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"NITRIC OXIDE REGULATION OF A LOW-AFFINITY CATIONIC AMINO ACID TRANSPORTER"

by
Ruifang Zheng
Pharmacology & Physiology Program
B.M., 1997, Weifang Medical College, China


Thesis Advisor: R. Daniel Peluffo, Ph.D.
Assistant Professor
Department of Pharmacology & Physiology

Monday, July 22, 2013
2:00 P.M., MSB H-609


Abstract

Supplementation with L-arginine (L-Arg), the substrate for nitric oxide (NO) biosynthesis by Nitric Oxide Synthase (NOS), has a beneficial effect on cardiac function. A possible underlying mechanism is that by increasing the availability of L-Arg the production of NO is increased, thus avoiding the formation of reactive oxygen species, which are harmful to cardiomyocytes. Cardiomyocytes lack L-Arg synthesis/rescue enzymes, as they depend solely on L-Arg transport from the extracellular space via cationic amino acid transporters (CATs). Therefore, CATs play a central role in maintaining adequate levels of L-Arg and NO within cardiac muscle. Our previous work identified high- and low-affinity CAT isoforms in rat cardiomyocytes, with kinetic properties that are consistent with the CAT1 and CAT2A members of the y+ family of transporters, respectively. NO acutely and directly inhibits the transport activity of these CATs in a non competitive manner in cardiac preparations. S-nitrosation is a direct modification of protein cysteine residues by NO. Thus, we propose that NO regulates CATs activity through S-nitrosation of cysteine residues in these proteins. To test this hypothesis and to identify the target residues, mammalian cell lines over-expressing Flag-tagged mouse CAT2A (Flag-mCAT2A) were used for site-directed mutagenesis studies. When cysteine residue 347 (Cys347) was mutated to alanine (Cys347Ala), the transport capacity (Vmax) of mCAT2A decreased by 50% compared to wild type (WT) without affecting membrane expression levels or the apparent affinity (KM) for the transported amino acid. Moreover, this Cys347Ala mutant became insensitive to inhibition by NO donors. Substituting Cys347 with serine (Cys347Ser), on the other hand, restored uptake levels to those of the WT while retaining the NO insensitivity. In cells co-expressing endothelial NOS (eNOS) and Flag-mCAT2A, exposure to extracellular L-Arg inhibited uptake activity of WT mCAT2A but not that of the Cys347Ser mutant. The inhibitory effect on the WT was prevented by applying NOS inhibitors.
Other cysteine residues that could be targets for NO inhibition, Cys171 and Cys427, were also mutated to Ala separately. However, these two mutants were found to be still sensitive to NO-donor inhibition. In addition, the Cys¡úAla substitution did not affect baseline transport activity.
Altogether, we conclude that the ¨CSH moiety of Cys347, but not Cys171 or Cys427, is uniquely responsible for the NO-inhibition of mCAT2A. This modulation of CAT2A activity via Cys347 ¨C NO interaction is likely to be a physiologically relevant mechanism, as endogenous NO produced by co-transfected eNOS blocks L-Arg uptake. Cys347 is also important for the basal transport activity of mCAT2A; although its requirement is not absolute since a Ser substitution restores CAT2A uptake levels.


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