title: Functional analysis of potassium channels in Kv7.2 G271V mutant causing early onset familial epilepsy.
abstract: Kv7 (KCNQ) channels underlying a class of voltage-gated K+ current are best known for regulating neuronal excitability. The first glycine (G) residue in the pore helix of Kv7.2 (KCNQ2) subunit is highly conserved among different classes of Kv7 channel family. A missense mutation causing the replacement of the corresponding G residues with a valine (p.G271V) in Kv7.2 was found in a large, four-generation pedigree. Here, we set out to examine the molecular pathomechanism of G271V mutants using patch clamp technology combined with biochemical and immunocytochemical techniques in transiently transfected human embryonic kidney (HEK) 293 cells. The expression of Kv7.2 protein had the same intensity for both wild type (WT) and G271V. In transfected HEK cells, G271V mutants induced large depolarizing shifts of the conductance-voltage relationships and marked slowing of current activation kinetics compared to WT. In addition, G271V mutants abolished currents in homomeric channels, and resulted in about 50% reduction of current in Kv7.2/G271V/Kv7.3 heteromultimeric condition, indicating a more severe functional defect. To test for G271V mutant channel expression in surface membrane, we performed fluorescence confocal microscopy imaging, which revealed no differences between the mutant and WT, suggesting that G271V channels fail to open in response to depolarization even though they are present in the membrane. Furthermore, pharmacologic intervention experiments revealed that upon specific incubation of transfected HEK 293 cells expressing G271V heteromultimeric channels in presence of Kv7 channel enhancer retigabine (ezogabine), the potassium currents increased significantly, suggesting the potential of retigabine as gene-specific therapy.
authors.affiliation: Department of Neonatology, First Affiliated Hospital of Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China; Ion Channel Disease Laboratory, Key Laboratory of Environment and Gene Associated Diseases, Ministry of Education, Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China.
authors.forename: Juanjuan
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Neonatology, First Affiliated Hospital of Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Neonatology, First Affiliated Hospital of Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China; Ion Channel Disease Laboratory, Key Laboratory of Environment and Gene Associated Diseases, Ministry of Education, Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China.
authors.forename: Zhiyan
authors.initials: Z
authors.lastname: Hui
authors.affiliation: Department of Neonatology, First Affiliated Hospital of Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Cao
authors.affiliation: Ion Channel Disease Laboratory, Key Laboratory of Environment and Gene Associated Diseases, Ministry of Education, Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China.
authors.forename: Ruiming
authors.initials: R
authors.lastname: Shi
authors.affiliation: Department of Neonatology, First Affiliated Hospital of Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China; Ion Channel Disease Laboratory, Key Laboratory of Environment and Gene Associated Diseases, Ministry of Education, Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Ion Channel Disease Laboratory, Key Laboratory of Environment and Gene Associated Diseases, Ministry of Education, Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China.
authors.forename: Limeng
authors.initials: L
authors.lastname: Geng
authors.affiliation: Department of Neonatology, First Affiliated Hospital of Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China; Ion Channel Disease Laboratory, Key Laboratory of Environment and Gene Associated Diseases, Ministry of Education, Xi'an Jiaotong University, No. 277, Yanta West Road, Xi'an, Shaanxi 710061, People's Republic of China. Electronic address: zhouxih@mail.xjtu.edu.cn.
authors.forename: Xihui
authors.initials: X
authors.lastname: Zhou
mesh_terms: D000927:Anticonvulsants / Q000494:pharmacology; D055592:Biophysical Phenomena / Q000187:drug effects / Q000235:genetics; D002219:Carbamates / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D005998:Glycine / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051658:KCNQ2 Potassium Channel / Q000235:genetics*; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics*; D008856:Microscopy, Fluorescence; D016296:Mutagenesis / Q000502:physiology; D009154:Mutation / Q000235:genetics*; D018408:Patch-Clamp Techniques; D010655:Phenylenediamines / Q000494:pharmacology; D013997:Time Factors; D014633:Valine / Q000235:genetics*
chemical_list: D000927:Anticonvulsants; D002219:Carbamates; D051658:KCNQ2 Potassium Channel; D010655:Phenylenediamines; C101866:ezogabine; D049452:Green Fluorescent Proteins; D014633:Valine; D005998:Glycine
keywords: Epilepsy; KCNQ2; Kv7.2; Patch-clamp electrophysiology; Voltage-gated potassium channel