Defective membrane expression of the Na(+)-HCO(3)(-) cotransporter NBCe1 is associated with familial migraine

Abstract

Homozygous mutations in SLC4A4, encoding the electrogenic Na(+)-HCO(3)(-) cotransporter NBCe1, have been known to cause proximal renal tubular acidosis (pRTA) and ocular abnormalities. In this study, we report two sisters with pRTA, ocular abnormalities, and hemiplegic migraine. Genetic analysis ruled out pathological mutations in the known genes for familial hemiplegic migraine, but identified a homozygous 65-bp deletion (Delta65bp) in the C terminus of NBCe1, corresponding to the codon change S982NfsX4. Several heterozygous members of this family also presented glaucoma and migraine with or without aura. Despite the normal electrogenic activity in Xenopus oocytes, the Delta65bp mutant showed almost no transport activity due to a predominant cytosolic retention in mammalian cells. Furthermore, coexpression experiments uncovered a dominant negative effect of the mutant through hetero-oligomer formation with wild-type NBCe1. Among other pRTA pedigrees with different NBCe1 mutations, we identified four additional homozygous patients with migraine. The immunohistological and functional analyses of these mutants demonstrate that the near total loss of NBCe1 activity in astrocytes can cause migraine potentially through dysregulation of synaptic pH.

Fig. 1.

Identification and characterization of the NBCe1 Δ65bp mutation. (A) The pedigree. Arrow: proband; squares: men; circles: women; slashes: deceased. Inner symbols: black—pRTA with hemiplegic migraine; gray—migraine with or without aura; dotted—convulsions; white—normal. Symbol borders: white—high-tension glaucoma; hatched—normal-tension glaucoma. −/−: homozygous mutation; +/−: heterozygous mutation; +/+: no mutation; question marks: no history available. (B) Current (I)/voltage (Vm) relationship of the NBCe1A constructs expressed in Xenopus oocytes. The data represent the mean ± SEM (n = 8 for each construct) obtained for wild-type NBCe1A (WT), the Δ65bp mutant (Δ65bp), or the H₂O injected Control (Cont). (C) Confocal images of GFP-tagged wild-type NBCe1A (WT) or the Δ65bp mutant in polarized MDCK cells. Green: GFP fluorescence; red: actin cytoskeletons; xy: front view; xz: side view. Scale bars, 10 μm. (D) The NBCe1B activity in C6 cells, analyzed by cell pH responses to high K⁺ (20 mM) solution. Confocal images of NBCe1B localization are at the bottom. (Scale bars, 10 μm.)

Fig. 2.

Association of wild-type NBCe1B and the Δ65bp mutant in C6 cells. (A) Localization of GFP-NBCe1B and GFP-Δ65bp. Green: GFP; red: PDI. (B) Immunoprecipitation with anti-Myc (Upper) or anti-GFP antibody (Lower). Control (Cont): cells transfected with vector alone. (C) Coexpression of Myc-NBCe1B together with GFP-NBCe1B or GFP-Δ65bp. Red: Myc; green: GFP.

Fig. 3.

Dominant negative effect of the Δ65bp mutant in HEK293 cells. (A) Original traces showing the Na⁺-dependent cell pH recovery for wild-type NBCe1B (WT) and the Δ65bp mutant. AML: amiloride (1 mM). (B) Summary data for the functional analyses. Control (Cont): cells transfected with vector alone.*P < 0.01 vs. Cont. ^#P < 0.01 vs. NBCe1B. Numbers observed were 11 (Control), 12 (Δ65bp), 16 (Δ65bp + NBCe1B), and 19 (NBCe1B). (C) Cell surface expression of NBCe1B. Blots with the anti-GFP antibody in biotinylated (2 μL per lane) and total fractions (15 μL per lane) were shown. Equal protein loading in the biotinylated fraction was confirmed by blots with the anti-Na/K pump antibody. Control (Cont): cells transfected with vector alone.

Fig. 4.

Association of NBCe1 mutations and migraine. (A) Topology of NBCe1 and mutations identified in pRTA patients. Red circles denote mutations associated with migraine and white circles mutations not associated with migraine. The dashed lines represent the specific regions of NBCe1B or NBCe1C, and the N and C denote the N and C terminus, respectively. (B) Confocal images of GFP-tagged wild-type NBCe1B (WT) and the mutants transiently expressed in C6 cells. Asterisks indicate mutations associated with migraine. (Scale bar, 10 μm.)