Ashutosh A. Kulkarni

Biophysical Evidence for His57 as a Proton-Binding Site in the Mammalian Intestinal Transporter hPepT1

AbstractPurpose. The objective of this study was to provide direct evidence of the relative importance of the His57 residue present in transmembrane domain 2 (TMD 2) and the His121 residue in TMD 4 as proton-binding sites in human PepT1 (hPepT1) by using a novel mutagenesis approach. Methods. His57 and His121 in hPepT1 were each mutated to alanine, arginine, or lysine individually to obtain H57A-, H57R-, H57K-, H121A-, H121R-, and H121K-hPepT1. H7A-hPepT1 was used as a negative control. [3H]Glycylsarcosine (Gly-Sar) uptake was measured 72 h posttransfection using HEK293 cells individually transfected with these mutated proteins. Steady-state I/V curves (−150 mV to +50 mV, holding potential −70 mV) were obtained by measuring 5 mM Gly-Sar-induced currents in oocytes expressing H57R- and H57K-hPepT1. Noninjected oocytes and wild-type hPepT1 (WT-hPepT1)-injected oocytes served as negative and positive controls, respectively. Results. At pH 6.0, H57K-, H57R-, H121K-, and H121R-hPepT1 led to a 97%, 90%, 45%, and 75% decrease in [3H]Gly-Sar uptake into HEK293 cells, respectively. At pH 7.4, uptake in cells transfected with H57K- and H57R-hPepT1 was not significantly different from that at pH 6.0, whereas cells expressing H121R- and H121K-hPepT1 showed 56% and 65% decrease, respectively, compared to that at pH 6.0. In oocytes expressing H57R-hPepT1, steady-state currents induced by 5 mM Gly-Sar increased with increasing pH (Imax= 300 nA at pH 8.5), suggesting the binding of protons to H57R. No such trend was observed in oocytes injected with H57K, H121R, and H121K cRNA. Conclusions. 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.

Pharmacological modulation of fluid secretion in the pigmented rabbit conjunctiva.

We determined net fluid secretion rate across the pigmented rabbit conjunctiva in the presence and absence of pharmacological agents known to affect active Cl- secretion and Na+ absorption. Fluid flow across a freshly excised pigmented rabbit conjunctiva mounted between two Lucite half chambers was measured by a pair of capacitance probes in an enclosed cabinet maintained at 37 degrees C and a relative humidity of 70%. Fluid transport was also measured in the presence of compounds known to affect active Cl- secretion (cAMP, UTP, and ouabain), Na+ absorption (D-glucose), or under the Cl--free condition on both sides of the tissue. Net fluid secretion rate across the pigmented rabbit conjunctiva in the serosal-to-mucosal direction at baseline was 4.3+/-0.2 microl/hr/cm2 (mean +/- s.e.m.). Net fluid secretion rate was increased approximately two-fold by mucosally applied 1 mM 8-Br cAMP (8.4+/-0.4 microl/hr/cm2) and 10 microM UTP (9.8+/-0.6 microl/hr/cm2), but was abolished by either serosally applied 0.5 mM ouabain (0.3+/-0.1 microl/hr/cm2) or under the Cl--free conditions (0.06+/-0.04 microl/hr/cm2). Mucosal addition of 20 mM D-glucose decreased net fluid secretion rate to 1.0+/-0.5 microl/hr/cm2. In conclusion, the pigmented rabbit conjunctiva appears to secrete fluid secondary to active Cl- secretion. This net fluid secretion is subject to modulation by changes in active Cl- secretion rate and in mucosal fluid composition such as glucose concentration.

Analysis of Transmembrane Segment 7 of the Dipeptide Transporter hPepT1 by Cysteine-scanning Mutagenesis

To investigate the involvement of transmembrane segment 7 (TMS7) of hPepT1 in forming the putative central aqueous channel through which the substrate traverses, we individually mutated each of the 21 amino acids in TMS7 to a cysteine and analyzed the mutated transporters using the scanning cysteine accessibility method. Y287C- and M292C-hPepT1 did not express at the plasma membrane. Out of the remaining 19 transporters, three (F293C-, L296C-, and F297C-hPepT1) showed negligible glycyl-sarcosine (gly-sar) uptake activity and may play an important role in defining the overall hPepT1 structure. K278C-hPepT1 showed ∼40% activity and the 15 other transporters exhibited more than 50% gly-sar uptake when compared with wild type (WT)-hPepT1. Gly-sar uptake for the 16 active transporters containing cysteine mutations was then measured in the presence of 2.5 mm 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 1 mm [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET). Gly-sar uptake was significantly inhibited for each of the 16 single cysteine mutants in the presence of 2.5 mm MTSEA. In contrast, significant inhibition of uptake was only observed for K278C-, M279C-, V280C-, T281C-, M284C-, L286C-, P291C-, and D298C-hPepT1 in the presence of 1 mm MTSET. MTSET modification of R282C-hPepT1 resulted in a significant increase in gly-sar uptake. To investigate this further, we mutated WT-hPepT1 to R282A-, R282E-, and R282K-hPepT1. R282E-hPepT1 showed a 43% reduction in uptake activity, whereas R282A- and R282K-hPepT1 had activities comparable with WT-hPepT1, suggesting a role for the Arg-282 positive charge in substrate translocation. Most of the amino acids that were MTSET-sensitive upon cysteine mutation, including R282C, are located toward the intracellular end of TMS7. Hence, our results suggest that TMS7 of hPepT1 is relatively solvent-accessible along most of its length but that the intracellular end of the transmembrane domain is particularly so. From a structure-function perspective, we speculate that the extracellular end of TMS7 may shift following substrate binding, providing the basis for channel opening and substrate translocation.

