Topology of NBCe1 protein transmembrane segment 1 and structural effect of proximal renal tubular acidosis (pRTA) S427L mutation.
In the kidney proximal tubule, NBCe1-A plays a critical role in absorbing HCO3(-) from cell to blood. NBCe1-A transmembrane segment 1 (TM1) is involved in forming part of the ion permeation pathway, and a missense mutation S427L in TM1 impairs ion transport, causing proximal renal tubular acidosis. In the present study, we examined the topology of NBCe1-A-TM1 in detail and its structural perturbation induced by S427L. We analyzed the N-terminal cytoplasmic region (Cys-389-Gln-424) of NBCe1-A-TM1 using the substituted cysteine scanning accessibility method combined with extensive chemical stripping, in situ chemical probing, and functional transport assays. NBCe1-A-TM1 was previously modeled on the anion exchanger 1 TM1 (AE1-TM1); however, our data demonstrated that the topology of AE1-TM1 differs significantly from NBCe1-A-TM1. Our findings revealed that NBCe1-A-TM1 is unusually long, consisting of 31 membrane-embedded amino acids (Phe-412 to Thr-442). The linker region (Arg-394-Pro-411) between the N terminus of TM1 and the cytoplasmic domain is minimally exposed to aqueous and is potentially folded in a helical structure that intimately interacts with the NBCe1-A cytoplasmic domain. In contrast, AE1-TM1 contains 25 amino acids connected to an aqueous-exposed cytoplasmic region. Based on our new NBCe1-A-TM1 model, Ser-427 resides in the middle of TM1. Leucine substitution at Ser-427 blocks the normal aqueous access to Thr-442, Ala-435, and Lys-404, implying a significant alteration of NBCe1-TM1 orientation. Our study provides novel structural insights into the pathogenic mechanism of S427L in mediating proximal renal tubular acidosis.
Department of Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1689. Electronic address: quzhu@mednet.ucla.edu.
Quansheng
Q
Zhu
Department of Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1689.
Weixin
W
Liu
Department of Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1689.
Liyo
L
Kao
Department of Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1689.
Rustam
R
Azimov
Department of Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1689.
Debra
D
Newman
Department of Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1689.
Natalia
N
Abuladze
Department of Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1689; Brain Research Institute, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1689.
Ira
I
Kurtz
D000141:Acidosis, Renal Tubular / Q000235:genetics* / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry; D001639:Bicarbonates / Q000737:chemistry; D001692:Biological Transport; D001710:Biotin / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D001639:Bicarbonates; D008051:Lipid Bilayers; D008301:Maleimides; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; C043592:maleimide; D001710:Biotin; D012964:Sodium