The major facilitator superfamily is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations including both the outward and inward facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.

To investigate possible effects of glucose concentration, dwell time, and peritoneal reabsorption on the combined diffusive and convective peritoneal solute transport, dialysate to plasma concentration ratios (D/P) and solute clearances were evaluated for 6-h peritoneal dwell studies with 1.36, 2.27, and 3.86% glucose solutions. The diffusive mass transport coefficient, KBD, and sieving coefficient, S, were estimated using the Babb-Randerson-Farrell model of peritoneal transport. Dialysate volumes over time and peritoneal reabsorption rates, KE, were assessed using radiolabeled iodinated serum albumin (RISA). The transport parameters were estimated with and without peritoneal reabsorption of solutes taken into account. To test the stability of the transport parameters throughout a single peritoneal dwell, KBD and S values were estimated for the initial 3-120 min, the final 120-360 min, and the entire 3-360 min dwell period for dialysis with 3.86% glucose solution. The transport parameters did not differ between the three dialysis fluids although clearances of small solutes were higher with the 3.86% solution. Values of KBD, but not S, were dependent on the correction for peritoneal reabsorption of solutes. Computer simulations showed that S could be estimated even with the 1.36% glucose solution. A significant change of the transport parameters, with increased values of KBD during the initial period of the dwell, was found for urea, potassium, sodium, and total protein during dialysis with the 3.86% solution. S values for urea and potassium were close to 1 during the initial period whereas unphysical (higher than 1) S values were found for the whole dwell period. The transient increase of KBD during the initial part of the dwell may reflect changes in the peritoneal barrier possibly induced by fresh dialysis fluid. In conclusion, the transport parameters KBD and S are not influenced by the concentration of glucose in the dialysis fluid. Moreover, the estimation of KBD but not of S is dependent on the assumed rate of peritoneal reabsorption. Finally, the current results challenge the assumption that KBD and S are constant throughout a peritoneal dialysis exchange.

During continuous ambulatory peritoneal dialysis (CAPD) treatment, loss of ultrafiltration capacity (UFC) is a common complication that can be associated with increased peritoneal fluid absorption rate. The aim of the present study was to investigate the relative importance of lymphatic absorption for total peritoneal fluid absorption in patients with permanent loss of UFC associated with a high peritoneal absorption rate (K_E, ml/min; high-K_E group, n = 4). Clinically stable CAPD patients (n = 23) as well as patients with loss of UFC associated with increased diffusive mass transport coefficients (K_BD, ml/min; high-K_BD group, n = 8) served as control groups. The patients were investigated with a 6-hour dwell study with 3.86% glucose solution. The total fluid absorption rate was estimated by the disappearance rate (K_E) of ¹³¹I-radioiodinated human serum albumin (RISA) from the peritoneal cavity, and the lymphatic absorption rate was estimated by the rate of RISA appearance in plasma (K_PP, ml/min). The values of K_E and K_PP in the high-K_E group (4.65 ± 0.93 and 0.42 ± 0.31 ml/min, respectively) were markedly higher than in the clinically stable CAPD patients (1.77 ± 0.60 and 0.15 ± 0.06 ml/min, respectively; both p < 0.001 vs. the high-K_E group). In the high-K_BD group, K_E was lower (2.19 ± 0.38 ml/min, p < 0.001) compared to the high-K_E group, whereas K_PP was similar (0.26 ± 0.09 ml/min, NS). The fraction of K_E which could be accounted for by K_PP was on average only 9 + 5% in the high-K_E group and did not differ from the fractions in the clinically stable patients or in the high-K_BD group (9 ± 5 and 12 ± 4%, respectively). In 5 patients in whom plasma RISA activity was measured for 24 h from the beginning of the 6-hour dwell study, a continuous increase of the RISA level in plasma was observed during this time period. We conclude that although K_PP was increased in patients with UFC loss associated with high K_E, it accounted for only a minor part of K_E. Furthermore, the relatively slow but prolonged appearance of RISA in plasma indicates that the interstitial compartment may serve as a reservoir of macromolecules which are slowly absorbed by local lymphatics. The present study supports previous findings that direct lymphatic absorption is only of relatively minor importance for the fluid absorption in peritoneal dialysis.