A charge pair interaction between Arg282 in transmembrane segment 7 and Asp341 in transmembrane segment 8 of hPepT1

To determine whether R282 in transmembrane segment 7 (TMS7) of hPepT1 forms a salt bridge with D341 in TMS8.

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.

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.

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.