title: Identification of dominant negative effect of L522P mutation in the electrogenic Na⁺-HCO₃⁻ cotransporter NBCe1.
abstract: Homozygous mutations in the electrogenic Na(+)-HCO3 (-) cotransporter NBCe1 cause proximal renal tubular acidosis (pRTA) associated with extrarenal manifestations such as ocular abnormalities and migraine. Previously, the NBCe1 cytosolic mutant S982NfsX4 was shown to have a dominant negative effect by forming hetero-oligomer complexes with wild type (WT), which might be responsible for the occurrence of glaucoma and migraine in the heterozygous family members. In this study, we investigated whether the NBCe1 L522P mutant has a similar dominant negative effect. Functional analyses in Xenopus oocytes and HEK293 cells revealed that the L522P mutant had no transport activity due to defective membrane expression. Furthermore, when coexpressed with WT, L522P significantly reduced the transport activity of WT. In HEK293 cells, the cytosolic mutant L522P reduced the membrane expression of NBCe1 by forming hetero-oligomer complexes with WT. Among the artificial Leu(522) mutants, L522I showed proper membrane expression and normal transport activity. However, the other mutants L522R, L522K, L522D, and L522E showed a predominant cytosolic retention. Moreover, L522R had a dominant negative effect, when coexpressed with WT. These results indicate that Leu(522) plays an important role in the structure and trafficking of NBCe1. They also suggest that the NBCe1 mutants retaining in cytoplasm may have the dominant negative effect in common, which may induce some clinical manifestations.
authors.affiliation: Department of Internal Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Yamazaki
authors.forename: Hideomi
authors.initials: H
authors.lastname: Yamada
authors.forename: Masashi
authors.initials: M
authors.lastname: Suzuki
authors.forename: Shoko
authors.initials: S
authors.lastname: Horita
authors.forename: Ayumi
authors.initials: A
authors.lastname: Shirai
authors.forename: Motonobu
authors.initials: M
authors.lastname: Nakamura
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Satoh
authors.forename: Toshiro
authors.initials: T
authors.lastname: Fujita
authors.forename: George
authors.initials: G
authors.lastname: Seki
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005799:Genes, Dominant; D005808:Genes, Recessive; D057809:HEK293 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D020125:Mutation, Missense; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
chemical_list: D027982:Sodium-Bicarbonate Symporters
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