Resonance assignment and secondary structure prediction of the N-terminal domain of hERG (Kv11.1)

Muskett, Frederick W.; Mitcheson, John S.

doi:10.1007/s12104-010-9256-3

Cited by 5 publications

(5 citation statements)

References 9 publications

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“…The EAG domain (residues 1–135) was subcloned into an Escherichia coli expression vector with a C terminus hexahistidine tag as described previously ( 29 ). All NMR experiments were performed on Bruker DRX or AvanceII spectrometers operating at either 600 or 800 MHz that were fitted with cryogenically cooled probe heads.…”

Section: Methodsmentioning

confidence: 99%

“…The samples were prepared in 95% H 2 O and 5% D 2 O. Detailed descriptions of the multidimensional experiments used to obtain the near complete sequence-specific 1 H, 13 C, and 15 N assignments have been reported previously ( 29 ). Additionally, 15 N-NOESY-HSQC ( 30 ) and 13 C-NOESY-HSQC ( 31 ) spectra were acquired with an NOE mixing period of 100 ms.…”

Section: Methodsmentioning

confidence: 99%

“…Two-dimensional 15 N/ 1 H-HSQC spectra were also acquired from a sample of the 15 N-labeled EAG domain dissolved in D 2 O to identify any slowly exchanging amide protons in the protein ( 33 ). All NMR data were processed essentially as described previously ( 29 ) using Topspin (Bruker Biospin Ltd.), and the resulting spectra were analyzed using the program Sparky (T. D. Goddard and D. G. Kneller, Sparky 3, University of California, San Francisco).…”

Section: Methodsmentioning

confidence: 99%

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Mechanistic Insight into Human ether-à-go-go-related Gene (hERG) K+ Channel Deactivation Gating from the Solution Structure of the EAG Domain

Muskett

Thouta

Thomson

et al. 2011

Journal of Biological Chemistry

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119

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Human ether-à-go-go-related gene (hERG) K+ channels have a critical role in cardiac repolarization. hERG channels close (deactivate) very slowly, and this is vital for regulating the time course and amplitude of repolarizing current during the cardiac action potential. Accelerated deactivation is one mechanism by which inherited mutations cause long QT syndrome and potentially lethal arrhythmias. hERG deactivation is highly dependent upon an intact EAG domain (the first 135 amino acids of the N terminus). Importantly, deletion of residues 2–26 accelerates deactivation to a similar extent as removing the entire EAG domain. These and other experiments suggest the first 26 residues (NT1–26) contain structural elements required to slow deactivation by stabilizing the open conformation of the pore. Residues 26–135 form a Per-Arnt-Sim domain, but a structure for NT1–26 has not been forthcoming, and little is known about its site of interaction on the channel. In this study, we present an NMR structure for the entire EAG domain, which reveals that NT1–26 is structurally independent from the Per-Arnt-Sim domain and contains a stable amphipathic helix with one face being positively charged. Mutagenesis and electrophysiological studies indicate that neutralizing basic residues and breaking the amphipathic helix dramatically accelerate deactivation. Furthermore, scanning mutagenesis and molecular modeling studies of the cyclic nucleotide binding domain suggest that negatively charged patches on its cytoplasmic surface form an interface with the NT1–26 domain. We propose a model in which NT1–26 obstructs gating motions of the cyclic nucleotide binding domain to allosterically stabilize the open conformation of the pore.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Mechanistic Insight into Human ether-à-go-go-related Gene (hERG) K+ Channel Deactivation Gating from the Solution Structure of the EAG Domain

Muskett

Thouta

Thomson

et al. 2011

Journal of Biological Chemistry

Self Cite

119

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“…The N-terminal region of all members of the ether-à-go-go K + channel family contains the cap sequence and the PAS domain that are collectively known as the eag domain [24] , [25] , [26] , [27] , [28] . In addition, the N-terminal region contains an N-linker region that connects the eag domain with the transmembrane S1 segment, with the Eag N-linker region being about 190 amino acids shorter than its counterpart in Erg.…”

