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First published online August 3, 2006
Journal of Experimental Biology 209, 3183-3198 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02374

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Molecular characterization of the first aromatic nutrient transporter from the sodium neurotransmitter symporter family

Ella A. Meleshkevitch¹, Poincyane Assis-Nascimento^1,2, Lyudmila B. Popova^1,3, Melissa M. Miller¹, Andrea B. Kohn¹, Elizabeth N. Phung¹, Anita Mandal⁴, William R. Harvey^1,5 and Dmitri Y. Boudko^1,*

¹ The Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Blvd., St Augustine, FL 32080, USA
² Barry University, FL 33161, USA
³ A. N. Belozersky Institute, Moscow State University, Russia
⁴ Department of Biology, University of North Florida, FL 32224, USA
⁵ Department of Physiology and Functional Genomics, College of Medicine, University of Florida, FL 32610, USA

View larger version (40K): [in a new window]   Fig. 1. Physical map of SNF and NAT members in the An. gambiae genome. agSNF (A) represents the relative chromosomal distribution of the entire SNF population. agNATs (B) show the exact location of all agNATs on chromosome 3, which is expanded by a virtual map of particular genes on the right. Values beneath chromosomes represent chromosome numbers and the number of genes belonging to the agSNF or agNAT cluster on the corresponding chromosomes. agSERT is a serotonin transporter, the location of which is presently unknown.

View larger version (81K): [in a new window]   Fig. 2. Alignment and reconstruction of agNAT8 structure. (A) Sequence/structure alignment of characterized insect transporters relative to the first crystallized bacterial NAT from A. aeolicus, LeuTAa (Yamashita et al., 2005). (B) 2D structure of agNAT8 based on structural homology with the LeuTAa protein sequence. NCBI Accession no.: LeuTAa, NP_214423 (PDB no., 2A65); msKAAT1, AAC24190; msCAATCH1, AAF18560; aeAAT1, AAR08269; agNAT8, AAN40409. Filled and open stars represent putative cationic gates at extra- and intracellular interfaces, respectively. Squares indicate putative substrate binding sites; red and blue spheres outline sites, which interact with the first and second sodium ion respectively. Red and yellow boxes show putative glycosylation motifs and disulfide bridges, respectively. The 12 transmembrane domains are numbered 1-12.

View larger version (40K): [in a new window]   Fig. 3. A comparative Bayesian evolutionary dendrogram of selected SNF members. This tree includes SNF members from three prokaryote and five eukaryote genomes and the most characterized SNF members from various insects; species abbreviations and color codes are given in the lower right corner insert. Branch lengths correspond to an evolutionary distance scale of 0.1 mutation per site (mps). The numbers at the nodes are posterior probabilities. Node values in the range 0.95-0.99 are shown, whereas nodes with a probability less then 0.95 were collapsed, and probability values of 1 are not shown. The red arrow indicates the position of cloned agNAT8. White and black triangles correspond with apparent gene expansion and conservation nodes. metNTT, metazoan neurotransmitter transporters; miNAT, mammalian-insect nutrient amino acid transporters (NAT); iNAT, insect NATs; ceNAT, Caenorhabditis elegans NATs, archNAT, archaean NATs; bacNAT, bacterial, Bacillus cereus NATs.

View larger version (22K): [in a new window]   Fig. 4. Profile of agNAT8-mediated amino acid-induced currents. The upper line combines typical currents of agNAT8-expressing oocytes upon a trial application of selected substrates at 2 mmol l-1 concentrations. The lower histogram incorporates mean values of induced responses upon application of 1 mmol l-1 concentration of selected substrates normalized to Phe induced currents (values are mean + s.d.; N 3 recordings for at least three different oocytes for each data point). Holding potentials for all recordings were -50 mV. Standard abbreviations of amino acids and neurotransmitters are used. Arrows highlight the D-isomer of phenylalanine.

View larger version (14K): [in a new window]   Fig. 5. Ion dependency of agNAT8. Representative data from a typical ion substitution experiment. Different coloured lines represent the response upon application of 2 mmol l-1 phenylalanine in oocyte medium with different ion concentrations: yellow, 100 mmol l-1 LiCl; red, 98 mmol l-1 KCl; blue, 98 mmol l-1 sodium gluconate; green, 98 mmol l-1 NaCl. Holding potentials were -50 mV.

View larger version (29K): [in a new window]   Fig. 6. Electrical properties of agNAT8 transporter. (A) Families of current traces induced by square pulse polarization of oocyte membranes in a 98 mmol l-1 NaCl solution (left) and after application of 2 mmol l-1 phenylalanine (right) (Vhold, holding voltage=-50 mV, first pulse=+30 mV, last pulse=-120 mV; increment=-10 mV). (B) Family of I/V plots upon ion substitution in the bathing media. Line colors and thickness represent different ion and substrate profiles, respectively (see inserts). (C) I/V profiles with subtracted `control' leak currents in the absence of substrate molecules. Phe concentration in B and C = 1 mmol l-1.

