Voltage- and substrate-dependent interactions between sites in putative re-entrant domains of a Na+-coupled phosphate cotransporter

A common structural feature characterises sodium-coupled inorganic phosphate cotransporters of the SLC34 family (NaPi-IIa/b/c): a pair of inverted regions in the N- and C-terminal halves of the protein. These regions are hypothesised to contain re-entrant domains that associate to allow alternating access of the substrates from either side of the membrane. To investigate if these domains interact during the NaPi-II transport cycle, we introduced novel cysteines at three functionally important sites associated with the predicted re-entrant domains of the flounder NaPi-IIb for the purpose of fluorescent labelling and cross-linking. Single and double mutants were expressed in Xenopus oocytes and their function analysed using electrophysiological and real-time fluorometric assays. The substitution at the cytosolic end of the first re-entrant domain induced a large hyperpolarizing shift in the voltage dependence of steady-state and presteady-state kinetics, whereas the two substitutions at the external face were less critical. By using Cu-phenanthroline to induce disulfide bridge formation, we observed a loss of transport activity that depended on the presence of sodium in the incubation medium. This suggested that external sodium increased the probability of NaPi-IIb occupying a conformation that favours interaction between sites in the re-entrant domains. Furthermore, voltage-dependent fluorescence data supported the hypothesis that a localised interaction between the two domains occurs that depends on the membrane potential and substrate present: we found that the fluorescence intensity reported by a labelled cysteine in one domain was dependent on the side chain substituted at a functionally critical site in the opposed domai

Conformational Dynamics of hSGLT1 during Na+/Glucose Cotransport

This study examines the conformations of the Na+/glucose cotransporter (SGLT1) during sugar transport using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes. The mutant exhibited similar steady-state and presteady-state kinetics as wild-type SGLT1, and labeling of Cys507 by tetramethylrhodamine-6-maleimide had no effect on kinetics. Our strategy was to record changes in charge and fluorescence in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin. In Na+ buffer, step jumps in membrane voltage elicited presteady-state currents (charge movements) that decay to the steady state with time constants τmed (3–20 ms, medium) and τslow (15–70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (ΔF). The charge vs. voltage (Q-V) and fluorescence vs. voltage (ΔF-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: zδ (apparent valence of voltage sensor) ≈ 1; and V0.5 (midpoint voltage) between −15 and −40 mV. Sugar induced an inward current (Na+/glucose cotransport), and reduced maximal charge (Qmax) and fluorescence (ΔFmax) with half-maximal concentrations (K0.5) of 1 mM. Increasing [αMDG]o also shifted the V0.5 for Q and ΔF to more positive values, with K0.5's ≈ 1 mM. The major difference between Q and ΔF was that at saturating [αMDG]o, the presteady-state current (and Qmax) was totally abolished, whereas ΔFmax was only reduced 50%. Phlorizin reduced both Qmax and ΔFmax (Ki ≈ 0.4 μM), with no changes in V0.5's or relaxation time constants. Simulations using an eight-state kinetic model indicate that external sugar increases the occupancy probability of inward-facing conformations at the expense of outward-facing conformations. The simulations predict, and we have observed experimentally, that presteady-state currents are blocked by saturating sugar, but not the changes in fluorescence. Thus we have isolated an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na+/sugar cotransport (saturating voltage and external Na+ and sugar concentrations) is the slow release of Na+ from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation

Kidney-synthesized erythropoietin is the main source for the hypoxia-induced increase in plasma erythropoietin in adult humans

PURPOSE Erythropoietin (EPO) is mainly synthesized within renal peritubular fibroblasts, and also other tissues such as the liver possess the ability. However, to what extent non-kidney produced EPO contributes to the hypoxia-induced increase in circulating EPO in adult humans remains unclear. METHODS We aimed to quantify this by assessing the distribution of EPO glycoforms which are characterized by posttranslational glycosylation patterns specific to the synthesizing cell. The analysis was performed on samples obtained in seven healthy volunteers before, during and after 1 month of sojourn at 3,454 m altitude. RESULTS Umbilical cord (UC) plasma served as control. As expected a peak (p < 0.05) in urine (2.3 ± 0.5-fold) and plasma (3.3 ± 0.5-fold) EPO was observed on day 1 of high-altitude exposure, and thereafter the concentration decreased for the urine sample obtained after 26 days at altitude, but remained elevated (p < 0.05) by 1.5 ± 0.2-fold above the initial sea level value for the plasma sample. The EPO glycoform heterogeneity, in the urine samples collected at altitude, did not differ from values at sea level, but were markedly lower (p < 0.05) than the mean percent migrated isoform (PMI) for the umbilical cord samples. CONCLUSION Our studies demonstrate (1) UC samples express a different glycoform distribution as compared to adult humans and hence illustrates the ability to synthesis EPO in non-kidney cells during fetal development (2) as expected hypoxia augments circulating EPO in adults and the predominant source here for remains being kidney derived

