Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1.
The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low Km) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low Km of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys160 of LAT1 and Cys110 of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl2 with a Ki of 0.56+/-0.11 microM. The inhibitory effect of HgCl2 for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl2 for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys88 and Cys439, altered amino acid transport. The Vmax was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys439 residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.
Boado
Ruben J
RJ
Department of Medicine, UCLA Warren Hall 13-164, 900 Veteran Ave., Los Angeles, CA 90024, USA. rboado@mednet.ucla.edu
Li
Jian Yi
JY
Chu
Chun
C
Ogoshi
Fumio
F
Wise
Petra
P
Pardridge
William M
WM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics*; D017931:DNA Primers; D007700:Kinetics; D027283:Large Neutral Amino Acid-Transporter 1 / Q000737:chemistry / Q000235:genetics*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000737:chemistry; D011817:Rabbits; D014982:Xenopus laevis
D017931:DNA Primers; D027283:Large Neutral Amino Acid-Transporter 1; D003545:Cysteine