137 research outputs found
Voltage Clamp Fluorometric Measurements on a Type II Na+-coupled Pi Cotransporter: Shedding Light on Substrate Binding Order
Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na+-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637–651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (ΔF), which suggested that this site lies in an environment that is affected by conformational change in the protein. ΔF was substrate dependent (no ΔF was detectable in 0 mM Na+) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1–18). In the new model, two (rather than one) Na+ ions precede Pi binding, and only the second Na+ binding transition is voltage dependent. Moreover, we show that Li+, which does not drive cotransport, interacts with the first Na+ binding transition. The results were incorporated in a new model of the transport cycle of type II Na+/Pi cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes
Transport Function of the Renal Type IIa Na+/Pi Cotransporter Is Codetermined by Residues in Two Opposing Linker Regions
Two highly similar regions in the predicted first intracellular (ICL-1) and third extracellular loop (ECL-3) of the type IIa Na+/Pi cotransporter (NaPi-IIa) have been shown previously to contain functionally important sites by applying the substituted cysteine accessibility method (SCAM). Incubation in methanethiosulfonate (MTS) reagents of mutants that contain novel cysteines in both loops led to full inhibition of cotransport activity. To elucidate further the role these regions play in defining the transport mechanism, a double mutant (A203C-S460C) was constructed with novel cysteines in each region. The effect of cysteine modification by different MTS reagents on two electrogenic transport modes (leak and cotransport) was investigated. MTSEA (2-aminoethyl MTS hydrobromide) and MTSES (MTS ethylsulfonate) led to full inhibition of cotransport and increased the leak, whereas incubation in MTSET (2-[trimethylammonium]ethyl MTS bromide) inhibited only cotransport. The behavior of other double mutants with a cysteine retained at one site and hydrophobic or hydrophilic residues substituted at the other site, indicated that most likely only Cys-460 was modifiable, but the residue at Ala-203 was critical for conferring the leak and cotransport mode behavior. Substrate interaction with the double mutant was unaffected by MTS exposure as the apparent Pi and Na+ affinities for Pi-induced currents and respective activation functions were unchanged after cysteine modification. This suggested that the modified site did not interfere with substrate recognition/binding, but prevents translocation of the fully loaded carrier. The time-dependency of cotransport loss and leak growth during modification of the double cysteine mutant was reciprocal, which suggested that the modified site is a kinetic codeterminant of both transport modes. The behavior is consistent with a kinetic model for NaPi-IIa that predicts mutual exclusiveness of both transport modes. Together, these findings suggest that parts of the opposing linker regions are associated with the NaPi-IIa transport pathway
Structure–Function Relations of the First and Fourth Extracellular Linkers of the Type IIa Na+/Pi Cotransporter: II. Substrate Interaction and Voltage Dependency of Two Functionally Important Sites
Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages
Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter: I. Cysteine Scanning Mutagenesis
The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport
The Tegument of the Human Parasitic Worm Schistosoma mansoni as an Excretory Organ: The Surface Aquaporin SmAQP Is a Lactate Transporter
Adult schistosomes are intravascular parasites that metabolize imported glucose largely via glycolysis. How the parasites get rid of the large amounts of lactic acid this generates is unknown at the molecular level. Here, we report that worms whose aquaporin gene (SmAQP) has been suppressed using RNAi fail to rapidly acidify their culture medium and excrete less lactate compared to controls. Functional expression of SmAQP in Xenopus oocytes demonstrates that this protein can transport lactate following Michaelis-Menten kinetics with low apparent affinity (Km = 41±5. 8 mM) and with a low energy of activation (Ea = 7.18±0.7 kcal/mol). Phloretin, a known inhibitor of lactate release from schistosomes, also inhibits lactate movement in SmAQP-expressing oocytes. In keeping with the substrate promiscuity of other aquaporins, SmAQP is shown here to be also capable of transporting water, mannitol, fructose and alanine but not glucose. Using immunofluorescent and immuno-EM, we confirm that SmAQP is localized in the tegument of adult worms. These findings extend the proposed functions of the schistosome tegument beyond its known capacity as an organ of nutrient uptake to include a role in metabolic waste excretion
The Formaldehyde Masers in NGC 7538 and G29.96-0.02: VLBA, MERLIN, and VLA Observations
The 6 cm formaldehyde (H2CO) maser sources in the compact HII regions NGC
7538-IRS1 and G29.96-0.02 have been imaged at high resolution (beams < 50 mas).
