XB-ART-46932Front Genet January 1, 2013; 4 54.
Dysfunction of the Heteromeric KV7.3/KV7.5 Potassium Channel is Associated with Autism Spectrum Disorders.
Heterozygous mutations in the KCNQ3 gene on chromosome 8q24 encoding the voltage-gated potassium channel KV7.3 subunit have previously been associated with rolandic epilepsy and idiopathic generalized epilepsy (IGE) including benign neonatal convulsions. We identified a de novo t(3;8) (q21;q24) translocation truncating KCNQ3 in a boy with childhood autism. In addition, we identified a c.1720C > T [p.P574S] nucleotide change in three unrelated individuals with childhood autism and no history of convulsions. This nucleotide change was previously reported in patients with rolandic epilepsy or IGE and has now been annotated as a very rare SNP (rs74582884) in dbSNP. The p.P574S KV7.3 variant significantly reduced potassium current amplitude in Xenopus laevis oocytes when co-expressed with KV7.5 but not with KV7.2 or KV7.4. The nucleotide change did not affect trafficking of heteromeric mutant KV7.3/2, KV7.3/4, or KV7.3/5 channels in HEK 293 cells or primary rat hippocampal neurons. Our results suggest that dysfunction of the heteromeric KV7.3/5 channel is implicated in the pathogenesis of some forms of autism spectrum disorders, epilepsy, and possibly other psychiatric disorders and therefore, KCNQ3 and KCNQ5 are suggested as candidate genes for these disorders.
PubMed ID: 23596459
PMC ID: PMC3627139
Article link: Front Genet
Species referenced: Xenopus
Genes referenced: ank1 ank3 cd4 kcnq2 kcnq3 kcnq4 kcnq5 map2 prpf31 tbx2 trh
Article Images: [+] show captions
|Figure 1. FISH mapping of translocation breakpoints reveals truncated KCNQ3 gene in patient A. (A) Schematic depiction of the 26 kb breakpoint region within chromosomal region 3q21.3 of patient A. The breakpoint region is located 10.5 kb downstream of the TRH gene and contains no genes. (B) Picture showing metaphase chromosomes from patient A (blue) hybridized with the chromosome 3 specific probe RP11-93K22 (green) that spans the breakpoint. The normal chromosome 3 as well as both derivative chromosomes are marked with white arrows and enlarged in the boxes to the right. (C) The 25 kb breakpoint region on chromosome 8 lies within intron 1 of the KCNQ3 gene. (D) The chromosome 8 specific probe RP11-213I2 (green) spans the breakpoint. The normal chromosome 8 as well as the two derivative chromosomes are marked with white arrows and enlarged.|
|Figure 2. c.1720C > T [p.P574S] variant detected in patient B, C and D. (A) The c.1720 C > T mutation in patient B was identified by Sanger sequencing. (B) The same mutation was identified in patient C and his mother, in patient D and his father and confirmed in patient B and his father by restriction enzyme cleavage of a PCR product encompassing the mutation.|
|Figure 3. Effect of KV7.3_P574S on KV7.2 and KV7.4 in X. laevis oocytes. Currents were activated by voltage-steps from −80 mV to +40 mV in 10 mV increments. Representative currents are shown as well as steady-state current plotted as a function of voltage. (A) KV7.2 was co-expressed with either KV7.3 WT (n = 6) or P574S (n = 5). (B) Effect of KV7.3_P574S on KV7.4. KV7.4 was co-expressed with either KV7.3 WT (n = 10) or P574S (n = 13) in X. laevis oocytes.|
|Figure 4. Effect of KV7.3_P574S on KV7.5. KV7.5 was expressed alone (n = 8) or co-expressed with either KV7.3 WT (n = 7), P547S (n = 6), or KV7.5 mixed with KV7.3 WT or KV7.3_P574S (in a 1:1 molar ratio, Hetero, n = 6) in X. laevis oocytes. Currents were activated by voltage-steps from −80 to +40 mV in 10 mV increments. (A) Representative currents are shown. (B) Steady-state current plotted as a function of voltage. Asterisks indicate statistical difference between Hetero and KV7.3/KV7.5. Comparison of the other points were left out for clarity and for voltages higher than 0 mV, all points were statistically different.|
|Figure 5. No effect of KV7.3_P574S on localization of the KV7.2/KV7.3 complex in HEK 293 cells. KV7.2 was transiently expressed in HEK 293 cells together with KV7.3 WT or KV7.3_P574S, and the localization of the complex was analyzed by immunocytochemistry and confocal microscopy. As illustrated, the P574S mutation is without effect on the localization of the complex that displays a primarily intracellular localization pattern. Scale bar 20 μm.|
|Figure 6. KV7.2/KV7.3_P574 complexes still target to the AIS of cultured hippocampal neurons. Confocal images of cultured rat hippocampal neurons (10 DIV) expressing KV7.3 WT or KV7.3_P574S (A) and co-transfected with KV7.2 (B). (A) KV7.3 is primarily intracellularly expressed when expressed on its own. No significant KV7.3 expression is observed at the AIS. (B) When co-expressing KV7.3 with KV7.2, both channel subunits are observed at the AIS. KV7.3_P574S displays the same localization characteristics. White arrowhead points to the location of the AIS. Ankyrin-G: marker of the axon initial segment, MAP2: marker of the somatodendritic region of neurons. Scale bars 50 and 20 μm, respectively.|
|Figure 7. CD4-KV7.3Cterm_P574 can target to the AIS. Confocal images of cultured rat hippocampal neurons (10 DIV) transfected with CD4 (two upper panels), CD4-KV7.3 C-term (two middle panels) and CD4-KV7.3_P574S C-term (two lower panels). The panels to the left illustrate the structure of the chimeric constructs analyzed. CD4-total reflects total CD4 staining in permeabilized cells. CD4-surface is a surface staining of the same cells where the CD4 antibody was applied before permeabilization. The somatodendritic marker MAP2 was included to indicate the location of the AIS (neurite which is MAP2 negative). As expected, CD4 distributes in a non-polarized manner on the surface of axon, soma and dendrites. When the C-terminal of KV7.3 is attached to CD4, the reporter redistributes to the axon initial segment, which illustrates that the KV7.3 C-terminal contains information for AIS localization. The C-terminus of KV7.3_P574S still has the ability to direct CD4 to the AIS. White arrowhead points to the location of the AIS. Scale bars 50 and 20 μm, respectively.|
|Figure 8. No effect of KV7.3_P574S on the localization of KV7.4 and KV7.5 containing complexes in HEK 293 cells. KV7.4 and KV7.5 were transiently expressed in HEK 293 cells together with KV7.3 or KV7.3_P574S and the localization of the expressed subunits analyzed by immunocytochemistry and confocal microscopy. As illustrated, the P574S mutation is without effect on the localization pattern of the complexes that displays a mixed surface and intracellular staining pattern. Scale bar 20 μm.|
|Figure 9. Localization and conservation of the SNP c.1720C > T [p.P574S] in KCNQ3. (A) The KV7 subunits consist of six transmembrane domains and a long intracellular carboxy-terminal that contains the subunit interaction domain (sid) (within the square). The P574 amino acid is located in the linker region between two coiled-coil regions. Adapted from (Wehling et al., 2007). (B) The c.1720C nucleotide is conserved between species. Adapted from the UCSC genome browser hg18. (C) Amino acid alignment of the si domains from all KV7 channels. The two coiled-coil domains are depicted as gray boxes and conserved amino acids are marked with an asterisk. The P574 amino acid is located in the linker region between two coiled-coil regions and is not conserved between KV7 members. Adapted from Wehling et al. (2007).|
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