Biophysical Evidence for His<sup>57</sup>as a Proton-Binding Site in the Mammalian Intestinal Transporter hPepT1

Uchiyama, Tomomi; Kulkarni, Ashutosh A.; Davies, Daryl L.; Lee, Vincent H.L.

doi:10.1023/b:pham.0000008036.05892.e9

Cited by 42 publications

(30 citation statements)

References 18 publications

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“…Up to date, histidyl residues have been reported extensively to be one of the most important amino acid residues in hPEPT1, which were revealed by DEPC modification 26,27,35,36) and site-directed mutagenesis studies. [28][29][30]37) DEPC modification showed non-competitive inhibition 26,27) and protection in the presence of substrates. 35,36) Site-directed mutagenesis studies revealed that His-57 in the second transmembrane domain (TMD2) is the most likely residue involved in the H ϩ binding/dissociation.…”

Section: Discussionmentioning

confidence: 97%

“…35,36) Site-directed mutagenesis studies revealed that His-57 in the second transmembrane domain (TMD2) is the most likely residue involved in the H ϩ binding/dissociation. [27][28][29][30]37) The site-directed mutagenesis studies affecting His-121 in TMD4 showed that it is the substrate-binding site. 28) His-57 and His-121 were hypothesized to be intimately located and form the substrate binding pockets.…”

Section: Discussionmentioning

confidence: 99%

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The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

Fujisawa

Tateoka

Nara

et al. 2006

Biological & Pharmaceutical Bulletin

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It has been estimated that more than 80% of amino acids found in the intestinal lumen after a protein meal are in the form of small peptides rather than free amino acids.1) Therefore, a majority of protein digestion products are obligatorily absorbed via an intestine oligo-peptide transporter called PEPT1. Of interest, PEPT1 also mediates the absorption of a huge number of pharmacologically important compounds including peptidomimetic drugs, 2,3) such as oral b-lactam antibiotics, 4-6) ACE-inhibitors like captopril, 7) or the peptidase inhibitor bestatine 8) and even compounds without an obvious peptide bond or equivalent, 9) such as d-amino levulinic acid.10) This surprisingly high tolerance for structural diversity of the substrate by PEPT1 has been expected to be used as a channel to increase the intestinal absorption of poorly absorbable drugs. 11)The transport of substrates via PEPT1 is coupled to the downward movement of a proton in accordance with its electrochemical gradient. 2,3,12) It has been demonstrated in intestinal membrane vesicles, 4,13,14) Xenopus oocytes 15) and culture cells 16) that the transport activity of PEPT1 shows a bellshaped pH dependence with an optimal pH 5.5 to 6.0. This optimal pH is physiologically collaborated with an acidic unstirred water layer (pH 5.5-6.0) in the intestinal lumen. 1,17) Hence, PEPT1 can function most efficiently in epithelial cells of the small intestine.As for the bell-shaped activity of PEPT1 vs. pH, the reason why the transport activity decreases with an increase in pH is obviously the decrease in the driving force of the proton electrochemical potential gradient. On the other hand, why does the activity decrease in a more acidic region although the driving force should increase? To answer this question, we will try to obtain a relationship between the transport activity and extracellular pH. However, there is a problem to be solved: The driving force changes along with the change in extracellular pH. The pH-dependent properties of PEPT1 should be determined without changing the driving force. Here, we employ the experimental condition that the proton concentration gradient across the membrane is dissipated by the addition of monensin and nigericin 18,19) ; then the driving force is solely the membrane potential. To perform this experiment, a cell line is necessary which shows high transport activity. First, we established stably transfected CHO cells which show high transport activities for various substrates with almost the same K m values as those obtained from those of a model human intestine epithelial cell line, Caco-2. [20][21][22] Thus, this CHO cell line over-expressing hPEPT1 (designated as CHO/hPEPT1) is useful and convenient for transport studies of hPEPT1. In the present study, we then examined the pH-dependency of transport activity by hPEPT1 using CHO/hPEPT1 cells. Human oligopeptide transporter (hPEPT1) translocates di/tri-peptide by coupling to movement of proton down the electrochemical gradient. This transporter has the characteristics ...

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

Fujisawa

Tateoka

Nara

et al. 2006

Biological & Pharmaceutical Bulletin

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“…8), suggesting that histidine residue of rMATE1 does not serve as substrate-binding site. In other H ϩ -coupled transporters such as H ϩ /peptide cotransporter 1 (Uchiyama et al, 2003) and Na ϩ /H ϩ exchanger (Cha et al, 2003), histidine residues function as an H ϩ -binding site. It is, therefore, suggested that histidine residue of the MATE family acts as a H ϩ -binding site for driving force.…”

Section: Discussionmentioning

confidence: 99%

Identification of Essential Histidine and Cysteine Residues of the H⁺/Organic Cation Antiporter Multidrug and Toxin Extrusion (MATE)

