Abstract 3398: Mechanistic Basis for the Pathogenesis of Long QT Syndrome Caused by a Splicing Mutation in KCNQ1 Gene
Background: 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.
Methods and Results: 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 (Δ7– 8: 0.1%, Δ8: 6.9%, of total KCNQ1 mRNA), the patients showed remarkable increases of exon-skipping mRNAs (Δ7: 23.5%, Δ7– 8: 16.8%, Δ8: 4.5%). Current recordings from Xenopus laevis oocytes heterologously expressing channels of full-length (FL) or exon-skipping KCNQ1 (Δ7, Δ7– 8, or Δ8) revealed that none of the mutants produced any measurable currents, and moreover they displayed mutant-specific degree of dominant-negative effects on FL currents, when co-expressed with FL. Oocytes injected with the cRNA ratios of the patients showed pronounced reduction in currents (0.67 μA) compared with those with the ratios of normal individuals (1.55 μA). Confocal microscopy analysis showed that fluorescent protein-tagged FL was predominantly expressed on the plasma membrane, whereas the mutants showed intracellular distribution. When FL was co-expressed with mutants, the majority of FL co-localized with the mutants in the intracellular space. Finally, we provide evidence showing direct protein-protein interactions between FL and the mutants, by using fluorescence resonance energy transfer. Thus, the mutants may exert their dominant-negative effects by trapping FL intracellularly and thereby interfering its translocation to the plasma membrane.
Conclusion: Our data provide a mechanistic basis for the pathogenesis of LQTS caused by a common splicing mutation in KCNQ1.