Determining the role of the S4-S5 Linker in hERG eag Domain Regulation of Channel Gating

Abstract

Human ether-a-go-go-related gene (hERG) potassium channels exhibit unique kinetic properties, including unusually slow deactivation kinetics, which help to specialize them for their role in the heart. Previously, we demonstrated that an N-terminal eag domain is essential in regulating channel deactivation kinetics. The mechanism by which the eag domain regulates gating remains unclear. Recent evidence suggests the intracellular loop between the S4 and S5 transmembrane domains (S4-S5 linker) may be important in regulating both activation and deactivation, and that modulation of gating by the eag domain may act via the S4-S5 linker. Here we sought to investigate the role of the S4-S5 linker using site-directed mutagenesis and a combination of electrophysiology and Forster Resonance Energy Transfer (FRET). We found that channels with alanine mutations in the S4-S5 linker exhibited altered gating. All the S4-S5 mutant channels caused an acceleration of deactivation kinetics, except for S543A which had significantly slowed deactivation. Co-expressing an eag domain gene fragment (N-eag) with S4-S5 mutant channels which additionally lacked a native eag domain (Δeag) failed to restore slow deactivation kinetics to the mutant channels. FRET analysis revealed that eag domains tagged with a CFP were in close proximity to each of the S4-S5 mutant channels tagged with a Citrine. Replacement of the entire S4-S5 linker with alanines (hERG[S4-S5]Ala) produced channels with altered gating, including fast deactivation and a far left-shifted steady-state activation curve. Co-expression of hERG Δeag[S4-S5]Ala channels with N-eag did not alter channel gating; however, FRET analysis revealed that N-eag was in close proximity to the mutant channels. Together, these findings suggest that an intact S4-S5 linker is necessary to transduce eag domain-dependent regulation of gating, but it is not required for the eag domain to bind to the channel.