View larger version (16K): [in a new window]   Fig. 7. Kinetic properties of agNAT8 transport mechanism. (A) The effects of increasing phenylalanine concentration on agNAT8-mediated currents; values are means ± s.e.m.; N 3. (B) The effects of increasing sodium ion concentration on phenylalanine-elicited, agNAT8-mediated currents; values are means ± s.e.m.; N 3, except for L-Met, N=1. (C) E0.5 for substrates with highest apparent transport velocity; values are means ± s.d.; N 3. (D) Estimated Hill coefficient for selected substrates; values are means ± s.d.; N 3. Lines in A and B represent iterative nonlinear regression of the data with three-parameter Hill function (E[S]0.5 and [S] are incorporated in the graph panels; see inserts).

View larger version (16K): [in a new window]   Fig. 8. Accumulation of isotope-labeled phenylalanine (A) and methionine (B) in deionized water-injected (empty circles and blue lines) and agNAT8 transcript-injected (black filled circles and red lines) oocytes. Values are means ± s.e.m. for at least three oocytes per data point. Lines are nonlinear regression fits of the data sets by a single rectangular function, f=a*x/(b+x). (C) pH dependency of phenylalanine-induced currents mediated by agNAT8. Values are means ± s.e.m. for at least three oocytes/experiment per data point. Data from different oocytes were normalized in each group relative to values at pH 7.3. The straight line represents a linear regression of the entire data set, f=y0+ax.

View larger version (90K): [in a new window]   Fig. 9. Spatial expression of agNAT8 transcript in the alimentary canal, CNS and PNS of An. gambiae larvae. (A) In situ hybridization of agNAT8-specific probes in the whole-mount midgut from 4th instar An. gambiae larvae. (B) Magnified image of the rectal gland. (C) Intensive hybridization in the salivary gland. (D) Stretched-opened esophageal epithelial infolding, cardia; this epithelial region is associated with secretion of the tubular peritrophic membrane. (E) Area-specific labeling in the isolated neuronal ventral cord of CNS; three thoracic ganglia are shown. (F,G) Dorsal and lateral views of agNAT8 hybridization pattern in the larval stemma (eye); this labeling is associated with primary photosensitive neurons (green arrows). (H) An example of hybridization of sensory neurons associated with vibration-sensitive hair sensilla on the larval head (black arrow). (I) Labeling in the basal part of the peg sensillum at the head capsule (white arrow); this structure has an unknown sensory role in larvae, but it is morphologically similar to temperature-sensitive structures in adults. (J) agNAT8-pecific hybridization of specific populations of chemosensory neurons in maxillary pulp (MP) and labium afferents (LB) (open arrows). AMG, anterior midgut; CA, cardia, a sub-esophageal invagination; GC, gastric caeca; MT, Malpighian tubules; PMG, posterior midgut. All images were acquired using a Hoffman contrast microscope except for A and E, which were acquired using a stereo microscope. Bars, 1000 µm (A), 200 µm (B-E), 100 µm (F-H,J), 50 µm (I).

View larger version (15K): [in a new window]   Fig. 10. Quantitative real-time PCR analysis of agNAT8 expression in distinct larval tissues and developmental stages of An. gambiae. (A) Relative expression levels of agNAT8 in tissues of 4th instar larvae. (B) Relative expression levels of agNAT8 at various developmental stages and in adult tissues. Note the different scales on the x axis for A and B. Data are represented as the mean of three averaged replicates of two independent experiments plus standard error. An. gambiae 18S ribosomal RNA was used as a reference gene. Results were normalized to values for whole larvae, which was set to a value of 1.0. CNS, central nervous system; SG, salivary glands; GC, gastric caeca; AM, anterior midgut; PM, posterior midgut; MT, Malpighian tubules; RG, rectal gland; rep org, reproductive organs.

View larger version (26K): [in a new window]   Fig. 11. Model of agNAT8 functions superimposed with patterns of its spatial expression and putative membrane localizations, along with specific physiological roles of particular epithelial areas and with a simplified Na+ ion recycling scheme. Light blue fill corresponds to epithelial areas with areas of hybridization and qPCR detection of agNAT8 transcripts. Dark blue outlines correspond with theoretically possible membrane localization of agNAT8. The posterior midgut is associated with apical Na+-dependent absorption of free amino acids from the midgut lumen into epithelial cells (green arrow). A complementary transporter for emitting amino acids via the basal membrane of the posterior midgut into the hemocoel is expected but presently unknown; also, it may not require an active transport mechanism since the intracellular concentration of free amino acids in this area is high vs the concentration in the hemolymph. In contrast, the cardia, salivary glands and gastric caeca clearly are secretory parts of the alimentary canal, which mediates the synthesis and apical secretion of various amino acids in the form of peptides and protein polymers. A basal accumulative transport mechanism is necessary here (green arrow), which is complemented by an apical secretion process. The disposable pool of sodium ions in the midgut lumen, which is involved in the transport process, is recycled via the area of anterior alkalinization (pH 11), shown as a pink gradient band in the anterior midgut lumen. PR, pharynx; SG, salivary glands; EO, oesophagus; CA, cardia; GC, gastric caeca; AMG, anterior midgut; PMG, posterior midgut; MT, Malpighian tubule; RG, rectal gland.