Asaka

Takato²,

Tsuda³

et al. 2007

Mol Pharmacol

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Multidrug and toxin extrusion 1 (MATE1) has been isolated as an H ϩ /organic cation antiporter located at the renal brushborder membranes. Previous studies using rat renal brushborder membrane vesicles indicated that cysteine and histidine residues played critical roles in H ϩ /organic cation antiport activity. In the present study, essential histidine and cysteine residues of MATE1 family were elucidated. When 7 histidine and 12 cysteine residues of rat (r)MATE1 conserved among species were mutated, substitution of His-385, Cys-62, and Cys-126 led to a significant loss of tetraethylammonium (TEA) transport activity. Cell surface biotinylation and immunofluorescence analyses with confocal microscopy indicated that rMATE1 mutant proteins were localized at plasma membranes.Mutation of the corresponding residues in human (h)MATE1 and hMATE2-K also diminished the transport activity. The transport of TEA via rMATE1 was inhibited by the sulfhydryl reagent p-chloromercuribenzenesulfonate (PCMBS) and the histidine residue modifier diethyl pyrocarbonate (DEPC) in a concentration-dependent manner. The PCMBS-caused inhibition of the transport via rMATE1 was protected by an excess of various organic cations such as TEA, suggesting that cysteine residues act as substrate-binding sites. In the case of DEPC, no such protective effects were observed. These results suggest that histidine and cysteine residues are required for MATE1 to function and that cysteine residues may serve as substraterecognition sites.

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“…Intriguingly, positioned close to this junction, but separated by the current position of helix H7, is a conserved histidine residue (His 61 ), which sits on helix H2 at the base of the extracellular cavity. Functional studies have identified the equivalent histidine residue in human PepT1 as the site of proton binding during the transport cycle [27]. The role of these residues in the transport cycle of PepT So , however, remain to be determined.…”

Section: Insights Into Conformational Flexibilitymentioning

confidence: 99%

Towards a structural understanding of drug and peptide transport within the proton-dependent oligopeptide transporter (POT) family

Newstead

2011

Biochemical Society Transactions

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One of the principal aims of modern drug design is the targeted delivery of drugs within the body, such as to the central nervous system, combined with their exclusion from the liver and kidneys, which break down foreign molecules and subsequently eliminate them. Many of the commonly prescribed drugs are transported into cells and across the plasma membrane via endogenous membrane transporters, whose principal roles are the uptake of essential nutrients for metabolism. In many cases, such drug transport is serendipitous as they are simply mistaken as 'natural' compounds. Many of these transporters could, however, be targeted more efficiently, improving drug absorption, distribution and retention. The molecular details of these drug-transporter interactions, however, are at best poorly understood, in large part through the absence of any high-resolution structural information. To address this issue, we recently determined the structure of a prokaryotic peptide transporter, PepTSo from Shewanella oneidensis, which shares a high degree of sequence similarity and functional characteristics with the human PepT1 and PepT2 proteins. PepT1 and PepT2 contribute significantly to the oral bioavailability and pharmacokinetic properties of a number of important drug families, including antibiotics, antivirals and anticancer agents. The crystal structure of PepTSo provides the first high-resolution model of a drug importer and provides the starting point for understanding drug and peptide transport within the human body.

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Biophysical Evidence for His⁵⁷as a Proton-Binding Site in the Mammalian Intestinal Transporter hPepT1

Abstract: H57R-hPepT1 is able to bind protons at a relatively basic pH, resulting in facilitation of transport of Gly-Sar by hPepT1 at higher pH. Our novel approach provides direct evidence that His57 is a principal proton-binding site in hPepT1.

Cited by 42 publications

References 18 publications

The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

Identification of Essential Histidine and Cysteine Residues of the H⁺/Organic Cation Antiporter Multidrug and Toxin Extrusion (MATE)

Towards a structural understanding of drug and peptide transport within the proton-dependent oligopeptide transporter (POT) family

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Biophysical Evidence for His57as a Proton-Binding Site in the Mammalian Intestinal Transporter hPepT1

Abstract: H57R-hPepT1 is able to bind protons at a relatively basic pH, resulting in facilitation of transport of Gly-Sar by hPepT1 at higher pH. Our novel approach provides direct evidence that His57 is a principal proton-binding site in hPepT1.

Cited by 42 publications

References 18 publications

The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

Identification of Essential Histidine and Cysteine Residues of the H+/Organic Cation Antiporter Multidrug and Toxin Extrusion (MATE)

Towards a structural understanding of drug and peptide transport within the proton-dependent oligopeptide transporter (POT) family

Contact Info

Product

Resources

About

Biophysical Evidence for His⁵⁷as a Proton-Binding Site in the Mammalian Intestinal Transporter hPepT1

Identification of Essential Histidine and Cysteine Residues of the H⁺/Organic Cation Antiporter Multidrug and Toxin Extrusion (MATE)