 13 years, 3 months ago (May 15, 2012)
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