title: Mechanistic basis for the pathogenesis of long QT syndrome associated with a common splicing mutation in KCNQ1 gene.
abstract: Mutations in KCNQ1, the gene encoding the delayed rectifier K(+) channel in cardiac muscle, cause long QT syndrome (LQTS). We studied 3 families with LQTS, in whom a guanine to adenine change in the last base of exon 7 (c.1032G>A), previously reported as a common splice-site mutation, was identified. We performed quantitative measurements of exon-skipping KCNQ1 mRNAs caused by this mutation using real-time reverse transcription polymerase chain reaction. Compared with normal individuals who have minor fractions of splicing variants (Delta7-8: 0.1%, Delta8: 6.9%, of total KCNQ1 transcripts), the affected individuals showed remarkable increases of exon-skipping mRNAs (Delta7: 23.5%, Delta7-8: 16.8%, Delta8: 4.5%). Current recordings from Xenopus laevis oocytes heterologously expressing channels of wild-type (WT) or exon-skipping KCNQ1 (Delta7, Delta7-8, or Delta8) revealed that none of the mutants produced any measurable currents, and moreover they displayed mutant-specific degree of dominant-negative effects on WT currents, when co-expressed with WT. Confocal microscopy analysis showed that fluorescent protein-tagged WT was predominantly expressed on the plasma membrane, whereas the mutants showed intracellular distribution. When WT was co-expressed with mutants, the majority of WT co-localized with the mutants in the intracellular space. Finally, we provide evidence showing direct protein-protein interactions between WT and the mutants, by using fluorescence resonance energy transfer. Thus, the mutants may exert their dominant-negative effects by trapping WT intracellularly and thereby interfering its translocation to the plasma membrane. In conclusion, our data provide a mechanistic basis for the pathogenesis of LQTS caused by a splicing mutation in KCNQ1.
authors.affiliation: Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Tsuji
authors.forename: Masaharu
authors.initials: M
authors.lastname: Akao
authors.forename: Takahiro M
authors.initials: TM
authors.lastname: Ishii
authors.forename: Seiko
authors.initials: S
authors.lastname: Ohno
authors.forename: Takeru
authors.initials: T
authors.lastname: Makiyama
authors.forename: Kotoe
authors.initials: K
authors.lastname: Takenaka
authors.forename: Takahiro
authors.initials: T
authors.lastname: Doi
authors.forename: Yoshisumi
authors.initials: Y
authors.lastname: Haruna
authors.forename: Hidetada
authors.initials: H
authors.lastname: Yoshida
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Nakashima
authors.forename: Toru
authors.initials: T
authors.lastname: Kita
authors.forename: Minoru
authors.initials: M
authors.lastname: Horie
mesh_terms: D000818:Animals; D001483:Base Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D019556:COS Cells; D002522:Chlorocebus aethiops; D004594:Electrophysiology; D005091:Exons / Q000235:genetics; D051657:KCNQ1 Potassium Channel / Q000235:genetics* / Q000378:metabolism*; D008133:Long QT Syndrome / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D018408:Patch-Clamp Techniques; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014982:Xenopus laevis
chemical_list: D051657:KCNQ1 Potassium Channel; D012333:RNA, Messenger