title: G418-mediated ribosomal read-through of a nonsense mutation causing autosomal recessive proximal renal tubular acidosis.
abstract: Autosomal recessive proximal renal tubular acidosis is caused by mutations in the SLC4A4 gene encoding the electrogenic sodium bicarbonate cotransporter NBCe1-A. The mutations that have been characterized thus far result in premature truncation, mistargeting, or decreased function of the cotransporter. Despite bicarbonate treatment to correct the metabolic acidosis, extrarenal manifestations persist, including glaucoma, cataracts, corneal opacification, and mental retardation. Currently, there are no known therapeutic approaches that can specifically target mutant NBCe1-A proteins. In the present study, we tested the hypothesis that the NBCe1-A-Q29X mutation can be rescued in vitro by treatment with aminoglycoside antibiotics, which are known for their ability to suppress premature stop codons. As a model system, we cloned the NBCe1-A-Q29X mutant into a vector lacking an aminoglycoside resistance gene and transfected the mutant cotransporter in HEK293-H cells. Cells transfected with the NBCe1-A-Q29X mutant failed to express the cotransporter because of the premature stop codon. Treatment of the cells with G418 significantly increased the expression of the full-length cotransporter, as assessed by immunoblot analysis. Furthermore, immunocytochemical studies demonstrated that G418 treatment induced cotransporter expression on the plasma membrane whereas in the absence of G418, NBCe1-A-Q29X was not expressed. In HEK293-H cells transfected with the NBCe1-A-Q29X mutant not treated with G418, NBCe1-A-mediated flux was not detectable. In contrast, in cells transfected with the NBCe1-A-Q29X mutant, G418 treatment induced Na(+)- and HCO(3)(-)-dependent transport that did not differ from wild-type NBCe1-A function. G418 treatment in mock-transfected cells was without effect. In conclusion, G418 induces ribosomal read-through of the NBCe1-A-Q29X mutation in HEK293-H cells. These findings represent the first evidence that in the presence of the NBCe1-A-Q29X mutation that causes proximal renal tubular acidosis, full-length functional NBCe1-A protein can be produced. Our results provide the first demonstration of a mutation in NBCe1-A that has been treated in a targeted and specific manner.
authors.affiliation: Division of Nephrology, David Geffen School of Medicine at UCLA, University of California-Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA.
authors.forename: Rustam
authors.initials: R
authors.lastname: Azimov
authors.forename: Natalia
authors.initials: N
authors.lastname: Abuladze
authors.forename: Pakan
authors.initials: P
authors.lastname: Sassani
authors.forename: Debra
authors.initials: D
authors.lastname: Newman
authors.forename: Liyo
authors.initials: L
authors.lastname: Kao
authors.forename: Weixin
authors.initials: W
authors.lastname: Liu
authors.forename: Nicholas
authors.initials: N
authors.lastname: Orozco
authors.forename: Piotr
authors.initials: P
authors.lastname: Ruchala
authors.forename: Alexander
authors.initials: A
authors.lastname: Pushkin
authors.forename: Ira
authors.initials: I
authors.lastname: Kurtz
mesh_terms: D000141:Acidosis, Renal Tubular / Q000151:congenital* / Q000188:drug therapy*; D000563:Amebicides / Q000627:therapeutic use*; D002460:Cell Line; D018389:Codon, Nonsense; D015870:Gene Expression; D005839:Gentamicins / Q000627:therapeutic use*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D012333:RNA, Messenger / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics*
chemical_list: D000563:Amebicides; D018389:Codon, Nonsense; D005839:Gentamicins; D012333:RNA, Messenger; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; C010680:antibiotic G 418