Functional characterization of a glutamate/aspartate transporter from the mosquito Aedes aegypti

Self Cite

Section: Preparation Of Crnamentioning

confidence: 99%

Section: Preparation Of Crnamentioning

confidence: 99%

Characterization of a blood-meal-responsive proton-dependent amino acid transporter in the disease vector, Aedes aegypti

Evans

Aimanova

Gill

2009

Self Cite

“…A total of more than a dozen Na + -coupled amino acid transporters have been cloned by Gill's group (Umesh et al, 2003), Boudko and Harvey's group and others (reviewed by Boudko et al, 2005b). When expressed in Xenopus oocytes, the five NATs from mosquito larvae exhibited characteristic profiles for uptake of the 20 structural amino acids.…”

Section: Drosophila Nhas Are Plasma Membrane Proteinsmentioning

confidence: 99%

Voltage coupling of primary H+ V-ATPases to secondary Na+- or K+-dependent transporters

Harvey

2009

SUMMARY This review provides alternatives to two well established theories regarding membrane energization by H+ V-ATPases. Firstly, we offer an alternative to the notion that the H+ V-ATPase establishes a protonmotive force (pmf) across the membrane into which it is inserted. The term pmf, which was introduced by Peter Mitchell in 1961 in his chemiosmotic hypothesis for the synthesis of ATP by H+ F-ATP synthases, has two parts, the electrical potential difference across the phosphorylating membrane, Δψ, and the pH difference between the bulk solutions on either side of the membrane, ΔpH. The ΔpH term implies three phases – a bulk fluid phase on the H+ input side, the membrane phase and a bulk fluid phase on the H+ output side. The Mitchell theory was applied to H+ V-ATPases largely by analogy with H+ F-ATP synthases operating in reverse as H+ F-ATPases. We suggest an alternative, voltage coupling model. Our model for V-ATPases is based on Douglas B. Kell's 1979 `electrodic view' of ATP synthases in which two phases are added to the Mitchell model – an unstirred layer on the input side and another one on the output side of the membrane. In addition, we replace the notion that H+ V-ATPases normally acidify the output bulk solution with the hypothesis, which we introduced in 1992, that the primary action of a H+ V-ATPase is to charge the membrane capacitance and impose a Δψ across the membrane; the translocated hydrogen ions (H+s) are retained at the outer fluid–membrane interface by electrostatic attraction to the anions that were left behind. All subsequent events, including establishing pH differences in the outside bulk solution, are secondary. Using the surface of an electrode as a model, Kell's`electrodic view' has five phases – the outer bulk fluid phase, an outer fluid–membrane interface, the membrane phase, an inner fluid–membrane interface and the inner bulk fluid phase. Light flash,H+ releasing and binding experiments and other evidence provide convincing support for Kell's electrodic view yet Mitchell's chemiosmotic theory is the one that is accepted by most bioenergetics experts today. First we discuss the interaction between H+ V-ATPase and the K+/2H+ antiporter that forms the caterpillar K+ pump, and use the Kell electrodic view to explain how the H+s at the outer fluid–membrane interface can drive two H+ from lumen to cell and one K+ from cell to lumen via the antiporter even though the pH in the bulk fluid of the lumen is highly alkaline. Exchange of outer bulk fluid K+ (or Na+) with outer interface H+ in conjunction with (K+ or Na+)/2H+ antiport, transforms the hydrogen ion electrochemical potential difference, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{{\mu}}_{\mathrm{H}}\) \end{document}, to a K+electrochemical potential difference, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{{\mu}}_{\mathrm{K}}\) \end{document} or a Na+electrochemical potential difference, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{{\mu}}_{\mathrm{Na}}\) \end{document}. The \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{{\mu}}_{\mathrm{K}}\) \end{document} or \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{{\mu}}_{\mathrm{Na}}\) \end{document} drives K+- or Na+-coupled nutrient amino acid transporters (NATs), such as KAAT1(K+ amino acid transporter 1), which moves Na+ and an amino acid into the cell with no H+s involved. Examples in which the voltage coupling model is used to interpret ion and amino acid transport in caterpillar and larval mosquito midgut are discussed.

“…gambiae larvae using standard differential centrifugation techniques (Umesh et al, 2003). The isolated membranes were treated with sample loading buffer NuPAGE ® LDS (Invitrogen, Carlsbad, CA, USA), 25 g were loaded per lane on a 4%-12% Bis-Tris polyacrylamide gradient gel (Invitrogen) and the proteins were separated by electrophoresis under reducing conditions.…”

Section: Western Blot Analysis Of Agnha1 Antibodymentioning

confidence: 99%

Molecular cloning, phylogeny and localization of AgNHA1: the first Na+/H+ antiporter (NHA) from a metazoan,Anopheles gambiae

Rheault

Okech

Keen

et al. 2007

SUMMARY We have cloned a cDNA encoding a new ion transporter from the alimentary canal of larval African malaria mosquito, Anopheles gambiae Giles sensu stricto. Phylogenetic analysis revealed that the corresponding gene is in a group that has been designated NHA, and which includes(Na+ or K+)/H+ antiporters; so the novel transporter is called AgNHA1. The annotation of current insect genomes shows that both AgNHA1 and a close relative, AgNHA2, belong to the cation proton antiporter 2 (CPA2) subfamily and cluster in an exclusive clade of genes with high identity from Aedes aegypti, Drosophila melanogaster, D. pseudoobscura, Apis mellifera and Tribolium castaneum. Although NHA genes have been identified in all phyla for which genomes are available, no NHA other than AgNHA1 has previously been cloned,nor have the encoded proteins been localized or characterized. The AgNHA1 transcript was localized in An. gambiae larvae by quantitative real-time PCR (qPCR) and in situ hybridization. AgNHA1 message was detected in gastric caeca and rectum, with much weaker transcription in other parts of the alimentary canal. Immunolabeling of whole mounts and longitudinal sections of isolated alimentary canal showed that AgNHA1 is expressed in the cardia, gastric caeca, anterior midgut, posterior midgut, proximal Malpighian tubules and rectum, as well as in the subesophageal and abdominal ganglia. A phylogenetic analysis of NHAs and KHAs indicates that they are ubiquitous. A comparative molecular analysis of these antiporters suggests that they catalyze electrophoretic alkali metal ion/hydrogen ion exchanges that are driven by the voltage from electrogenic H+ V-ATPases. The tissue localization of AgNHA1 suggests that it plays a key role in maintaining the characteristic longitudinal pH gradient in the lumen of the alimentary canal of An. gambiae larvae.