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A Charge Pair Interaction Between Arg282 in Transmembrane Segment 7 and Asp341 in Transmembrane Segment 8 of hPepT1

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Abstract

Purpose

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.

Conclusion

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.

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Acknowledgments

This work was supported by National Institutes of Health grants GM59297 (VHLL & ISH) and AA013890 (DLD).

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, California, 90089-9121, USA

    Ashutosh A. Kulkarni, Jennifer S. Links, Leena N. Patel, Vincent H. L. Lee & Ian S. Haworth

  2. Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, California, 90089-9121, USA

    Daryl L. Davies

  3. Department of Ophthalmology, University of Southern California, Los Angeles, California, 90089-9121, USA

    Vincent H. L. Lee

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  1. Ashutosh A. Kulkarni
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Correspondence to Ian S. Haworth.

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Kulkarni, A.A., Davies, D.L., Links, J.S. et al. A Charge Pair Interaction Between Arg282 in Transmembrane Segment 7 and Asp341 in Transmembrane Segment 8 of hPepT1. Pharm Res 24, 66–72 (2007). https://doi.org/10.1007/s11095-006-9119-x

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  • DOI: https://doi.org/10.1007/s11095-006-9119-x

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