Using the VLBA and MERLIN, we find the angular sizes of the NGC 7538 masers to
be ~10 mas (30 AU) corresponding to brightness temperatures ~10^8 K. The
angular sizes of the G29.96-0.02 masers are ~20 mas (130 AU) corresponding to
brightness temperatures ~10^7 K. Using the VLA, we detect 2 cm formaldehyde
absorption from the maser regions. We detect no emission in the 2 cm line,
indicating the lack of a 2 cm maser and placing limits on the 6 cm excitation
process. We find that both NGC 7538 maser components show an increase in
intensity on 5-10 year timescales while the G29.96-0.02 masers show no
variability over 2 years. A search for polarization provides 3-sigma upper
limits of 1% circularly polarized and 10% linearly polarized emission in NGC
7538 and of 15% circularly polarized emission in G29.96-0.02. A pronounced
velocity gradient of 28 km/s/arcsecond (1900 km/s/pc) is detected in the NGC
7538 maser gas.Comment: accepted to ApJ, 15 figures, 11 table
Improving the normalization of complex interventions: measure development based on normalization process theory (NoMAD): study protocol
<b>Background</b> Understanding implementation processes is key to ensuring that complex interventions in healthcare are taken up in practice and thus maximize intended benefits for service provision and (ultimately) care to patients. Normalization Process Theory (NPT) provides a framework for understanding how a new intervention becomes part of normal practice. This study aims to develop and validate simple generic tools derived from NPT, to be used to improve the implementation of complex healthcare interventions.<p></p>
<b>Objectives</b> The objectives of this study are to: develop a set of NPT-based measures and formatively evaluate their use for identifying implementation problems and monitoring progress; conduct preliminary evaluation of these measures across a range of interventions and contexts, and identify factors that affect this process; explore the utility of these measures for predicting outcomes; and develop an online users’ manual for the measures.<p></p>
<b>Methods</b> A combination of qualitative (workshops, item development, user feedback, cognitive interviews) and quantitative (survey) methods will be used to develop NPT measures, and test the utility of the measures in six healthcare intervention settings.<p></p>
<b>Discussion</b> The measures developed in the study will be available for use by those involved in planning, implementing, and evaluating complex interventions in healthcare and have the potential to enhance the chances of their implementation, leading to sustained changes in working practices
Translation of evidence-based Assistive Technologies into stroke rehabilitation: Users' perceptions of the barriers and opportunities
Background: Assistive Technologies (ATs), defined as "electrical or mechanical devices designed to help people recover movement", demonstrate clinical benefits in upper limb stroke rehabilitation; however translation into clinical practice is poor. Uptake is dependent on a complex relationship between all stakeholders. Our aim was to understand patients', carers' (P&Cs) and healthcare professionals' (HCPs) experience and views of upper limb rehabilitation and ATs, to identify barriers and opportunities critical to the effective translation of ATs into clinical practice. This work was conducted in the UK, which has a state funded healthcare system, but the findings have relevance to all healthcare systems. Methods. Two structurally comparable questionnaires, one for P&Cs and one for HCPs, were designed, piloted and completed anonymously. Wide distribution of the questionnaires provided data from HCPs with experience of stroke rehabilitation and P&Cs who had experience of stroke. Questionnaires were designed based on themes identified from four focus groups held with HCPs and P&Cs and piloted with a sample of HCPs (N = 24) and P&Cs (N = 8). Eight of whom (four HCPs and four P&Cs) had been involved in the development. Results: 292 HCPs and 123 P&Cs questionnaires were analysed. 120 (41%) of HCP and 79 (64%) of P&C respondents had never used ATs. Most views were common to both groups, citing lack of information and access to ATs as the main reasons for not using them. Both HCPs (N = 53 [34%]) and P&C (N = 21 [47%]) cited Functional Electrical Stimulation (FES) as the most frequently used AT. Research evidence was rated by HCPs as the most important factor in the design of an ideal technology, yet ATs they used or prescribed were not supported by research evidence. P&Cs rated ease of set-up and comfort more highly. Conclusion: Key barriers to translation of ATs into clinical practice are lack of knowledge, education, awareness and access. Perceptions about arm rehabilitation post-stroke are similar between HCPs and P&Cs. Based on our findings, improvements in AT design, pragmatic clinical evaluation, better knowledge and awareness and improvement in provision of services will contribute to better and cost-effective upper limb stroke rehabilitation. © 2014 Hughes et al.; licensee BioMed Central Ltd
Imputing HIV treatment start dates from routine laboratory data in South Africa: a validation study
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