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Differences in the expression pattern of HCN isoforms among mammalian tissues: sources and implications

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Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a critical role in a broad range of cell types, but the expression of the various HCN isoforms is still poorly understood. In the present study we have compared the expression of HCN isoforms in rat excitable and non-excitable tissues at both the mRNA and protein levels. Real-time PCR and Western blot analysis revealed distinct expression patterns of the four HCN isoforms in brain, heart, pituitary and kidney, with inconsistent mRNA-protein expression correlation. The HCN2 was the most abundant mRNA transcript (95.6, 78.0 and 59.0 % in kidney heart and pituitary, respectively) except in the brain (42.0 %) whereas HCN4 was the most abundant protein isoform. Our results suggest that HCN channels are mostly produced by the HCN4 isoform in heart, which contrasts with the sharp differences in the isoform stoichiometry in pituitary (15 HCN4:2 HCN2:1 HCN1:1 HCN3), kidney (24 HCN4:2 HCN3:1 HCN2:1 HCN1) and brain (3 HCN4:2 HCN2:1 HCN1:1 HCN3). Moreover, deviations of the electrophoretic molecular weight (MW) of the HCN isoforms relative to the theoretical MW were observed, suggesting that N-glycosylation and enzymatic proteolysis influences HCN channel surface expression. We hypothesize that selective cleavage of HCN channels by membrane bound metalloendopeptidases could account for the multiplicity of properties of native HCN channels in different tissues.

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References

  1. Postea O, Biel M (2011) Exploring HCN channels as novel drug targets. Nat Rev Drug Discov 10:903–914. doi:10.1038/nrd3576

    CAS  PubMed  Google Scholar 

  2. Biel M, Wahl-Schott C, Michalakis S, Zong X (2009) Hyperpolarization-activated cation channels: from genes to function. Physiol Rev 89:847–885. doi:10.1152/physrev.0.0029.2008

    Article  CAS  PubMed  Google Scholar 

  3. Brown HF, DiFrancesco D, Noble SJ (1979) How does adrenaline accelerate the heart? Nature 280:235–236

    Article  CAS  PubMed  Google Scholar 

  4. Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER, Siegelbaum SA, Tibbs GR (1998) Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 93:717–729. doi:10.1016/S0092-8674(00)81434-8

    Article  CAS  PubMed  Google Scholar 

  5. Monteggia LM, Eisch AJ, Tang MD, Kaczmarek LK, Nestler EJ (2000) Cloning and localization of the hyperpolarization-activated cyclic nucleotide-gated channel family in rat brain. Brain Res Mol Brain Res 81:129–139. doi:10.1016/S0169-328X(00),00155-8

    Article  CAS  PubMed  Google Scholar 

  6. Kaupp UB, Seifert R (2001) Molecular diversity of pacemaker ion channels. Annu Rev Physiol 63:235–257. doi:10.1146/annurev.physiol.63.1.235

    Article  CAS  PubMed  Google Scholar 

  7. Zhong N, Beaumont V, Zucker RS (2004) Calcium influx through HCN channels does not contribute to cAMP-enhanced transmission. J Neurophysiol 92:644–647. doi:10.1152/jn.0.0112.2004

    Article  CAS  PubMed  Google Scholar 

  8. Herrmann S, Stieber J, Ludwig A (2007) Pathophysiology of HCN channels. Pflugers Arch 454:517–522. doi:10.1007/s00424-007-0224-4

    Article  CAS  PubMed  Google Scholar 

  9. Robinson RB, Siegelbaum SA (2003) Hyperpolarization-activated cation currents: from molecules to physiological function. Annu Rev Physiol 65:453–480. doi:10.1146/annurev.physiol.65.092101.142734

    Article  CAS  PubMed  Google Scholar 

  10. Accili EA, Proenza C, Baruscotti M, DiFrancesco D (2002) From funny current to HCN channels: 20 years of excitation. News Physiol Sci 17:32–37

    CAS  PubMed  Google Scholar 

  11. Hegle AP, Nazzari H, Roth A, Angoli D, Accili EA (2010) Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression. Am J Physiol Cell Physiol 298:C1066–C1076. doi:10.1152/ajpcell.00389.2009

    Article  CAS  PubMed  Google Scholar 

  12. Much B, Wahl-Schott C, Zong X, Schneider A, Baumann L, Moosmang S, Ludwig A, Biel M (2003) Role of subunit heteromerization and N-linked glycosylation in the formation of functional hyperpolarization-activated cyclic nucleotide-gated channels. J Biol Chem 278:43781–43786. doi:10.1074/jbc.M306958200

