Abstract
The proton-coupled di- and tripeptide transporter PepT1 (SLC15a1) is the major route by which dietary nitrogen is taken up from the small intestine, as well as being the route of entry for important therapeutic (pro)drugs such as the β-lactam antibiotics, angiotensin-converting enzyme inhibitors and antiviral and anti-cancer agents. PepT1 is a member of the major facilitator superfamily of 12 transmembrane domain transporter proteins. Expression studies in Xenopus laevis on rabbit PepT1 that had undergone site-directed mutagenesis of a conserved arginine residue (arginine282 in transmembrane domain 7) to a glutamate revealed that this residue played a role in the coupling of proton and peptide transport and prevented the movement of non-coupled ions during the transporter cycle. Mutations of arginine282 to other non-positive residues did not uncouple proton–peptide cotransport, but did allow additional ion movements when substrate was added. By contrast, mutations to positive residues appeared to function the same as wild-type. These findings are discussed in relation to the functional role that arginine282 may play in the way PepT1 operates, together with structural information from the homology model of PepT1 based on the Escherichia coli lactose permease crystal structure.
Highlights
The uptake of di- and tripeptides by the protoncoupled transporters of the SLC15 family is widely accepted to be the major route of dietary nitrogen absorption from the small intestine and nitrogen reabsorption from the glomerular filtrate in the renal proximal tubule
The rabbit PepT1 (707 amino acids) and human PepT1 (708 amino acids) share an overall identity of 81 per cent at the amino acid level, with the major areas of difference being in the large extracellular loop between transmembrane spanning domains (TMDs) 9 and 10, and the intracellular C-terminus
One explanation for the lack of proton stimulation for the mutant transporters could be that R282E is changing the protein environment of the crucial histidine in TMD2 (H57), long implicated as the residue in PepT1 protonated as the first step in the transport cycle so that it is always protonated (Meredith & Boyd 1995; Temple et al 1996)
Summary
The uptake of di- and tripeptides by the protoncoupled transporters of the SLC15 family is widely accepted to be the major route of dietary nitrogen absorption from the small intestine (via SLC15a1/ PepT1) and nitrogen reabsorption from the glomerular filtrate in the renal proximal tubule (via PepT1 and SLC15a2/PepT2) (see Meredith & Boyd 2000 and Daniel & Kottra 2004 for reviews). One explanation for the lack of proton stimulation for the mutant transporters could be that R282E is changing the protein environment of the crucial histidine in TMD2 (H57), long implicated as the residue in PepT1 protonated as the first step in the transport cycle so that it is always protonated (Meredith & Boyd 1995; Temple et al 1996) Support from this comes from the finding that if the rate of transport is normalized to the amount of protein expressed in the oocyte membrane using luminometry (Panitsas et al 2006), the rate of transport by R282E-rbPepT1 is the same as the wild-type at pHout 5.5, but unlike the wild-type is not slower at pHout 7.4. The lower stoichiometry seen when the proton electrochemical gradient is lower (pHout 7.4 versus 5.5) does suggest that the ion movement is electrodiffusive in nature
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