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Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating.

ABSTRACT: A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG) potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET) analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF) conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.

SUBMITTER: Fernandez-Trillo J

PROVIDER: S-EPMC3174182 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating.

Fernández-Trillo Jorge J Barros Francisco F Machín Angeles A Carretero Luis L Domínguez Pedro P de la Peña Pilar P

PloS one 20110915 9

A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG) potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the p ...[more]

PMID: 21935437

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Project description:Human ether-á-go-go (eag)-related gene (hERG) potassium channel kinetics are characterized by rapid inactivation upon depolarization, along with rapid recovery from inactivation and very slow closing (deactivation) upon repolarization. These factors combine to create a resurgent hERG current, where the current amplitude is paradoxically larger with repolarization than with depolarization. Previous data showed that the hERG N-terminal eag domain regulated deactivation kinetics by making a direct interaction with the C-terminal region of the channel. A primary mechanism for fast inactivation depends on residues in the channel pore; however, inactivation was also shown to be slower after deletion of a large N-terminal region. The mechanism for N-terminal region regulation of inactivation is unclear. Here, we investigated the contributions of the large N-terminal domains (amino acids 1-354), including the eag domain (amino acids 1-135), to hERG channel inactivation kinetics and steady-state inactivation properties. We found that N-deleted channels lacking just the eag domain (?2-135) or both the eag domain and the adjacent proximal domain (?2-354) had less rectifying current-voltage (I-V) relationships, slower inactivation, faster recovery from inactivation, and lessened steady-state inactivation. We coexpressed genetically encoded N-terminal fragments for the eag domain (N1-135) or the eag domain plus the proximal domain (N1-354) with N-deleted hERG ?2-135 or hERG ?2-354 channels and found that the resulting channels had more rectifying I-V relationships, faster inactivation, slower recovery from inactivation, and increased steady-state inactivation, similar to those properties measured for wild-type (WT) hERG. We also found that the eag domain-containing fragments regulated the time to peak and the voltage at the peak of a resurgent current elicited with a ramp voltage protocol. The eag domain-containing fragments effectively converted N-deleted channels into WT-like channels. Neither the addition of the proximal domain to the eag domain in N1-354 fragments nor the presence of the proximal domain in hERG ?2-135 channels measurably affected inactivation properties; in contrast, the proximal region regulated steady-state activation in hERG ?2-135 channels. The results show that N-terminal region-dependent regulation of channel inactivation and resurgent current properties are caused by a direct interaction of the eag domain with the rest of the hERG channel.

Dataset Information

Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating.

Publications

Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating.

Similar Datasets

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