Abstract
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.
Highlights
By using NBCe1-A-5CϪ as the mutagenesis template, 8 pRTA residues were individually substituted with cysteines
It is known that 3 pRTA missense mutations (R510H, L522P, and R881C) result in the retention of mutant NBCe1-A intracellularly [11, 12, 14, 16], we performed immunocytochemistry analysis with an antibody that recognizes the region between the two glycosylated sites in the proposed NBCe1-A extracellular loop 3 (Ab-162) to determine whether cysteine substitution of pRTA residues have defects in protein plasma membrane trafficking
To quantitatively determine the amount of protein processed to the plasma membrane, we performed surface labeling of the cysteine-substituted pRTA constructs with sulfo-NHS-SS-biotin
Summary
It is known that 3 pRTA missense mutations (R510H, L522P, and R881C) result in the retention of mutant NBCe1-A intracellularly [11, 12, 14, 16], we performed immunocytochemistry analysis with an antibody that recognizes the region between the two glycosylated sites in the proposed NBCe1-A extracellular loop 3 (Ab-162) to determine whether cysteine substitution of pRTA residues have defects in protein plasma membrane trafficking. To quantitatively determine the amount of protein processed to the plasma membrane, we performed surface labeling of the cysteine-substituted pRTA constructs with sulfo-NHS-SS-biotin.
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