    Article  CAS  PubMed  Google Scholar 

  13. Lewis AS, Estep CM, Chetkovich DM (2010) The fast and slow ups and downs of HCN channel regulation. Channels 4:215–231. doi:10.4161/chan.4.3.11630

    Article  CAS  PubMed  Google Scholar 

  14. Bolívar JJ, Tapia D, Arenas G, Castañón-Arreola M, Torres H, Galarraga E (2008) A hyperpolarization-activated, cyclic nucleotide-gated, (Ih-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells. Am J Physiol Cell Physiol 294:C893–C906. doi:10.1152/ajpcell.0.0616.2006

    Article  PubMed  Google Scholar 

  15. Hurtado R, Bub G, Herzlinger D (2010) The pelvis–kidney junction contains HCN3, a hyperpolarization-activated cation channel that triggers ureter peristalsis. Kidney Int 77:500–508. doi:10.1038/ki.2009

    Article  CAS  PubMed  Google Scholar 

  16. Varghese A, Tenbroek EM, Coles J Jr, Sigg DC (2006) Endogenous channels in HEK cells and potential roles in HCN ionic current measurements. Prog Biophys Mol Biol 90:26–37. doi:10.1016/j.pbiomolbio.2005.05.002

    Article  CAS  PubMed  Google Scholar 

  17. El-Kholy W, MacDonald PE, Fox JM, Bhattacharjee A, Xue T, Gao X, Zhang Y, Stieber J, Li RA, Tsushima RG, Wheeler MB (2007) Hyperpolarization-activated cyclic nucleotide-gated channels in pancreatic beta-cells. Mol Endocrinol 21:753–764. doi:10.1210/me.2006-0258

    Article  CAS  PubMed  Google Scholar 

  18. Kretschmannova K, Kucka M, Gonzalez-Iglesias AE, Stojilkovic SS (2012) The expression and role of hyperpolarization-activated and cyclic nucleotide-gated channels in endocrine anterior pituitary cells. Mol Endocrinol 26:153–164. doi:10.1210/me.2011-1207

    Article  CAS  PubMed  Google Scholar 

  19. He P, Deng J, Zhong X, Zhou Z, Song B, Li L (2012) Identification of a hyperpolarization-activated cyclic nucleotide-gated channel and its subtypes in the urinary bladder of the rat. Urology 79:1411e7–1411e13. doi:10.1016/j.urology.2012.01.037

    Article  Google Scholar 

  20. Xue L, Li Y, Han X, Yao L, Yuan J, Qin W, Liu F, Wang H (2012) Investigation of hyperpolarization-activated cyclic nucleotide-gated channels in interstitial cells of Cajal of human bladder. Urology 224:e13–e18. doi:10.1016/j.urology.2012.04.005

    Google Scholar 

  21. Aponte Y, Lien CC, Reisinger E, Jonas P (2006) Hyperpolarization-activated cation channels in fast-spiking interneurons of rat hippocampus. J Physiol 574:229–243. doi:10.1113/jphysiol.2005.104042

    Article  CAS  PubMed  Google Scholar 

  22. Cho HJ, Furness JB, Jennings EA (2011) Postnatal maturation of the hyperpolarization-activated cation current, I(h), in trigeminal sensory neurons. J Neurophysiol 106:2045–2056. doi:10.1152/jn.00798.2010

    Article  CAS  PubMed  Google Scholar 

  23. Greener ID, Monfredi O, Inada S, Chandler NJ, Tellez JO, Atkinson A, Taube MA, Billeter R, Anderson RH, Efimov IR, Molenaar P, Sigg DC, Sharma V, Boyett MR, Dobrzynski H (2011) Molecular architecture of the human specialised atrioventricular conduction axis. J Mol Cell Cardiol 50:642–651. doi:10.1016/j.yjmcc.2010.12.017

    Article  CAS  PubMed  Google Scholar 

  24. Horwitz GC, Lelli A, Géléoc GS, Holt JR (2010) HCN channels are not required for mechanotransduction in sensory hair cells of the mouse inner ear. PLoS One 5:e8627. doi:10.1371/journal.pone.0008627

    Article  PubMed Central  PubMed  Google Scholar 

  25. Marionneau C, Couette B, Liu J, Li H, Mangoni ME, Nargeot J, Lei M, Escande D, Demolombe S (2005) Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J Physiol 562:223–234. doi:10.1113/jphysiol.2004.074047

