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. 2009 Nov 10;4(11):e7771.
doi: 10.1371/journal.pone.0007771.

A novel TRPC6 mutation that causes childhood FSGS

Saskia F Heeringa  1 , Clemens C Möller, Jianyang Du, Lixia Yue, Bernward Hinkes, Gil Chernin, Christopher N Vlangos, Peter F Hoyer, Jochen Reiser, Friedhelm Hildebrandt
Affiliations

A novel TRPC6 mutation that causes childhood FSGS

Saskia F Heeringa et al. PLoS One. .

Abstract

Background: TRPC6, encoding a member of the transient receptor potential (TRP) superfamily of ion channels, is a calcium-permeable cation channel, which mediates capacitive calcium entry into the cell. Until today, seven different mutations in TRPC6 have been identified as a cause of autosomal-dominant focal segmental glomerulosclerosis (FSGS) in adults.

Methodology/principal findings: Here we report a novel TRPC6 mutation that leads to early onset FSGS. We identified one family in whom disease segregated with a novel TRPC6 mutation (M132T), that also affected pediatric individuals as early as nine years of age. Twenty-one pedigrees compatible with an autosomal-dominant mode of inheritance and biopsy-proven FSGS were selected from a worldwide cohort of 550 families with steroid resistant nephrotic syndrome (SRNS). Whole cell current recordings of the mutant TRPC6 channel, compared to the wild-type channel, showed a 3 to 5-fold increase in the average out- and inward TRPC6 current amplitude. The mean inward calcium current of M132T was 10-fold larger than that of wild-type TRPC6. Interestingly, M132T mutants also lacked time-dependent inactivation. Generation of a novel double mutant M132T/N143S did not further augment TRPC6 channel activity.

Conclusions: In summary, our data shows that TRPC6 mediated FSGS can also be found in children. The large increase in channel currents and impaired channel inactivation caused by the M132T mutant leads to an aggressive phenotype that underlines the importance of calcium dose channeled through TRPC6.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Segregation and Evolutionary conservation of M132T.
(A) Pedigree of FSGS Family F505 with affecteds III-2 and IV-1 is compatible with autosomal/dominant inheritance. Filled symbols denote individuals affected with FSGS. Age at onset (years) is shown in parentheses. (B) TRPC6 mutation in F505. Nucleotide and amino acid change for M132T are given above sequence traces. The normal control (NC) sequence is shown below the mutated sequence. Reading frame is indicated by underlining all codon triplets in the chromatogram. (C) Evolutionary conservation of TRPC6 The Met132 is highly conserved through evolution down to Drosophila Melanogaster.
Figure 2
Figure 2. Gain of function of TRPC6 mutants M132T and M132T/N143S.
A, C, E, and G: Representative currents elicited by voltage-ramp protocol ranging from −120 to +100 mV in the presence of 100 µM Carbachol (CCh) in wt TRPC6 (A), M132T (C), N143S (E), and M132T/143S (G). No activation was observed before 100 µM CCh stimulation. B, D, F, and H: Time-dependent changes of inward current measured at −120 mV and outward current measured at +100 mV of wt TRPC6 (B), M132T (D), N143S (F), and M132T/143S (H). TRPC6 and the mutants were only activated after application of 100 µM CCH. In both panel D and H, N-methyl D-glucamine (NMDG) was applied to confirm that there was no leak current. For panel B and F, there was no significant difference of the sustained current amplitude between wt TRPC6 and N143S.
Figure 3
Figure 3. Averaged current amplitude of wt TRPC6 and the mutants.
A: Outward current amplitude measured at +100 mV (mean±SEM, n = 10). The outward current amplitude of M132T and M132T/N143S was significantly bigger than that of wt TRPC6. B: Inward current amplitude measured at −120 mV (mean±SEM, n = 10). The inward current amplitude of N143S, M132T, and M132T/N143S was statistically larger than that of wt TRPC6. C: The ratio of inward/outward current amplitude. Note the current amplitude of wt TRPC6 and N143S was measured at the peak inward and outward current amplitude (as shown in Figure 2). (*: p<0.05, **: p<0.01, in comparison with wt TRPC6).
Figure 4
Figure 4. Increase in Ca2+ current in mutantsN143S, M132T and M132T/N143S.
A: Inward current of wt TRPC6 and the mutants measured at −120 mV in the presence of 100 µM CCH. Inward current level was close to 0 pA level in NMDG solution, and increased after the cell was exposed to 10 mM calcium/NMDG solutions. Note the significant larger calcium current amplitude of M132T and M132T/N143S in comparison with wt TRPC6 and M143S. B: Averaged current amplitude of wt TRPC6, N143S, M132T, and M132T/N143S at −120 mV (mean±SEM, n = 4−7). (**: p<0.01 in comparison with wt TRPC6).
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