Functional characterization and cloning of amino acid transporter B^0,+ (ATB^0,+) in primary cultured rat pneumocytes

Abstract

Cationic amino acid transport in primary cultured rat pneumocytes exhibiting characteristics of alveolar epithelial type I-like cells are described. Asymmetry and activator ion dependency of (3)H-L-arginine uptake were characterized from the apical or basolateral fluid of pneumocytes grown on permeable support. Substrate specificity of transport was evaluated as a function of (3)H-L-arginine uptake inhibition in the presence of other amino acids. Transepithelial transport studies estimated (3)H-L-arginine flux in the apical-to-basolateral and basolateral-to-apical directions. Full length cDNA of rat amino acid transporter B(0,+) (rATB(0,+)) was cloned and its relative expression level studied. Results indicate that uptake of (3)H-L-arginine from apical fluid is dependent on Na(+) and Cl(-). Zwitterionic and cationic amino acids (excluding L-proline and anionic amino acids) inhibited uptake of (3)H-L-arginine from apical, but not basolateral incubation fluid. Apical-to-basolateral transepithelial flux of (3)H-L-arginine was 20x higher than basolateral-to-apical transport. Kinetic studies of (3)H-L-arginine uptake from apical fluid revealed maximal velocity (V(max)) and Michaelis-Menten constants (K(t)) of 33.32 +/- 2.12 pmol/mg protein/15 min and 0.50 +/- 0.11 mM, respectively, in a cooperative process having a coupling ratio of 1.18 +/- 0.16 with Na(+) and 1.11 +/- 0.13 with Cl(-). Expression of rATB(0,+) mRNA was identified by RT-PCR and Northern analysis. Corresponding cloned 3.2 kb rATB(0,+) cDNA sequence exhibits pronounced homology in deduced amino acid sequence to mouse (95% identity and 97% similarity) and human (89% identity and 95% similarity) ATB(0,+) homologues. We conclude that rat pneumocytes express ATB(0,+), which may partly contribute towards recovering cationic and neutral amino acids from alveolar luminal fluid.

Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation

“…With respect to amino acid selectivity, we found that amino acids with hydrophobic side chain, such as Trp, Leu, Met, Phe, and Ala generally interact with SLC6A14 with the highest apparent affinities, whereas glycine and positively charged amino acids generate the largest transport current at saturating substrate concentrations. These results on substrate selectivity are in agreement with previous reports ( Sloan and Mager, 1999 ; Ugawa et al, 2001 ; Umapathy et al, 2004 ; Karunakaran et al, 2008 ) and highlight the broad recognition range of amino acid substrates for the SLC6A14 family member ( Uchiyama et al, 2008 ).…”

A three-sodium-to-glycine stoichiometry shapes the structural relationships of ATB0,+ with GlyT2 and GlyT1 in the SLC6 family

“…In the lung, ATB 0,+ plays a similar scavenging role on the apical side of the airway epithelium ( Sloan and Mager, 1999 ; Sloan et al, 2003 ; Uchiyama et al, 2008 ; Paola et al, 2017 ), thus depleting essential nutrients and limiting bacteria growth in the thin and sterile airway surface layer (ASL) ( Mager and Sloan, 2003 ; Paola et al, 2017 ; Ruin et al, 2020 ). Short circuit currents evoked by amino acids and dependent on Na + have been recorded in cultured human bronchial epithelial cells and mouse trachea epithelium ( Galietta et al, 1998 ; Sloan et al, 2003 ; Ahmadi et al, 2019 ), although the [Na + ] and [Cl − ] in the thin ASL are not precisely known as two opposing models predict either a low salt condition (~50 mM) or near isotonic concentrations ( Boucher, 1999 ; Jayaraman et al, 2001 ; Song et al, 2003 ).…”

Update on SLC6A14 in lung and gastrointestinal physiology and physiopathology: focus on cystic fibrosis

“…Several studies showed that SLC6A14 plays a primary role as an amino acid transporter in various epithelial cells and models [32,[48][49][50][51][52]. Taken together, SLC6A14 expression data in human and genetic studies suggest that SLC6A14 may have an important role in the lung and intestinal pathophysiology of CF patients (see Parts 1 and 2).…”