Transmembrane segment 5 of the dipeptide transporter hPepT1 forms a part of the substrate translocation pathway

This study is the first systematic attempt to investigate the role of transmembrane segment 5 of hPepT1, the most conserved segment across different species, in forming a part of the aqueous substrate translocation pathway. We used cysteine-scanning mutagenesis in conjunction with the sulfhydryl-specific reagents, MTSEA and MTSET. Neither of these reagents reduced wild-type-hPepT1 transport activity in HEK293 cells and Xenopus oocytes. Twenty-one single cysteine mutations in hPepT1 were created by replacing each residue within TMS5 with a cysteine. HEK293 cells were then transfected with each mutated protein and the steady-state protein level, [3H]Gly-Sar uptake activity, and sensitivity to the MTS reagents were measured. S164C-, L168C-, G173C-, and I179C-hPepT1 were not expressed on the plasma membrane. Y167C-, N171C-, and S174C-hPepT1 showed </=25% Gly-Sar uptake when compared with WT-hPepT1. P182C-hPepT1 showed approximately 40% specific activity whereas all the remaining transporters, although still sensitive to single cysteine mutations, exhibited more than 50% specific activity when compared to WT-hPepT1. The activity of F166C-, L176C-, S177C-, T178C-, I180C-, T181C-, and P182C-hPepT1 was partially inhibited, while the activity of F163C- and I170C-hPepT1 was completely inhibited by 2.5mM MTSEA. F163C, I165C, F166C, A169C, I170C, S177C, T181C, and P182C were clearly accessible to 1mM MTSET. Overall, these results suggest that TMS5 lines the putative aqueous channel and is slightly tilted from the vertical axis of the channel, with the exofacial half forming a classical amphipathic alpha-helix and the cytoplasmic half being highly solvent accessible.

Mutagenesis and Cysteine Scanning of Transmembrane Domain 10 of the Human Dipeptide Transporter

PurposeThe human dipeptide transporter (hPEPT1) facilitates transport of dipeptides and drugs from the intestine into the circulation. The role of transmembrane domain 10 (TMD10) of hPEPT1 in substrate translocation was investigated using cysteine-scanning mutagenesis with 2-Trimethylammonioethyl methanethiosulfonate (MTSET).MethodsEach amino acid in TMD10 was mutated individually to cysteine, and transport of [3H]Gly-Sar was evaluated with and without MTSET following transfection of each mutant in HEK293 cells. Similar localization and expression levels of wild type (WT) hPEPT1 and all mutants were confirmed by immunostaining and biotinylation followed by western blot analysis.ResultsE595C- and G594C-hPEPT1 showed negligible Gly-Sar uptake. E595D-hPEPT1 showed similar uptake to WT-hPEPT1, but E595K- and E595R-hPEPT1 did not transport Gly-Sar. Double mutations E595K/R282E and E595R/R282E did not restore uptake. G594A-hPEPT1 showed similar uptake to WT-hPEPT1, but G594V-hPEPT1 eliminated uptake. Y588C-hPEPT1 showed uptake of 20% that of WT-hPEPT1. MTSET modification supported a model of TMD10 with an amphipathic helix from I585 to V600 and increased solvent accessibility from T601 to F605.ConclusionsOur results suggest that G594 and E595 in TMD10 of hPEPT1 have key roles in substrate transport and that Y588 may have an important secondary mechanistic role.