Section: Introductionmentioning

confidence: 99%

The Eag Domain Regulates the Voltage-Dependent Inactivation of Rat Eag1 K+ Channels

Lin¹,

Jow

Fang³

et al. 2014

PLoS ONE

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Eag (Kv10) and Erg (Kv11) belong to two distinct subfamilies of the ether-à-go-go K+ channel family (KCNH). While Erg channels are characterized by an inward-rectifying current-voltage relationship that results from a C-type inactivation, mammalian Eag channels display little or no voltage-dependent inactivation. Although the amino (N)-terminal region such as the eag domain is not required for the C-type inactivation of Erg channels, an N-terminal deletion in mouse Eag1 has been shown to produce a voltage-dependent inactivation. To further discern the role of the eag domain in the inactivation of Eag1 channels, we generated N-terminal chimeras between rat Eag (rEag1) and human Erg (hERG1) channels that involved swapping the eag domain alone or the complete cytoplasmic N-terminal region. Functional analyses indicated that introduction of the homologous hERG1 eag domain led to both a fast phase and a slow phase of channel inactivation in the rEag1 chimeras. By contrast, the inactivation features were retained in the reverse hERG1 chimeras. Furthermore, an eag domain-lacking rEag1 deletion mutant also showed the fast phase of inactivation that was notably attenuated upon co-expression with the rEag1 eag domain fragment, but not with the hERG1 eag domain fragment. Additionally, we have identified a point mutation in the S4–S5 linker region of rEag1 that resulted in a similar inactivation phenotype. Biophysical analyses of these mutant constructs suggested that the inactivation gating of rEag1 was distinctly different from that of hERG1. Overall, our findings are consistent with the notion that the eag domain plays a critical role in regulating the inactivation gating of rEag1. We propose that the eag domain may destabilize or mask an inherent voltage-dependent inactivation of rEag1 K+ channels.

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“…While one mammalian voltage-gated potassium channel, Kv1.2 [15], has been crystallized, the crystal structure is limited to the transmembrane domains. We turned to the human Ether-a-go-go-related gene (HERG) potassium channel, for which a nuclear magnetic resonance (NMR) structure exists for the first 23 residues of the intracellular N-terminal domain [16][17][18]. Importantly, the first half of this domain is unstructured, whereas the second half is alpha helical.…”

Section: Introductionmentioning

confidence: 99%

Binding interface of cardiac potassium channel proteins identified by hydrogen deuterium exchange of synthetic peptides

et al. 2012

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Three synthetic peptides, derived from the human potassium channel proteins Ether-a-go-go-related gene (HERG), KCNQ1, and KCNE1, were investigated by hydrogen deuterium exchange coupled with electron-transfer dissociation mass spectrometry at single residue resolution. Each amino acid residue in the first half of the HERG peptide incorporated deuterons with a higher rate than those in the second half of the peptide, consistent with the nuclear magnetic resonance structure of this peptide, with amino acids 1-10 being a flexible coil, whereas amino acids 11-24 are a stable amphipathic helix. The binding interface of KCNQ1 and KCNE1 was determined by comparing the difference of sequential fragment ions before and after binding. The residues determined to be involved in binding were consistent with a cysteine cross-linking study and confirmed by double mutant cycle analysis.

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Resonance assignment and secondary structure prediction of the N-terminal domain of hERG (Kv11.1)

Cited by 5 publications

References 9 publications

Mechanistic Insight into Human ether-à-go-go-related Gene (hERG) K+ Channel Deactivation Gating from the Solution Structure of the EAG Domain

Mechanistic Insight into Human ether-à-go-go-related Gene (hERG) K+ Channel Deactivation Gating from the Solution Structure of the EAG Domain

The Eag Domain Regulates the Voltage-Dependent Inactivation of Rat Eag1 K+ Channels

Binding interface of cardiac potassium channel proteins identified by hydrogen deuterium exchange of synthetic peptides

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