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First published online May 1, 2009
Journal of Experimental Biology 212, 1559-1567 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.027383
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Cloning and functional expression of the first eukaryotic Na+–tryptophan symporter, AgNAT6

Ella A. Meleshkevitch1, Marvin Robinson2, Lyudmila B. Popova2,3, Melissa M. Miller2, William R. Harvey2 and Dmitri Y. Boudko1,*

1 Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA
2 Whitney Laboratory for Marine Bioscience, University of Florida, St Augustine, FL 32080, USA
3 A. N. Belozersky Institute, Moscow State University, Moscow, 119899, Russia


Figure 1
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  		Fig. 1. Alignment of AgNAT6 with selected insect and bacterial nutrient amino acid transporters (NATs). Transmembrane domains (TMD1–12) and conserved structural features including substrate interaction sites were identified after alignment of AgNAT6 with selected insect and prokaryotic NATs including LeuTAa sequence (this figure), which is supported by structural alignment, and substrate docking (not shown). The alignment was generated by Geneious Pro 4.5 software (Biomatters, Auckland, New Zealand) with a minor manual improvement. Increasing background intensity indicates an increase in sequence similarity. Green triangles indicate substrate-binding sites, red and brown rhombuses are first and second Na+ interacting sites, purple squares represent Cl–-binding sites. Dark red arrows are transmembrane helices; yellow bars are sub-membrane (EL, extracellular loop; IL, intracellular loop) helices.

 

Figure 2
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  		Fig. 2. Phylogenetic position of AgNAT6 in the solute carrier family 6 (SLC6) tree. The tree includes 98 SLC6 members from seven completed genomes including one mammalian, three dipteran insect, one nematode and two prokaryotic genomes; two characterized lepidopteran NAT sequences were also added. The evolutionary history was inferred using the UPGMA method (Sneath and Sokal, 1973Go). The optimal tree with a sum of branch length=63.23 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) is shown next to the branches (Felsenstein, 1985Go). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965Go) and are in units of the number of amino acid substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter=1). All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (pairwise deletion option). There were a total of 566 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007Go). Lines show NCBI accession numbers followed by arbitrary definitions of obvious orthologs and cloned transporters (shown in bold). Abbreviations: Ae, Aedes aegypti; Ag, Anopheles gambiae; Ce, Caenorhabditis elegance; Dm, Drosophila melanogaster; Mm, Mus musculus; Ms, Manduca sexta; NTTs, neurotransmitter transporters. Discussed transporters are underlined. Invertebrate NATs are depicted by different font colors.

 

Figure 3
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  		Fig. 3. Electrochemical properties of AgNAT6 mechanism expressed in Xenopus laevis oocytes. (A) An example of substrate-induced currents obtained from a representative oocyte. All substrates were superfused at 1 mmol l–1 concentrations in 98 mmol l–1 NaCl media at –30 mV holding transmembrane voltage potential. (B) Normalized substrate-induced currents (bars are means ± s.d. for N>3 experiments and oocytes). (C) Unusual positive responses, which often but not always were observed after application of L-Phe and L-DOPA at 6–8 days after cRNA injection. (D) Ion dependency of a Trp-induced current. Millimolar concentrations of the major salt component of perfusion solutions are shown in the order of current traces above. (E)I–V plots of AgNAT6 interactions with Trp at specific compositions of inorganic ions; plots represent current–voltage relationships following subtraction of the Trp-independent current component. I–V plots relating to perfusion solutions containing different major salts are depicted by different line styles (see key). (F) Relative uptake ratios calculated after a 10 min exposure of the AgNAT6-injected (filled bars) and distilled water (DW)-injected (open bar) oocytes to specified isotope-labeled substrate (bars are normalized mean uptake ratio ±s.d., N=3).

 

Figure 4
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  		Fig. 4. Kinetic properties and pH dependency of AgNAT6. AgNAT6-mediated currents as a function of concentration of selected organic substrates (A) and sodium ions at 0.3 and 3 mmol l–1 concentrations of tryptophan (C). Curves are non-linear regression of the data calculated from the Hill equation: f=ax{eta}/(K0.5{eta}+x{eta}); 3 mmol l–1 Trp (filled circles), {eta}=0.98±0.14, K0.5=25.27±6.38 and 0.3 mmol l–1 Trp (open circles), {eta}=1.90±0.21, K0.5=38.46±3.47 in C. (B) Estimated affinity constants (bars are means ± s.d., N>3) and Hill constants (circles are means ± s.d., N>3) for selected organic substrates. (D) pH dependency of AgNAT6 (circles are means ± s.d., N>3).

 

Figure 5
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  		Fig. 5. AgNAT6 transcription in the alimentary canal and neuronal system of mosquito larvae. (A) In situ hybridization of AgNAT6 in the anterior and posterior portions of the whole-mount gut of 4th instar larvae. (B) Pattern of AgNAT6 hybridization in the larval head. (C) Strong hybridization signal in the neuropile of a larval abdominal ganglion. (D) More intense hybridization was detected in the gut from earlier 3rd instar larvae. The blue color represents the intensity of the hybridization signal. Scale bars are 200 µm. (E) Result of quantitative PCR assay of AgNAT6 transcript across different tissues and developmental stages of An. gambiae (data were normalized relative to values in whole larvae represented by the black bar; bars are means ± s.d.; N=3 for each data point). Abbreviations: amg, anterior midgut; ca, cardia; gc, gastric caeca; pmg, posterior midgut, a putative nutrient amino acid absorption site; Mt, Malpighian tubules; sg, salivary glands; r, rectum; cns, central nervous system; l, larvae; a, adult. The l-sg samples also include heart; a-integ samples include the ventral nerve cord and a-gut samples include the reproductive organs. Vertical scale is a log of relative transcript density.

 

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© The Company of Biologists Ltd 2009