Site-directed mutagenesis of Arginine282 suggests how protons and peptides are co-transported by rabbit PepT1.
Site-directed mutagenesis of Arginine282 suggests how protons and peptides are co-transported by rabbit PepT1.
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精氨酸282的位置定向诱变表明质子和肽是如何通过兔Pept1共同传输的。
DOI：
10.1016/j.biocel.2007.10.010
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发表时间：
2008
影响因子：
4
通讯作者：
Meredith, David
中科院分区：
文献类型：
--
作者：
Pieri, Myrtani;Hall, Dashiell;Price, Richard;Bailey, Patrick;Meredith, David
关键词：
The mammalian proton-coupled peptide transporter PepT1 is the major route of uptake for dietary nitrogen, as well as the oral absorption of a number of drugs, including β-lactam antibiotics and angiotensin-converting enzyme inhibitors. Here we have used site-directed mutagenesis to investigate further the role of conserved charged residues in transmembrane domains. Mutation of rabbit PepT1 arginine282 (R282, transmembrane domain 7) to a positive (R282K) or physiologically titratable residue (R282H), resulted in a transporter with wild-type characteristics when expressed in Xenopus laevis oocytes. Neutral (R282A, R282Q) or negatively charged (R282D, R282E) substitutions gave a transporter that was not stimulated by external acidification (reducing pHout from 7.4 to 5.5) but transported at the same rate as the wild-type maximal rate (pHout 5.5); however, only the R282E mutation was unable to concentrate substrate above the extracellular level. All of the R282 mutants showed trans-stimulation of efflux comparable to the wild-type, except R282E-PepT1 which was faster. A conserved negatively charged residue, aspartate341 (D341) in transmembrane domain 8 was implicated in forming a charge pair with R282, as R282E/D341R- and R282D/D341R-PepT1 had wild-type transporter characteristics. Despite their differences in ability to accumulate substrate, both R282E- and R282D-PepT1 showed an increased charge:peptide stoichiometry over the wild-type 1:1 ratio for the neutral dipeptide Gly-l-Gln, measured using two-electrode voltage clamp. This extra charge movement was linked to substrate transport, as 4-aminobenzoic acid, which binds but is not translocated, did not induce membrane potential depolarisation in R282E-expressing oocytes. A model is proposed for the substrate binding/translocation process in PepT1.
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DOI：
10.1085/jgp.109.6.703
发表时间：
1997-06
期刊：
The Journal of general physiology
影响因子：
--
作者：
Yao Y;Tsien RY
通讯作者：
Tsien RY
影响因子：
64.8
作者：
FEI, YJ;KANAI, Y;HEDIGER, MA
通讯作者：
HEDIGER, MA
DOI：
10.1046/j.1432-1327.2000.01405.x
发表时间：
2000-06-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Meredith, D;Temple, CS;Bailey, PD
通讯作者：
Bailey, PD
影响因子：
5.5
作者：
Meredith, D;Boyd, CAR;Temple, CS
通讯作者：
Temple, CS
影响因子：
2.9
作者：
Covitz, KMY;Amidon, GL;Sadée, W
通讯作者：
Sadée, W