Abstract
One of the major goals in membrane transporter research is to understand how transporter proteins work at the molecular level. Ideally, this research would be carried out with a detailed knowledge of the three-dimensional structure of the protein. However, in the absence of protein crystals of transporters suitable for X-ray diffraction analysis other molecular tools need to be employed. In vitro site-directed mutagenesis is one method that has the capacity to provide both structural information and identification of the role of individual residues and/or regions of a protein that are involved in function.
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References
Kaback, H. R., Sahin-Toth, M., and Weinglass, A. B. (2001) The kamikaze approach to membrane transport. Nature Rev./Mol. Cell. Biol. 2, 610–620.
Buck, K. and Amara, S. G. (1994) Chimeric dopamine—norepinephrine transporters delineate structural domains influencing selectivity for catecholamines and 1-methyl-4-phenylpyridinium. Proc. Natl. Acad. Sci. USA 91, 12,584–12,588.
Mitrovic, A. D., Amara, S. G., Johnston, G. A. R., et al. (1998) Identification of functional domains of glutamate transporters J. Biol. Chem. 273, 14,698–14,706.
Seal, R. P. and Amara, S. G. (1998) A reentrant loop domain in the glutamate carrier EAAT1 participates in substrate binding and translocation. Neuron 21, 1487–1498.
Grunewald, M., Bendahan, A., and Kanner, B. I. (1998) Biotinylation of single cysteine mutants of the glutamate transporter GLT-1 from rat brain reveals its unusual topology. Neuron 21, 623–632.
Monks, S. A., Needleman, D. J., and Miller, C. (1999) Helical structure and packing orientation of the S2 segment in the Skaker K+ channel. J. Gen. Physiol. 113, 415–423.
Arriza, J. L., Fairman, W. A., Wadiche, J. I., et al. (1994) Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex. J. Neurosci. 14, 5559–5569.
Kreig, P. A. and Melton, D. A. (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 12, 7057–7070.
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© 2003 Humana Press Inc., Totowa, NJ
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Ryan, R.M., Mitrovic, A.D., Vandenberg, R.J. (2003). Site-Directed Mutagenesis in the Study of Membrane Transporters. In: Yan, Q. (eds) Membrane Transporters. Methods in Molecular Biology, vol 227. Humana Press. https://doi.org/10.1385/1-59259-387-9:97
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DOI: https://doi.org/10.1385/1-59259-387-9:97
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