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First published online January 16, 2009
Journal of Experimental Biology 212, 347-357 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.026047
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NHEVNAT: an H+ V-ATPase electrically coupled to a Na+:nutrient amino acid transporter (NAT) forms an Na+/H+ exchanger (NHE)

William R. Harvey1,2,*, Dmitri Y. Boudko1,{dagger}, Mark R. Rheault1,{ddagger} and Bernard A. Okech1,2

1 Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Boulevard, St Augustine, FL 32080, USA
2 Department of Physiology and Functional Genomics, Department of Epidemiology and Biostatistics and Emerging Pathogens Institute, University of Florida, Gainesville, FL 32610, USA


Figure 1
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  		Fig. 1. Predicted secondary structure of K+ amino acid transporter 1 (KAAT1) polypeptide, showing the location of mutations that were made to investigate function (blue, mutation; red, location) and tyrosine 147 (yellow Y in lower box) that is conserved among related transporters; from Liu et al. (Liu et al., 2003Go).

 

Figure 2
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  		Fig. 2. Characteristics of Aedes aegypti amino acid transporter 1 (AeAAT1i). Reciprocal amino acid sequence identity matrix (left); hydropathy plot and predicted transmembrane topology (right); TopPed II parameters were: GES-hydrophobicity scales: 1.0 upper and 0.5 lower cutoffs; 10 and 5; core and wage window sizes, 60; critical loop length, 2 for critical transmembrane spacer. Hydropathy values (vertical scale) and distribution of transmembrane spanning domain (insert) are aligned relative to amino acid position (horizontal scale). (Modified from Boudko et al., 2005aGo.)

 

Figure 3
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  		Fig. 3. Phylogram of sodium neurotransmitter symporter family (SNF) showing the relationship of nutrient amino acid transporters (NATs) to neurotransmitter transporters (NTTS). NATs are Na+-coupled amino acid± (neutral amino acid) symporters. Accession numbers for the seven Anopheles gambiae NATs are shown in blue font (Boudko et al., 2005bGo).

 

Figure 4
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  		Fig. 4. Alignment and reconstruction of Anopheles gambiae nutrient amino acid transporter 8 (AgNAT8) structure: (A) sequence/structure alignment of characterized insect transporters relative to the first crystallized bacterial nutrient amino acid transporters (NAT) from A. aeolicus, LeuTAa (Yamashita et al., 2005). (B) 2-D structure of AgNAT8 based on structural homology with the LeuTAa protein sequence. NCBI Accession no.: LeuTAa, NP_214423 (PDB no., 2A65); Manduca sexta K+ amino acid transporter 1 (MsKAAT1), AAC24190; M. sexta cation amino acid transporter channel 1 (MsCAATCH1), AAF18560; Aedes aegypti amino acid transporter 1 (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 that 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; from Meleshkevitch et al. (Meleshkevitch et al., 2006Go).

 

Figure 5
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  		Fig. 5. When expressed in Xenopus oocytes Anopheles gambiae nutrient amino acid transporter 8 (AgNAT8) exhibits large inward currents showing that it is electrophoretic (Meleshkevitch et al., 2006Go).

 

Figure 6
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  		Fig. 6. Immunolabeling of Anopheles gambiae nutrient amino acid transporters (AgNATs) in frozen sections of the larval alimentary with epitope-specific purified antibodies (green channel) along with actin (TRITCPhalloidin, red channel) and nuclei (DRAQ-5, blue channel) viewed by confocal microscopy. Actin and nuclei were not visualized in a few sections to improve overall clarity. The red channel generally represents actin in the muscular envelope around the alimentary canal and corresponds to the location of the basal membrane except in salivary glands and Malpighian tubules where actin reveals the location of microvilli on the apical membrane. The location of the apical membrane is indicated by white arrows. Approximate positions of individual sections are shown at the insert diagram. Abbreviations are: SG, salivary gland; CA, cardia; GC, gastric caeca; AMG, anterior midgut; PMG, posterior midgut; MT, Malpighian tubes. Control sections of the AMG (control AMG) and PMG (control PMG) incubated with pre-bleed serum shown at the left bottom corner insert. Scale bar, 50 µm (Okech et al., 2008bGo).

 

Figure 7
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  		Fig. 7. Current/voltage (I/V) relationships of four (Na+ or K+):amino acid± symporters (NATs). In all four cases the current is voltage dependent. In Manduca sexta K+ amino acid transporter 1(MsKAAT1) and Aedes aegypti amino acid transporter 1 (AeAAT1) the conductance is linear from 0 to ~–60 mV then increases at voltages more negative than –70 mV. These results demonstrate that all four NATs are voltage driven (electrophoretic): (A) Manduca sexta K+ amino acid transporter 1 (MsKAAT1) (Castagna et al., 1998Go): (B) M. sexta cation amino acid transporter channel 1 (MsCAATCH1) (Feldman et al., 2000Go); (C) Ae. aegypti amino acid transporter 1 (AeAAT1) (Boudko et al., 2005aGo): (D) An. gambiae nutrient amino acid transporter 8 (AgNAT8) (Meleshkevitch et al., 2006Go).

 

Figure 8
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  		Fig. 8. An H+ V-ATPase (V) is electrically coupled by the membrane potential (Vm) to AgNAT8, a Na+:amino acid transporter (NAT; N) in the apical plasma membrane of posterior midgut (PMG) constituting an NHEVNAT. AgNHA1, a Na+/nH+ Antiporter (NHA; A), is also present on the apical membrane. A Na+/K+ ATPase is present in the basal membrane along with Anopheles gambiae nutrient amino acid transporter 6 (AgNAT6) (modified from Okech et al., 2008aGo; Okech et al., 2008bGo).

 

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