Physiological, biochemical, anthropometric and biomechanical influences on exercise economy in humans

Inter-individual variation in running and cycling exercise economy (EE) remains unexplained although studied for more than a century. This study is the first to comprehensively evaluate the importance of biochemical, structural, physiological, anthropometric, and biomechanical influences on running and cycling EE within a single study. In 22 healthy males (VO2 max range 45.5 to 72.1 ml.min(-1) .kg(-1) ) no factor related to skeletal muscle structure (% slow twitch fibre content, number of capillaries per fibre), mitochondrial properties (volume density, oxidative capacity, or mitochondrial efficiency) or protein content (UCP3 and MFN2 expression) explained variation in cycling and running EE among subjects. In contrast, biomechanical variables related to vertical displacement correlated well with running EE, but were not significant when taking body weight into account. Thus, running EE and body weight were correlated (R(2) = 0.94; P < 0.001), but was lower for cycling EE (R(2) = 0.23; P < 0.023). To separate biomechanical determinants of running EE we contrasted individual running and cycling EE considering that during cycle ergometer exercise the biomechanical influence on EE would be small because of the fixed movement pattern. Differences in cycling and running exercise protocols, e.g., related to biomechanics, play however only a secondary role in determining EE. There was no evidence for an impact of structural or functional skeletal muscle variables on EE. Body weight was the main determinant of EE explaining 94% of variance in running EE, although more than 50% of the variability of cycling EE remains unexplained

Using lithium to probe sequential cation interactions with GAT1

Li(+) interacts with the Na(+)/Cl(-)-dependent GABA transporter, GAT1, under two conditions: in the absence of Na(+) it induces a voltage-dependent leak current; in the presence of Na(+) and GABA, Li(+) stimulates GABA-induced steady-state currents. The amino acids directly involved in the interaction with the Na(+) and Li(+) ions at the so-called "Na2" binding site have been identified, but how Li(+) affects the kinetics of GABA cotransport has not been fully explored. We expressed GAT1 in Xenopus oocytes and applied the two-electrode voltage clamp and (22)Na uptake assays to determine coupling ratios and steady-state and presteady-state kinetics under experimental conditions in which extracellular Na(+) was partially substituted by Li(+). Three novel findings are: 1) Li(+) reduced the coupling ratio between Na(+) and net charge translocated during GABA cotransport; 2) Li(+) increased the apparent Na(+) affinity without changing its voltage dependence; 3) Li(+) altered the voltage dependence of presteady-state relaxations in the absence of GABA. We propose an ordered binding scheme for cotransport in which either a Na(+) or Li(+) ion can bind at the putative first cation binding site (Na2). This is followed by the cooperative binding of the second Na(+) ion at the second cation binding site (Na1) and then binding of GABA. With Li(+) bound to Na2, the second Na(+) ion binds more readily GAT1, and despite a lower apparent GABA affinity, the translocation rate of the fully loaded carrier is not reduced. Numerical simulations using a nonrapid equilibrium model fully recapitulated our experimental findings

Lactate oxidation in human skeletal muscle mitochondria

Lactate is an important intermediate metabolite in human bioenergetics and is oxidized in many different tissues including the heart, brain, kidney, adipose tissue, liver, and skeletal muscle. The mechanism(s) explaining the metabolism of lactate in these tissues, however, remains unclear. Here, we analyze the ability of skeletal muscle to respire lactate by using an in situ mitochondrial preparation that leaves the native tubular reticulum and subcellular interactions of the organelle unaltered. Skeletal muscle biopsies were obtained from vastus lateralis muscle in 16 human subjects. Samples were chemically permeabilized with saponin, which selectively perforates the sarcolemma and facilitates the loss of cytosolic content without altering mitochondrial membranes, structure, and subcellular interactions. High-resolution respirometry was performed on permeabilized muscle biopsy preparations. By use of four separate and specific substrate titration protocols, the respirometric analysis revealed that mitochondria were capable of oxidizing lactate in the absence of exogenous LDH. The titration of lactate and NAD(+) into the respiration medium stimulated respiration (P ≤ 0.003). The addition of exogenous LDH failed to increase lactate-stimulated respiration (P = 1.0). The results further demonstrate that human skeletal muscle mitochondria cannot directly oxidize lactate within the mitochondrial matrix. Alternately, these data support previous claims that lactate is converted to pyruvate within the mitochondrial intermembrane space with the pyruvate subsequently taken into the mitochondrial matrix where it enters the TCA cycle and is ultimately oxidized