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Interactions between the thiol-group reagent N-ethylmaleimide and neutral and basic amino acid transporter-related amino acid transport.

ABSTRACT: The neutral and basic amino acid transport protein (NBAT) expressed in renal and jejunal brush-border membranes is involved in amino acid and cystine absorption. NBAT mutations result in Type 1 cystinuria. A C-terminal myc-tagged NBAT (NBATmyc) retains the amino acid transport and protein-protein interaction properties of NBAT when expressed in Xenopus oocytes. Neutral amino acid (Ala, Phe)-cationic amino acid (Arg) heteroexchanges related to NBATmyc expression in oocytes are inactivated by treatment with the thiol-group reagent N-ethylmaleimide (NEM), although significant Arg-Arg and Ala-Ala homoexchanges persist. Inactivation of heteroexchange activity by NEM is accompanied by loss of >85% of alanine and cystine uptake, with smaller (<50%) inhibition of arginine and phenylalanine uptake. NEM-sensitive cystine uptake and arginine-alanine heteroexchange (system b(0,+) activity) are not expressed by an NBAT truncation mutant (NBATmyc-Sph1) lacking the 13 C-terminal amino acid residues, but the mutant expresses NEM-resistant transport activity (system y(+)L-like) equivalent to that of full-length NBATmyc. The deleted region of NBATmyc-Sph1 contains two cysteine residues (671/683) which may be the targets of NEM action. The synthetic amino acid 2-trifluoromethylhistidine (TFMH) stimulated alanine efflux at pH 7.5 and arginine at pH 5.5, but not vice versa, establishing the existence of distinct pathways for cationic and neutral amino acid homoexchange (TFMH is zwitterionic at pH 7.5 and cationic at pH 5.5). We suggest that NBAT expresses a combination of system b(0,+) and y(+)L-like activities, possibly by interacting with different light-chain subunits endogenous to oocytes (as does the homologous 4F2hc protein). The C-terminus of NBAT may also have an additional, direct role in the mechanism of System b(0,+) transport (the major transport activity that is defective in Type 1 cystinuria).

SUBMITTER: Peter GJ

PROVIDER: S-EPMC1220538 | biostudies-other | 1999 Oct

REPOSITORIES: biostudies-other

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Project description:BackgroundSkeletal muscle mass and function are partly maintained by the supply of amino acids, altered amino acid transport is an important cause of frailty that can lead to decreased independence with increasing age and slow trauma recovery. The system-A sodium coupled neutral amino acid transporter (SNAT)-2 coded by gene family SLC38A2 generates a 506 amino acid 56 kDa protein that is an important transporter of amino acids in skeletal muscle. Ageing is associated with a decrease in expression of SNAT2 transporters.MethodsIn this study, we used the C2C12 cell line, using myoblast cells and cells differentiated into myotubes. We investigated if the expression of SNAT2 DNA would enhance intracellular amino acid levels and increase their availability for protein synthesis.ResultsIn control myoblasts and myotubes, we found significantly decreased expression of SNAT2 (6.5× decrease, n = 4 per group, P < 0.05) in myotubes than found in myoblasts. After transfection with a SNAT2-eGFP cDNA plasmid, C2C12 myoblasts significantly increased perinuclear punctate SNAT2-eGFP expression that persisted and was more cytoplasmic after differentiation into myotubes. Interestingly, transfected cells were significantly more responsive to the hormone 5α-dihydrotestosterone (DHT, 4.5 nM, by 1.6×, n = 3 per group, P < 0.04). Starvation significantly enhanced the amino acid C14 -MeAIB transport (1.7×, n = 3 per group, P < 0.05) indicating increased function of SNAT2. Inhibiting SNAT2 with high concentrations of MeAIB (3.3 or 5 mM) significantly reduced C14 -Isoleucine transport by L-type amino acid transporter (LAT2, 52.8% and 77%, respectively, n = 3 per group, P < 0.05). However, there was no increase in the LAT2 transport of C14 -isoleucine detectable in SNAT2-eGFP transfected cells after DHT (4.5 nM) exposure. This indicated that small amino acid availability was not rate limiting to LAT2 function in myoblasts.ConclusionsOverall, these data show that transfection of SNAT2-eGFP expression enhanced its function following starvation and treatment with physiological levels of DHT. Enhanced SNAT2 expression in muscle cells offers a viable epigenetic target in pathological conditions associated with altered amino acid transport.

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Interactions between the thiol-group reagent N-ethylmaleimide and neutral and basic amino acid transporter-related amino acid transport.

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