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To test whether the rabbit proton-coupled peptide transporter PepT1 is a multimer, we have employed a combination of transport assays, luminometry and site-directed mutagenesis. A functional epitope-tagged PepT1 construct (PepT1-FLAG) was co-expressed in Xenopus laevis oocytes with a non-functional but normally trafficked mutant form of the same transporter (W294F-PepT1). The amount of PepT1-FLAG cRNA injected into the oocytes was kept constant, while the amount of W294F-PepT1 cRNA was increased over the mole fraction range of 0 to 1. The uptake of [(3)H]-D: -Phe-L: -Gln into the oocytes was measured at pH(out) 5.5, and the surface expression of PepT1-FLAG was quantified by luminometry. As the mole fraction of injected W294F-PepT1 increased, the uptake of D: -Phe-L: -Gln decreased. This occurred despite the surface expression of PepT1-FLAG remaining constant, and so we can conclude that PepT1 must be a multimer. Assuming that PepT1 acts as a homomultimer, the best fit for the modelling suggests that PepT1 could be a tetramer, with a minimum requirement of two functional subunits in each protein complex. Western blotting also showed the presence of higher-order complexes of PepT1-FLAG in oocyte membranes. It should be noted that we cannot formally exclude the possibility that PepT1 interacts with unidentified Xenopus protein(s). The finding that PepT1 is a multimer has important implications for the molecular modelling of this protein.

We test the hypothesis that lysinuric protein intolerance (LPI), a rare autosomal recessive defect of cationic amino acid transport, results from the absence of the recently described y+L amino acid transporter. We compare fluxes of lysine (1 microM) into erythrocytes of normal subjects with those of patients homozygous for the LPI mutation. No significant differences in fluxes through system y+L in normal or LPI cells were found, excluding the possibility that system y+L cannot be expressed in patients with LPI. Reasons for supposing that there may be tissue-specific processing of two recently described genes encoding the y+L transporter are discussed. Polymerase chain reaction measurement of expression of these two genes in an erythroleukemic cell line suggests that alternatively there may be an as-yet-unidentified additional member of this gene family.

The proton dependence of the transport of three labelled, hydrolysis-resistant synthetic dipeptides carrying a net charge of -1, 0 or +1 has been investigated in a brush border membrane vesicle preparation obtained from rat renal cortex. Cross-inhibition studies are consistent with the transport of all peptides studied being through a single system. The extent and time course of uptake in response to an inwardly directed electrochemical gradient of protons differed for each peptide. For the cationic peptide D-Phe-L-Lys this gradient did not stimulate the initial rate of uptake, while for the neutral dipeptide D-Phe-L-Ala and the anionic peptide D-Phe-L-Glu stimulation was observed. However, the effect on D-Phe-L-Glu was more marked than that on D-Phe-L-Ala and the proton activation differed for these two peptides. The calculated Hill coefficients for the two proton-dependent peptides were 1.14 +/- 0.16 and 2.15 +/- 0.10 for D-Phe-L-Ala and D-Phe-L-Glu, respectively, providing evidence that the stoichiometry of proton:peptide cotransport is different for each peptide (0:1, 1:1 and 2:1 for D-Phe-L-Lys, D-Phe-L-Ala and D-Phe-L-Glu respectively); studies on energetics are compatible with this conclusion. The physiological and molecular implications of this model are discussed, as are the applicability of the conclusions to secondary active transport systems more generally.

We have studied chloride influx and efflux in a highly purified preparation of type II cells freshly isolated from adult guinea-pig lung using 36Cl-. Chloride uptake was time-dependent, saturable (Km < 10 mM) and was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; Ki approximately 80 microM). In the absence of external chloride (substituted by gluconate), 36Cl- uptake exhibited an overshoot above equilibrium. The rate of 36Cl- entry was strongly inhibited by addition of external nitrate; sulphate was a weaker inhibitor. 36Cl- efflux was stimulated by external bromide > bicarbonate > or = chloride > or = citrate; and was inhibited by propionate > acetate > oxalate. Although the "chloride channel blocker" 4-nitro-2-(3-phenylpropylamino)benzoate (0.14 mM) caused an inhibition, 36Cl- influx did not appear to be electrogenic. These data are compatible with the existence of a substantial electroneutral anion-exchange pathway for chloride transport in freshly isolated adult type II pneumocytes.

Lysine influx (2 microM) into activated human B and T lymphocytes through transport systems y+ and y+L was measured. In activated T cells very substantial activation of system y+ was detected; system y+L was also activated in these cells but with a slower time course and to a smaller extent. No stimulation of either system was found in activated B cells. The time course of activation of system y+ precisely matched the expression of the T cell early antigen gene described by MacLeod et al, 1990. The functional significance of these observations with respect to L-arginine transport and nitric oxide synthesis in activated T lymphocytes is discussed.

36Cl uptake by microvillus brush border membrane vesicles prepared from normal term human placenta has been studied using an ion exchange column assay and a novel method to correct for vesicle recovery. Uptake of this anion, which was largely into an osmotically active space, was time dependent with a half-time of approximately 4 min at 4 degrees C. DIDS at a maximal inhibitory concentration inhibited 36Cl uptake by approximately 40%; furosemide (10(-4)M) showed a similar degree of inhibition and the effects of the two drugs appeared not to be additive. Anions including Cl, Br, I, NO3, salicylate and SCN reduced 36Cl uptake, the latter two being the most potent; gluconate was without effect. Inhibition by maximal inhibitory concentrations of DIDS and the anions SCN and salicylate was not additive. Neither the DIDS-sensitive nor the SCN-sensitive component of 36Cl uptake was influenced by membrane potential; in contrast the inhibitor-insensitive component was increased by an inside positive membrane potential. These findings suggest that under the conditions employed an electroneutral anion exchange system is responsible for approximately half of the chloride flux across this plasma membrane.