A charge pair interaction between Arg282 in transmembrane segment 7 and Asp341 in transmembrane segment 8 of hPepT1.
OBJECTIVE
To determine whether R282 in transmembrane segment 7 (TMS7) of hPepT1 forms a salt bridge with D341 in TMS8.


METHODS
Mutated hPepT1 transporters containing point mutations at R282 and/or D341 were transiently transfected into HEK293 cells. Their steady state expression and functional activity were measured using immunoprecipitation and 3H-gly-sar uptake, respectively. Gly-sar uptake by cysteine mutants (R282C and D341C) was also measured in the presence and absence of cysteine-modifying MTS reagents.


RESULTS
The reverse-charge mutants R282D-hPepT1 and D341R-hPepT1 showed significantly reduced gly-sar uptake, but the double mutant (R282D/D341R-hPepT1) has functionality comparable to that of wild-type hPepT1. Gly-sar uptake by R282C-hPepT1 is reduced, but pre-incubation with 1 mM MTSET, a positively charged cysteine-modifying reagent, restored function to wild-type levels. Similarly, pre-incubation of D341C-hPepT1 with 10 mM MTSES, a negatively charged cysteine-modifying reagent, increased gly-sar uptake compared to unmodified D341C-hPepT1. In contrast, MTSET modification of D341C-hPepT1 (giving a positive charge at position 341) resulted in significant reduction in gly-sar uptake, compared to D341C-hPepT1.


CONCLUSIONS
Our results are consistent with a salt bridge between R282 and D341 in hPepT1, and we use these and other data to propose a role for the R282-D341 charge pair in the hPepT1 translocation mechanism.
Kulkarni
Ashutosh A
AA
Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90089-9121, USA.
Davies
Daryl L
DL
Links
Jennifer S
JS
Patel
Leena N
LN
Lee
Vincent H L
VH
Haworth
Ian S
IS
D019943:Amino Acid Substitution; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D047468:Immunoprecipitation; D049449:Luminescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D000074011:Peptide Transporter 1; D027981:Symporters / Q000737:chemistry* / Q000235:genetics; D014162:Transfection
D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters