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Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells.
J Pharm Sci 2019; 108(10):3416-3424JP

Abstract

PHT2, a member of the proton-coupled oligopeptide transporter family, participates in the transportation of small peptides and histidine from lysosomes to the cytosol. It facilitates maintenance of intracellular peptide homeostasis. However, it remains a challenge to elucidate the functional properties of PHT2 due to its localization in the lysosomal membrane. The aim of this study was to explore the transport function and substrate properties of human PHT2 (hPHT2) by transfecting Madin-Darby canine kidney cells with hPHT2 mutants to obtain stably expressed protein in the cell membrane. Using this cell model, we found that the transport activity of hPHT2 reached a maximum capacity when the extracellular pH was 5.5. hPHT2 showed relatively low affinity for Gly-Sar and relatively high affinity for d3-L-histidine, with Km values of 428 ± 88 μM and 66.9 ± 5.7 μM, respectively. Several typical substrates or inhibitors of PEPT1 and PEPT2, including valacyclovir, Gly-Gly-Gly, and cefadroxil but not 5-aminolevulinic acid or captopril, were proven to be substrates of hPHT2. However, hPHT2 showed low affinity for valacyclovir with a Km value of 5350 ± 1234 μM. In conclusion, this study established a suitable and efficient cell model to explore the function of hPHT2 in vitro and provided important information on the transport activity and substrate properties of hPHT2.

Authors+Show Affiliations

Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China; Translational Medicine Research Center, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, Zhejiang 310058, P. R. China.Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China.Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China.Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China.Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China.Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China; Translational Medicine Research Center, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, Zhejiang 310058, P. R. China.Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China.Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China. Electronic address: hdjiang@zju.edu.cn.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31254495

Citation

Wang, Yuqing, et al. "Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells." Journal of Pharmaceutical Sciences, vol. 108, no. 10, 2019, pp. 3416-3424.
Wang Y, Li P, Song F, et al. Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells. J Pharm Sci. 2019;108(10):3416-3424.
Wang, Y., Li, P., Song, F., Yang, X., Weng, Y., Ma, Z., ... Jiang, H. (2019). Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells. Journal of Pharmaceutical Sciences, 108(10), pp. 3416-3424. doi:10.1016/j.xphs.2019.06.016.
Wang Y, et al. Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells. J Pharm Sci. 2019;108(10):3416-3424. PubMed PMID: 31254495.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells. AU - Wang,Yuqing, AU - Li,Ping, AU - Song,Feifeng, AU - Yang,Xi, AU - Weng,Yayun, AU - Ma,Zhiyuan, AU - Wang,Lu, AU - Jiang,Huidi, Y1 - 2019/06/26/ PY - 2019/02/14/received PY - 2019/06/04/revised PY - 2019/06/20/accepted PY - 2019/6/30/pubmed PY - 2019/6/30/medline PY - 2019/6/30/entrez KW - cell model KW - hPHT2 KW - substrates KW - transport function SP - 3416 EP - 3424 JF - Journal of pharmaceutical sciences JO - J Pharm Sci VL - 108 IS - 10 N2 - PHT2, a member of the proton-coupled oligopeptide transporter family, participates in the transportation of small peptides and histidine from lysosomes to the cytosol. It facilitates maintenance of intracellular peptide homeostasis. However, it remains a challenge to elucidate the functional properties of PHT2 due to its localization in the lysosomal membrane. The aim of this study was to explore the transport function and substrate properties of human PHT2 (hPHT2) by transfecting Madin-Darby canine kidney cells with hPHT2 mutants to obtain stably expressed protein in the cell membrane. Using this cell model, we found that the transport activity of hPHT2 reached a maximum capacity when the extracellular pH was 5.5. hPHT2 showed relatively low affinity for Gly-Sar and relatively high affinity for d3-L-histidine, with Km values of 428 ± 88 μM and 66.9 ± 5.7 μM, respectively. Several typical substrates or inhibitors of PEPT1 and PEPT2, including valacyclovir, Gly-Gly-Gly, and cefadroxil but not 5-aminolevulinic acid or captopril, were proven to be substrates of hPHT2. However, hPHT2 showed low affinity for valacyclovir with a Km value of 5350 ± 1234 μM. In conclusion, this study established a suitable and efficient cell model to explore the function of hPHT2 in vitro and provided important information on the transport activity and substrate properties of hPHT2. SN - 1520-6017 UR - https://www.unboundmedicine.com/medline/citation/31254495/Substrate_Transport_Properties_of_the_Human_Peptide/Histidine_Transporter_PHT2_in_Transfected_MDCK_Cells L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-3549(19)30386-7 DB - PRIME DP - Unbound Medicine ER -