    Article  CAS  PubMed  Google Scholar 

  26. Poller WC, Bernard R, Derst C, Weiss T, Madai VI, Veh RW (2011) Lateral habenular neurons projecting to reward-processing monoaminergic nuclei express hyperpolarization-activated cyclic nucleotide-gated cation channels. Neuroscience 19:205–216. doi:10.1016/j.neuroscience.2011.07.013

    Article  Google Scholar 

  27. Ye B, Nerbonne JM (2009) Proteolytic processing of HCN2 and co-assembly with HCN4 in the generation of cardiac pacemaker channels. J Biol Chem 284:25553–25559. doi:10.1074/jbc.M109.007583

    Article  CAS  PubMed  Google Scholar 

  28. Altomare C, Terragnim B, Brioschi C, Milanesi R, Pagliuca C, Viscomi C, Moroni A, Baruscotti M, DiFrancesco D (2003) Heteromeric HCN1–HCN4 channels: a comparison with native pacemaker channels from the rabbit sinoatrial node. J Physiol 549:347–359. doi:10.1113/jphysiol.2002.027698

    Article  CAS  PubMed  Google Scholar 

  29. Ulens C, Tytgat J (2001) Functional heteromerization of HCN1 and HCN2 pacemaker channels. J Biol Chem 276:6069–6072. doi:10.1074/jbc.C000738200

    Article  CAS  PubMed  Google Scholar 

  30. Michels G, Er F, Khan I, Südkamp M, Herzig S, Hoppe UC (2005) Single-channel properties support a potential contribution of hyperpolarization-activated cyclic nucleotide-gated channels and if to cardiac arrhythmias. Circulation 111:399–404. doi:10.1161/01.CIR.0000153799.65783.3A

    Article  CAS  PubMed  Google Scholar 

  31. Whitaker GM, Angoli D, Nazzari H, Shigemoto R, Accili EA (2007) HCN2 and HCN4 isoforms self-assemble and co-assemble with equal preference to form functional pacemaker channels. J Biol Chem 282:22900–22909. doi:10.1074/jbc.M610978200

    Article  CAS  PubMed  Google Scholar 

  32. Proenza C, Tran N, Angoli D, Zahynacz K, Balcar P, Accili EA (2002) Different roles for the cyclic nucleotide binding domain and amino terminus in assembly and expression of hyperpolarization-activated, cyclic nucleotide-gated channels. J Biol Chem 277:29634–29642. doi:10.1074/jbc.M200504200

    Article  CAS  PubMed  Google Scholar 

  33. Turner AJ, Tanzawa K (1997) Mammalian membrane metallopeptidases: NEP, ECE, KELL, and PEX. FASEB J 11:355–364

    CAS  PubMed  Google Scholar 

  34. Korovkina VP, Stamnes SJ, Brainard AM, England SK (2009) Nardilysin convertase regulates the function of the maxi-K channel isoform mK44 in human myometrium. Am J Physiol Cell Physiol 296:C433–C440. doi:10.1152/ajpcell.00357.2008

    Article  CAS  PubMed  Google Scholar 

  35. Horton P, Nakai K (1997) Better prediction of protein cellular localization sites with the k nearest neighbors classifier. Proc Int Conf Intell Syst Mol Biol 5:147–152

    CAS  PubMed  Google Scholar 

  36. Schechter I, Berger A (1968) On the active site of proteases 3. Mapping the active site of papain; specific peptide inhibitors of papain. Biochem Biophys Res Commun 32:898–902

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Portuguese Foundation of Science and Technology of the Portuguese Ministry of Sciences, Technology, and High Education (Bilateral Agreement between Portugal and Slovenia—Proc.º 441.00 ESLOVENIA,SFRH/BPD/14677/2003, SFRH/BPD/26611/2006, SFRH/BD/41217/2007 and SFRH/BD/47868/2008) and by the Ministry of Higher Education, Sciences and Technology of the Republic of Slovenia (BI-PT-10-11).

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Authors and Affiliations

  1. Departamento de Biologia & CESAM, Universidade de Aveiro, 3810-193, Aveiro, Portugal

    Ana I. Calejo, Marisa Reverendo, Virgília S. Silva, Patrícia M. Pereira, Manuel A. S. Santos & Paula P. Gonçalves

  2. Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia

    Robert Zorec

  3. Celica Biomedical Center, Tehnološki Park 24, 1000, Ljubljana, Slovenia

    Robert Zorec

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  1. Ana I. Calejo
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Correspondence to Paula P. Gonçalves.

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Calejo, A.I., Reverendo, M., Silva, V.S. et al. Differences in the expression pattern of HCN isoforms among mammalian tissues: sources and implications. Mol Biol Rep 41, 297–307 (2014). https://doi.org/10.1007/s11033-013-2862-2

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