PMC

Mg²⁺ uptake from the pro-urine via the epithelial Mg²⁺ channel TRPM6 is primarily driven by the negative potential across the luminal membrane. This luminal membrane potential is maintained by an apical K⁺ efflux via Kv1.1 energized by the action of the Na⁺/K⁺-ATPase. At the basolateral membrane, Mg²⁺ extrusion to the blood side occurs via an unknown mechanism. The identified N255D mutation results in a nonfunctional Kv1.1 channel and thereby decreases the driving force for Mg²⁺ influx, thus resulting in renal Mg²⁺ wasting.

(A) Staining for Kv1.1 (green) and TRPM6 (red) of mouse serial kidney sections (right panels: overview of a cortical region; left panels: magnified images of immunopositive tubules). The asterisks indicate the same distal tubules on serial sections intensively stained for Kv1.1 and TRPM6. (B) Mouse kidney sections were costained for Kv1.1 (green) and calbindin D_28K (red) (lower panels: immunopositive tubules; upper panel: merged differential interference contrast [DIC] image). (C) Costaining of Kv1.1 (green) and AQP2 (red) in mouse kidney sections (lower panels: immunopositive tubules; upper panel: merged DIC image). G, glomerulus; 28K, calbindin D_28K. Scale bars: 50 μm (A, left panels), 80 μm (A, right panels; C, bottom panels), 20 μm (B, top panel), 40 μm (B, bottom panels; C, top panel).

(Kv1.1 WT), or Kv1.1 N255D. (A) Representative original traces of outward K⁺ currents of cells transfected with mock plasmid, Kv1.1 WT, Kv1.1 N255D, or Kv1.1 WT and mock plasmid elicited by voltage steps from –100 to +50 mV in 10-mV increments, applied from a holding potential of –80 mV, every 10 seconds. (B) The I-V relationships of cells transfected with mock plasmid (circles, n = 5), Kv1.1 WT (squares, n = 11), Kv1.1 N255D (triangles, n = 10), and Kv1.1 WT and mock plasmid (diamonds, n = 9). (C) Histogram presenting averaged current densities at +50 mV of cells transfected with mock plasmid (n = 5), Kv1.1 WT (n = 11), Kv1.1 WT and mock plasmid (n = 9), Kv1.1 WT and Kv1.1 N255D (n = 9), and Kv1.1 N255D (n = 10). *P < 0.05, compared with mock; ^#P < 0.05, compared with Kv1.1 WT. (D) Cell surface biotinylation of HEK293 cells transfected with mock plasmid, Kv1.1 WT, Kv1.1 WT and mock plasmid, Kv1.1 WT and Kv1.1 N255D, and Kv1.1 N255D. Kv1.1 expression was analyzed by immunoblotting for plasma membrane fraction (PM) and input from the total cell lysates (IP). Representative immunoblot of 4 independent experiments is shown. (E) Histogram presenting averaged current densities at +80 mV after 200 seconds of cells cotransfected with TRPM6 and mock plasmid (n = 26), TRPM6 and Kv1.1 WT (n = 26), and TRPM6 and Kv1.1 N255D (n = 25). (F) Membrane potential of cells transfected with mock plasmid, Kv1.1 WT, and Kv1.1 N255D upon acute application of DTX-K (10 nM), measured in current clamp mode of whole-cell patch clamp configuration. Representative recordings of 8 independent experiments are shown. The error bars denote SEM.

(A) Pedigree with 5 generations (I–V) of a Brazilian family with autosomal dominant hypomagnesemia. Affected family members are indicated in black; males and females are indicated by squares and circles, respectively. A diagonal line indicates that the individual is deceased. Numbers in red indicate individuals included in the Sequence Tagged Site (STS) mapping. (B) 10K SNP array–based haplotyping analysis was performed that showed linkage to a 14.3-cM region between SNP rs717596 and rs252028 on the short arm of chromosome 12. This region was confirmed and narrowed down with STS markers to a 11.6-cM region between markers D12S1626 and D12S1623 containing 31 genes, including KCNA1. (C) KCNA1 encodes the voltage-gated potassium channel Kv1.1. Mutation analysis of KCNA1 revealed a heterozygous A763G missense mutation in affected individual III-1 that results in a N255D amino acid substitution (underlined). (D) Multiple alignment analysis shows conservation of the N255 amino acid (red bar) among species and Kv1 family members. Mutated amino acids in other families with the Kv1.1 genotype are indicated by dark blue dots (, , , ). Blue and black letters represent conserved and nonconserved amino acids, respectively. (E) Schematic representation of the Kv1.1 channel, which consists of a voltage sensor in transmembrane segment S4 and a pore-forming region (S5 and S6). Localization of the newly identified N255D mutation is denoted by the red dot, while other nearby mutations are indicated by dark blue dots.