Structural study on the N-terminus of the vanilloid receptor TRPV1
A.Samad1, E. Jindrova1, V. Vlachova2, J. Teisinger2, R. Ettrich1
1Laboratory of High Performance Computing, Institute of Systems Biology and Ecology ASCR and Institute of Physical Biology USB, Zámek 136, CZ-373 33 Nové Hrady, Czech Republic.
2Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeòská 1083, CZ-14220 Praha 4
Transient receptor potential vanilloid receptor-1 (TRPV1) is a cation channel present in sensory nerve endings capable of sensing pain-producing stimuli  and believed to play a central role in promoting neurogenic inflammation. To date, mammalian TRPV1 has been cloned and characterized from human , rat , guinea pig , rabbit , and mouse . Members of TRPV1 family respond to a variety of selective activators including chemical compounds such as capsaicin, resiniferatoxin, olvanil and N- oleoyldopamine , as well as physiological conditions such as changes in extracellular pH , temperature above 42 C and direct phosphorylation via protein kinase C (PKC) . The transient receptor potential vanilloid 1 (TRPV1) is predicted to have six transmembrane (TM) domains and a short, pore- forming hydrophobic stretch between the fifth and sixth TM domains . Like many other TRP channels, TRPV1 has a long amino terminus containing three ankyrin-repeat domains and a carboxyl terminus containing a TRP domain close to sixth TM . It has been recently shown that calmodulin interacts strongly with the C-terminal domain of TRPV1, similarly, a histidine triad-like protein, PKCi, was identified as interacting partner of the N-terminus of TRPV1  but these findings are still not enough to propose their putative role in TPV1 function. Efforts have been made to unravel the structure of TRPV1 [13,14], but the possible contributions of terminal regions to vanilloid receptor function are still a question. Our aim is to resolve the structure and functional properties of the N-terminal domain of TRPV1. We have cloned a DNA fragment of 775 bp encoding the N-terminal region of TRPV1 in pET32a (+) with EcoR V and Hind III restriction sites and its expression under the control of strong bacteriophage T7 transcription , in fusion with genes encoding His – tag S – tag hook and purified by using affinity chromatography. Purified protein will be used for measuring by vibrational spectroscopy. Experimental results will be combined with homology and energetic modeling techniques to propose a three – dimensional structure of the N – terminus.
This research was supported by the Ministry of Education, Youth and Sports of the Czech Republic (MSM6007665808, LC06010) and by the Academy of Sciences of the Czech Republic (Institutional research concept AVOZ60870520)
 A. Toth, J. Boczan, N. Kedei, E. Lizanecz, Z. Bagi, Z. Papp, I. Edes, L. Csiba, P. M. Blumberg, Molecular Brain Researh, 135 (2005) 162.168
 P. Hayes, H. J. Meadows, M. J. Gunthrope, M. H. Harries, D. M. Duckworth, W. Cairns, D. C. Harrison, C. E. Clarke, K, Ellington, R. K. Prinhja, A. J. Baarton, A. D. Medhurst, G.D. Smith, S. Topp, P. Murdock, G. J. Sanger, J. Terrett, O. Jenkins, C. D. Behham, A. D. Randall, I. S. Gloger, J.B. Davis, Pain, 88 (2000) 205-215.
 M. J. Caterina, M. A. Schumbacher, M. Tominaga, T. A. Rosen, J. D. Levine, D. Julius, Nature, 389 (1997) 816-824.
 J. Savidge, C. Davis, K. Shah, S. Colley, E.Philip, S. Ranasinghe, J. Winter, P. Kotsonis, H. Range, P. Mclntyre, Neuropharmacology, 43 (2002) 450-456.
 N. R. Gavva, L. Klionsky, X. Qu, L. shi, R. Tamir, S. Edenson, T. J. Zhang, V. N. Viswandhan, A. Toeth, L.V. Pearse, T. W. Vanderah, F. Précis, P. M. Blumberg, J. Lile, Y. Sun, K. Wild, J.C. Louis, J. Treanor, J. Biol. Chem., 279 (2004) 20283-20295.
 C. Cornell, P. T. Phelps, J. A. Anthes, Neurosci. Lett., 370 (2004) 55-60.
 C. J. Chu, S. M. Huang, L. De Petrocellis, T. Bisongo, S. A Ewing, J. D. Milleer, R. E. Zipkin, N. Daddario, G. Appendino, V. Di Marzo, J. M. Walker, J. Boil. Chem., 278 (2003) 13633-13639.
 M. Tominaga, M. J. Caternia, A. B. Malmberg, T. A. Rosen, H. Gilbert, K. Skinner, B. E. Raumann, A. I. Basbaum, D. Julius, Neuron, 21 (1998) 531-543.
 M. Numazaki, T. Toyooka, M. Tominaga, J. Biol. Chem., 277 (2002) 13375-13378.
 M. J. Caterina, M.A. Schumacher, M. Tominaga, T.A Rosen, J. D. Levine, D. Julius, Nature, 298 (1997) 816-824.
 S. G. Sedgwick, S. J. Smerdon, Tredns Biochem. Sci., 24 (1999) 311-316.
 C. Morenilla-Palao, R. Planells- Cases, N. Garcia-Sanz, A. Fernandez-Carvajal, A. Ferrer-Monteil, Abstr-Soc. Neurosci., 27 (2001) 56.2.
 V. Vlachova, J. Teisinger, K. Susankova, A. Lyfenko, R. Ettrich, and L. Vyklicky, J. Neurosci., 23(4) (2003) 1340-1350.
 N. García-Sanz, A. Fernandez-Carvajal, C. Morenilla-Palao, R. Planells-Cases, E. Fajardo- Sanchez, G. Fernandez-Ballester, and A. Ferrer-Montiel, J. Neurosci., 24 (23) (2004) 5307-5314.
 H. Lu, Y. Zang, Y. Ze, et. al. in Protein Expression and Purification, 43 (2005) 126–132.