Functional characterization of the LQT2-causing mutation R582C and the associated voltage-dependent fluorescence signal

Background The R582C mutation is one of many Long-QT Syndrome type 2 (LQT2)-causing mutations localized to the human ether-a-go-go related gene (hERG) channel's S5-P linker subdomain, yet its specific mechanism of dysfunction has not been examined. Objective This study sought to characterize the biophysical properties of the congenital LQT2-causing mutation, R582C, and utilize this mutation to provide the first report of voltage-dependent fluorescence from the S5-P linker. Methods Properties of the R582C channels were characterized by heterologous expression in both HEK293 cells and Xenopus oocytes using a combination of patch-clamp, 2-electrode voltage-clamp, immunoblot assay, and voltage-clamp fluorimetry. Results Expression of hERG R582C was found to be deficient in HEK293 cells, yet was amenable to rescue by incubation at reduced temperature or by treatment with dofetilide. Rescued channels expressed at levels comparable to wild type (WT) channels. Kinetic differences result in decreased outward repolarizing current evoked by an action potential clamp protocol. Voltage-clamp fluorimetry experiments utilized the introduced cysteine to covalently attach a fluorescent probe (tetramethylrhodamine-5-maleimide) to the S5-P linker to directly observe conformational changes occurring due to inactivation. Conclusion The major mechanism underlying pathogenicity of the R582C mutation is a trafficking deficiency, although channels also exhibit kinetic deficiencies, perhaps reflecting the position of the mutation in the pore turret. Voltage clamp fluorescence signals from R582C channels provide evidence that the hERG turret undergoes distinct conformational changes during inactivation.