Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen

The interaction of the N-type calcium channel 3 subunit with the 1B subunit alters the activation/inactivation kinetics and the maximal conductance of the channel. The defined protein-protein interaction of the human 1B and 3 subunits provides a target for small-molecule modulation of N-type channel activity. We describe a high throughput screen based on a counterseiection yeast two-hybrid assay, which was used to identify small molecules that disrupt 1B-3 subunit interactions and inhibit N-type calcium channel activity. These small molecules may be a new class of calcium channel antagonists with therapeutic potential.

1. Dunlap, K., Luebke, J.L, and Turner, T.J.1995 Exocytotic Ca2+ channels in mammalian central neurons. Trends Neurosci. 18: 89−98. | Article | PubMed  | ISI | ChemPort |
2. Newcomb, R. and Palma, A. Effects of diverse omega-conopeptldes on the in vivo release of glutamic and gamma-aminobutyric acids. Brain Res. 638: 95−102. (1994). | Article | PubMed  | ISI | ChemPort |
3. Valentino, K., Newcomb, R., Gadbois, T., Singh, T., Bowersox, S., Bitner, S. et al. 1993. Selective N-type calcium channel antagonist protects against neuronal loss after global cerebral ischemia. Proc. Natl. Acad. Sci. USA 90: 7894−7897. | PubMed  | ChemPort |
4. Qin, N., Platano, D., Olcese, R., Stefani, E., and Birnbaumer, L. 1997. Direct interaction of G / with a C-terminal G /-binding domain of the Ca2+ channel 1 subunit is responsible for channel inhibition by G protein-coupled receptors. Proc. Natl. Acad. Sci. USA 94: 8866−8871. | Article | PubMed  | ChemPort |
5. Walker, D., Bichet, D., Campbell, K.P., and De Waard, M. 1998. A 4 isoform-specific interaction site in the carboxyl-terminal region of the voltage-dependent Ca2+ channel 1 A subunit. J. Biol. Chem. 273: 2361−2367. | Article | PubMed  | ISI | ChemPort |
6. Page, K.M., Canti, C., Stephens, G.J., Berrow, N.S., and Dolphin, A.C. 1998. Identification of the amino terminus of neuronal Ca2+ channel 1 submits 1B and 1E as an essential determinant of G-protein modulation. J. Neurosci. 18: 4815−4824. | PubMed  | ISI | ChemPort |
7. Zamponi, G.W., Bourinet, E., Nelson, D., Nargeiot, J., and Snutch, T.P. 1997. Crosstalk between G-proteins and protein kinase C mediated by the calcium channel a1 subunit. Nature 385: 442−446. | Article | PubMed  | ISI | ChemPort |
8. De Waard, M., Liu, H., Walker, D., Scott, V.E., Scott, S., CA.et al. 1997. Direct binding of G-protein / complex to voltage-dependent calcium channels. Nature. 385: 446−450. | Article | PubMed  | ChemPort |
9. Walker, D. and De Waard, M. 1998. Subunit interaction sites in voltage-dependent Ca+2 channels: role in channel function. Trends Neurosci. 21: 148−154. | Article | PubMed  | ISI | ChemPort |
10. Stea, A., Dubel, S.J., Pragnell, M., Leonard, J.P., Campbell, K., and Snutch, T.P.A -subunit normalizes the electrophysiological properties of a cloned N-type Ca2+ channel 1,-subunit.Neuropharmacology 32: 1103−1116. | PubMed  | ChemPort |
11. Anwyl, R. 1991. Modulation of vertebrate neuronal calcium channels by transmitters. Brain Res 16: 265−281. | Article | ISI | ChemPort |
12. Hille, B. 1994. Modulation of ion-channel function by G-protein-coupled receptors. Trends Neurosci. 17: 531−536. | Article | PubMed  | ISI | ChemPort |
13. De Waard, M., Pragnell, M., and Campbell, K. 1994. Ca2+ channel regulation by a conserved subunit domain. Neuron 13: 495−593. | Article | PubMed  | ChemPort |
14. Pragnell, M., De Waard, M., Mori, Y., Tanabe, T., Snutch, T.P., and Campbell, K., 1997. Calcium channel -subunit binds to conserved motif in the l-ll cytoplasmic linker. Nature 368: 67−70. | Article |
15. De Waard, M., Witcher, D.R., Pragnell, M., Liu, H., and Campbell, K. 1995. Properties of the ,- anchoring site in voltage-dependent Ca2+ channels. J. Biol. Chem. 270: 12056−12064. | Article | PubMed  | ChemPort |
16. Fields, S. and Song, O. 1989. A novel genetic system to detect protein-protein interactions. Nature 340: 245−256. | Article | PubMed  | ISI | ChemPort |
17. Bean, B.P. 1989. Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence. Nature340: 153−155. | Article | PubMed  | ISI | ChemPort |
18. Mintz, I.M. and Bean, B.P. 1993. Block of calcium channels in rat neurons by synthetic omega-aga-iva. Neuropharmacology 32: 1161−1169. | Article | PubMed  | ISI | ChemPort |
