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Received for publication October 29, 2004.
Revised January 18, 2005.
Accepted for publication January 19, 2005.
To identify functionally relevant amino acids in the rat organic cation transporter rOCT1 eighteen consecutive amino acids in the presumed 4th transmembrane 
-helix (TMH) were mutated and functionally characterized after expression in oocytes of Xenopus laevis. After mutation of three amino acids on successive turns of the -helix KM values for tetraethylammonium (TEA) and/or 1-methyl-4-phenylpyridinium (MPP) were decreased. After replacement of W218 by tyrosine (W218Y) and Y222 by leucine (Y222L) the KM values for both TEA and MPP were decreased. In mutant Y222F only the KM for TEA, and in mutant T226A only the KM for MPP was decreased. The data suggest that amino acids W218 and Y222 participate in binding of both, TEA and MPP, whereas T226 is only involved in binding of MPP. Using the crystal structure of the lactose permease LacY from Escherichia coli that belongs to the same major facilitator superfamily as rOCT1, we modelled the tertiary structure of the presumed 12 transmembrane -helices. The validity of the model was suggested since seven amino acids that have been shown to participate in binding of cations by mutagenesis experiments (4th TMH W218, Y222, T226 (this paper); 10th TMH A443, L447, Q448 (accompanying paper); 11th TMH D475 (previous report)) are located in one region surrounding a large cleft that opens to the intracellular side. The dimensions of TEA in comparison to the interacting amino acids in the modelled cleft suggest that more than one TEA molecule can bind in parallel to the modelled conformation of the transporter.
Key words:
Biogenic Amine, Organic anion, Structure-activity relationships and modeling, Mutagenesis/Chimeric approaches
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