Cysteine scanning of transmembrane domain three of the human dipeptide transporter: Implications for substrate transport

The human intestinal dipeptide transporter (hPepT1) transports dipeptides and pharmacologically active drugs from the intestine to the blood. The role of transmembrane domain 3 (TMD3) of hPepT1 was studied using cysteine-scanning mutagenesis and methane thiosulfonate (MTS) cysteine modification. Each amino acid in TMD3 was individually mutated to a cysteine and Gly–Sar uptake by each mutated and modified transporter was determined relative to wild-type hPepT1. Uptake data for mutated transporters modified with the lipid-insoluble cysteine-modifying reagent MTSET suggested tilting of TMD3 relative to the substrate translocation pathway; the extracellular region of TMD3 showed little MTSET reactivity, indicative of solvent inaccessibility, whereas the intracellular part of TMD3 was relatively solvent accessible. Modification at 10 positions of TMD3 with MTSEA, a lipid-soluble cysteine-modifying reagent, gave unusual and statistically significant increases in Gly–Sar uptake relative to untreated mutants. We interpret these data in terms of the spatial properties of the hPepT1 substrate translocation channel and possible interactions of TMD3 with other transmembrane domains.

Nucleoside and nucleotide stimulation of fluid secretion in the pigmented rabbit conjunctiva.

Pigmented rabbit conjunctival epithelium is capable of secreting fluid at a rate of 4.3 ± 0.2 µl/hr/cm2.1 Active chloride secretion across the epithelium, which in the case of conjunctiva is modulated by second messengers such as cAMP,2 Ca2+,2 protein kinase C (PKC),2,3 and purine and pyrimidine nucleotides,4 is proposed to be a major driving force for this vectorial fluid movement.5 Several of these second messengers also stimulate fluid secretion across the corneal epithelium,6 retinal pigment epithelium,7 and tracheal epithelium.8 Further, we have reported previously that 8Br-cAMP is an efficient stimulant to fluid secretion in the pigmented rabbit conjunctiva.1

Inhibitors of the Neutral Amino Acid Transporters ASCT1 and ASCT2 Are Effective in In Vivo Models of Schizophrenia and Visual Dysfunction

The N-methyl-d-aspartate receptor coagonist d-serine is a substrate for the neutral amino acid transporters ASCT1 and ASCT2, which may regulate its extracellular levels in the central nervous system (CNS). We tested inhibitors of ASCT1 and ASCT2 for their effects in rodent models of schizophrenia and visual dysfunction, which had previously been shown to be responsive to d-serine. L-4-fluorophenylglycine (L-4FPG), L-4-hydroxyPG (L-4OHPG), and L-4-chloroPG (L-4ClPG) all showed high plasma bioavailability when administered systemically to rats and mice. L-4FPG showed good brain penetration with brain/plasma ratios of 0.7–1.4; however, values for L-4OHPG and L-4ClPG were lower. Systemically administered L-4FPG potently reduced amphetamine-induced hyperlocomotion in mice, whereas L-4OHPG was 100-fold less effective and L-4ClPG inactive at the doses tested. L-4FPG and L-4OHPG did not impair visual acuity in naive rats, and acute systemic administration of L-4FPG significantly improved the deficit in contrast sensitivity in blue light–treated rats caused by retinal degeneration. The ability of L-4FPG to penetrate the brain makes this compound a useful tool to further evaluate the function of ASCT1 and ASCT2 transporters in the CNS.

Ethanol Inhibits Functional Activity of the Human Intestinal Dipeptide Transporter hPepT1 Expressed in Xenopus Oocytes

BACKGROUND The pathological effects of high alcohol (ethanol) consumption on gastrointestinal and hepatic systems are well recognized. However, the effects of ethanol intake on gastric and intestinal absorption and transport systems remain unclear. The present study investigates the effects of ethanol on the human peptide transporter 1 (hPepT1) which mediates the transport of di-and tripeptides as well as several orally administered peptidomimetic drugs such as beta-lactam antibiotics (e.g., penicillin), angiotensin-converting enzyme inhibitors, the anti-neoplastic agent bestatin, and prodrugs of acyclovir. METHODS Xenopus oocytes were injected with hPepT1 cRNA and incubated for 3 to 10 days. Currents induced by glycyl-sarcosine (Gly-Sar), Ala-Ala (dipeptides), penicillin and enalapril measured in the presence or absence of ethanol were determined using an 8-channel 2-electrode voltage clamp system, with a membrane potential of -70 mV and 11 voltage steps of 100 milliseconds (from +50 mV to -150 mV in -20 mV increments). RESULTS Ethanol (200 mM) inhibited Gly-Sar and Ala-Ala currents by 42 and 30%, respectively, with IC(50)s of 184 and 371 mM, respectively. Ethanol reduced maximal transport capacity (I(max)) of hPepT1 for Gly-Sar without affecting Gly-Sar binding affinity (K(0.5) and Hill coefficient). Penicillin- and enalapril-induced currents were significantly less than those induced by dipeptides and were not inhibited by ethanol. CONCLUSION Ethanol significantly reduced transport of dipeptides via a reduction in transport capacity, rather than competing for binding sites in hPepT1. Ethanol inhibition or alteration of transport function may be a primary causative factor contributing to both the nutritional deficits as well as the immunological deficiencies that many alcoholics experience including alcohol liver disease and brain damage.