19. Herlitze, S., Garcia, D.E., Mackie, K., Hille, B., Scheuer, T., and Catterall, W.A., 1996. Modulation of Ca2+ channels by G-protein subunits. Nature380: 258−261. | Article | PubMed  | ISI | ChemPort |
20. Ikeda, S.R. 1996. Voltage-dependent modulation of N-type calcium channels by G-protein subunits. Nature 380: 255−258. | Article | PubMed  | ISI | ChemPort |
21. Huang, J. and Schreiber, S. 1997. A yeast genetic system for selection of small molecule inhibitors of protein-protein interaction in nanodroplets.Proc. Natl. Acad. Sci. USA 97: 13396−13401. | Article |
22. Vidal, M., Braun, P., Chen, E., Boeke, J.D., and Harlow, E. 1996. Genetic characterization of a mammalian protein-protein interaction by using a yeast reverse two-hybrid system. Proc. Natl. Acad. Sci. USA 93: 10321−10326. | Article | PubMed  | ChemPort |
23. Leanna, M. and Hannick, M. 1996. The reverse two-hybrid system: a genetic scheme for selection against specific protein-protein interactions. Nucleic Acids Res 24: 3341−3347. | Article | PubMed  | ISI | ChemPort |
24. Shih, H-M. Godman, P.S., DeMaggis, A.J., Hollenberg, S.M., Goodman, K.H., and Hoekdstra, M.F. 1997. A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with the co-activator CBP. Proc. Natl. Acad. Sci. USA 93: 13896−13901. | Article |
25. Manlatis, T., Fritsch, E.F., and Sambrook, J. 1992. Molecular cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
26. Finney, M. 1993. Current protocols in molecular biology. Wiley & Sons, New York.
27. Ellinor, P.T., Zhang, J.F., Randall, A.D., Zhou, M., Schwarz, T.L., Tsien, R.W. et al. 1993. Functional expression of a rapidly inactivating neuronal calcium channel. Nature 363: 455−458. | Article | PubMed  | ISI | ChemPort |
28. Durfee, T., Becherer, K., Chen, P.-L., Yeh, S.-H., Yang, Y., Kilburn, A.E., Lee, W.-H., and Elledge, S.J., 1993. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7: 555−569. | PubMed  | ISI | ChemPort |
29. Collin, T., Lory, P., Taviaux, S., Courtieu, C., Guilbault, P., Berta, P. et al. 1994. Cloning, chromosomal location and functional expression of the human voltage-dependent calcium channel 3 subunit. Eur. J. Biochem 220: 257−262. | PubMed  | ISI | ChemPort |
30. Castellano, A., Wei, X., Birnbaumer, L., and Perez-Reyes, E., 1993. Cloning and expression of a third calcium channel subunit. J. Biol. Chem 268: 3450−3455. | PubMed  | ISI | ChemPort |
31. Hullin, R., Singer-Lahat, D., Freichel, M., Biel, M., Oascal, N., Hofmann, F. et al. 1992. Calcium channel subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain. EMBO J.11: 885−890. | PubMed  | ISI | ChemPort |
32. Harper, J.W., Adami, G.R., Wei, N., Kayomarsi, K., and Elledge, S.J. 1993. The p21 Cdk interacting protein Cip1 is a potent inhibitor of G1 cyclin dependent kinases. Cell 75: 805−816. | Article | PubMed  | ISI | ChemPort |
33. Elledge, S.J. and Davis, R.W. 1988. A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene 70: 303−312. | Article | PubMed  | ISI | ChemPort |
34. Kaufer, N.F., Fried, H.M., Schwindinger, W.F., Jasin, M., and Warner, J.R. Cyclohexlmide resistance in yeast; the gene and its protein. Nucleic Acids Res. 11: 3123−3235. | PubMed  | ChemPort |
35. Yocum, R.R., Hanley, S., West, R., and Ptashne, M. 1984. Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol. Cell. Biol. 4: 1985−1988. | PubMed  | ISI | ChemPort |
36. Price, L., Kajkowski, E.M., Hadcock, J.R., Ozenberger, B.A., and Pausch, M.A. 1995. Functional coupling of a mammalian somatostatin receptor to the yeast pheromone response pathway. Mol. Cell Biol. 15: 6188−6195. | PubMed  | ISI | ChemPort |
37. Hill, J.E., Myers, A.M., Koerner, T.J., and Tzagoloff, A. 1986. Yeast/E.coli shuttle vectors with multiple unique restriction sites. Yeast 2: 163−167. | PubMed  | ISI | ChemPort |
38. Bai, C. and Elledge, S.J. 1997. Gene identification using the two-hybrid system. Methods Enzymol. 283: 141−156. | Article | PubMed  | ISI | ChemPort |
39. Ozenberger, B.A. and Young, K.H. 1995. Functional interacton of ligands and receptors o the hematopoietic superfamily n yeast. Mol. Endocrinol. 9: 1321−1329. | Article | PubMed  | ISI | ChemPort |
40. Rose, M.D. Winston, R. and Young, K.H. 1990. Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, NY.
41. Plummer, M.R., Rittenhouse, A., Kanevsky, M., and Hess, P. 1991. Neurotransmitter modulation of calcium channels in rat sympathetic neurons. J. Neuroscience.11: 2339−2348. | ISI | ChemPort |

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