Topological location and structural importance of the NBCe1-A residues mutated in proximal renal tubular acidosis.
NBCe1-A electrogenically cotransports Na(+) and HCO(3)(-) across the basolateral membrane of renal proximal tubule cells. Eight missense mutations and 3 nonsense mutations in NBCe1-A cause severe proximal renal tubular acidosis (pRTA). In this study, the topologic properties and structural importance of the 8 endogenous residues mutated in pRTA and the in situ topology of NBCe1-A were examined by the substituted cysteine accessibility method. Of the 55 analyzed individually introduced cysteines, 8 were labeled with both membrane permeant (biotin maleimide (BM)) and impermeant (2-((5(6)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate (MTS-TAMRA)) sulfhydryl reagents, 4 with only BM, and 3 with only MTS-TAMRA. The location of the labeled and unlabeled introduced cysteines clearly indicates that the transmembrane region of NBCe1-A contains 14 transmembrane segments (TMs). In this in situ based NBCe1-A topology, residues mutated in pRTA (pRTA residues) are assigned as: Ser(427), TM1; Thr(485) and Gly(486), TM3; Arg(510) and Leu(522), TM4; Ala(799), TM10; and Arg(881), TM12. Substitution of pRTA residues with cysteines impaired the membrane trafficking of R510C and R881C, the remaining membrane-processed constructs had various impaired transport function. Surprisingly, none of the membrane-processed constructs was accessible to labeling with BM and MTS-TAMRA, nor were they functionally sensitive to the inhibition by (2-aminoethyl)methanethiosulfonate. Functional analysis of Thr(485) with different amino acid substitutions indicated it resides in a unique region important for NBCe1-A function. Our findings demonstrate that the pRTA residues in NBCe1-A are buried in the protein complex/lipid bilayer where they perform important structural roles.
Zhu
Quansheng
Q
Division of Nephrology, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, USA.
Kao
Liyo
L
Azimov
Rustam
R
Newman
Debra
D
Liu
Weixin
W
Pushkin
Alexander
A
Abuladze
Natalia
N
Kurtz
Ira
I
D000141:Acidosis, Renal Tubular / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; D008051:Lipid Bilayers; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters