Identification and characterization of Nutrient Amino Acid Transporters of the SLC6 family in Caenorhabditis elegans

The project presented here focused on a transporterfamily whose members comply with both the apical docking necessary for absorption of amino acids from the alimentary canal and the electrochemical coupling to the universal motive force of the Na+ gradient supported by the Na +/K+ ATPase, namely SoLute Carrier family 6 (SLC6), which includes the majority of sodium-dependent transporters for the essential amino acids (Boudko, 2010).;Several members of the SLC6 family were initially identified as transporters of monoamine and GABA neurotransmitters, resulting in the original naming of the family as the NeuroTransmitter Transporter (NTT) or Neurotransmitter Sodium Symporter (NSS) family. However, recent studies showed that a basal population of orphan transporters in this family exhibits the capability to transport amino acids, including the precursors of the aforementioned neurotransmitters. As with their NTT brethren, the Nutrient Amino acid Transporters (NATs) use the Na+ or K+ ion gradient, and can utilize Cl- anion as transport modulator. A majority of NAT members have been shown to exhibit substrate profiles resembling the B0 system and are designated as Broad-spectrum neutral (0 charge) amino acid transporters. Others are more specialized as selective systems for the absorption of the most underrepresented amino acids in the dietary environment such as aromatic L-Phe and L-Trp, or the sulfur containing L-Met, L-Cys and L-hCys. Despite the growing studies elucidating aspects of SLC6-NAT function in terms of transport properties, little is known about the biological requirement for these transporters for maintenance of organismal metabolism and survival. This, perhaps, is due to the complexity of the mammalian and insect models from which characterized members of the SLC6-NAT system were cloned and analyzed more intensely. Thus, there lies hope in gaining insight to the role of SLC6-NAT function in Metazoan physiology through study in a more simple model, that of the nematode Caenorhabditis elegans.;In these studies, we identified the putative NAT-SLC6 subfamily within the C. elegans genome. It contains five transporters paralogous to the previously identified NAT-SLC6 subfamily in insects. Subsequently, we cloned most of the members of the C. elegans NAT-SLC6 subfamily, as well as functionally expressed and characterized its first representative, named SNF-5 following the canonical convention for nomenclature of genes and proteins in C. elegans. SNF-5 represents a broad-spectrum neutral amino acid transporter with the unique additional capacity to transport acidic amino acids, classifying it as the first B0,- transporter. We demonstrated that SNF-5 utilizes 1 Amino Acid (A.A.) : 1 Na+ stoichiometry for the majority of its transported substrates, except for L-Pro, for which it exhibits 1 A.A : 2 Na+ stoichiometry, and L-Glu, 1 A.A. : 3 Na+. The adaptive ability of the transporter to adjust transport stoichiometry in response to the size or charge of the transported amino acid is remarkable, and is unique amongst characterized SLC6-NATs.;We also elucidated the expression profile of an SNF-5 promoter driven GFP construct, an indicator of likely regions of SNF-5 expression, in vivo. As expected for the NAT subfamily of SLC6 transporters, SNF-5 is expressed in the alimentary canal of the nematode, which corresponds to the requirement for the active absorption of nutrient amino acids from digested food. More surprisingly, SNF-5 expression was detected in a subset of sensory amphid neurons (ASI, ASK, ADF) responsible for chemotaxis to amino acid substrates and regulation and maintenance of nematode metabolism, including dauer diapause state.;Our studies of the SNF-5 knockout strain HKK479 corroborated the neuronal role of SNF-5. Specifically, we found that nematodes deprived of SNF-5 function show no apparent morphological defects, but do exhibit increased dauer formation that coincides with a decreased ability to maintain the dauer stage during extended starvation. This result not only implicates a direct role for SNF-5 in the regulation of dauer stage, but also provides preliminary insight into mechanisms for the query of systemic and environmental amino acid concentrations and availability.;The recently published crystal structure of a bacterial NAT-SLC6 transporter (Yamashita et al., 2005) allows us to explore the structural features of SNF-5 and other transporters in this family. Using a combined in silico modeling and site-directed mutagenesis approach, we examined an aspect of the SNF-5 Substrate Binding Pocket believed to convey to it the unique ability to transport acidic amino acid substrates, namely a Histidine residue within the SNF-5 amino acid sequence at position 377. Mutation of this residue was predicted to alter the transporter substrate profile in our in silico ligand interaction studies. Heterologous expression of mutated SNF-5 constructs corroborated this data, directly implicating the residue at position 377 as a key contributor to the unique substrate selectivity of this transporter. (Abstract shortened by UMI.).