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Probing the outer mouth structure of the HERG channel with peptide toxin footprinting and molecular modeling.

ABSTRACT: Previous studies have shown that the unusually long S5-P linker lining human ether a-go-go related gene's (hERG's) outer vestibule is critical for its channel function: point mutations at high-impact positions here can interfere with the inactivation process and, in many cases, also reduce the pore's K+ selectivity. Because no data are available on the equivalent region in the available K channel crystal structures to allow for homology modeling, we used alternative approaches to model its three-dimensional structure. The first part of this article describes mutant cycle analysis used to identify residues on hERG's outer vestibule that interact with specific residues on the interaction surface of BeKm-1, a peptide toxin with known NMR structure and a high binding affinity to hERG. The second part describes molecular modeling of hERG's pore domain. The transmembrane region was modeled after the crystal structure of KvAP pore domain. The S5-P linker was docked to the transmembrane region based on data from previous NMR and mutagenesis experiments, as well as a set of modeling criteria. The models were further restrained by contact points between hERG's outer vestibule and the bound BeKm-1 toxin molecule deduced from the mutant cycle analysis. Based on these analyses, we propose a working model for the open conformation of the outer vestibule of the hERG channel, in which the S5-P linkers interact with the pore loops to influence ion flux through the pore.

SUBMITTER: Tseng GN

PROVIDER: S-EPMC1853143 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Probing the outer mouth structure of the HERG channel with peptide toxin footprinting and molecular modeling.

Tseng Gea-Ny GN Sonawane Kailas D KD Korolkova Yuliya V YV Zhang Mei M Liu Jie J Grishin Eugene V EV Guy H Robert HR

Biophysical journal 20070209 10

Previous studies have shown that the unusually long S5-P linker lining human ether a-go-go related gene's (hERG's) outer vestibule is critical for its channel function: point mutations at high-impact positions here can interfere with the inactivation process and, in many cases, also reduce the pore's K+ selectivity. Because no data are available on the equivalent region in the available K channel crystal structures to allow for homology modeling, we used alternative approaches to model its three ...[more]

PMID: 17293393

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Project description:Most voltage-gated K(+) currents are relatively insensitive to extracellular Na(+) (Na(+)(o)), but Na(+)(o) potently inhibits outward human ether-a-go-go-related gene (HERG)-encoded K(+) channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429-30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na(+) (Na(+)(i)) and extracellular Ba(2+) (Ba(2+)(o)), to define a site where Na(+)(o) interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na(+)(o) was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K(+) selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na(+)(o). Quantitatively similar effects of Na(+)(i) to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na(+)(o) sensitivity, this result suggested that Na(+)(o) and Na(+)(i) act at different sites. Extracellular Ba(2+) (Ba(2+)(o)) blocks K(+) channel pores, and thereby serves as a useful probe of K(+) channel structure. HERG channel inactivation promotes relief of Ba(2+) block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265-278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na(+)(o) action. A remote allosteric model predicts that Na(+)(o) will speed relief of Ba(2+)(o) block by promoting inactivation. Instead, Na(+)(o) slowed Ba(2+) egress and Ba(2+) relieved Na(+)(o) inhibition, consistent with Na(+)(o) binding to an outer pore site. The apparent affinities of the outer pore for Na(+)(o) and K(+)(o) as measured by slowing of Ba(2+) egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na(+)(o) inhibition. Na(+)(o) inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.

Dataset Information

Probing the outer mouth structure of the HERG channel with peptide toxin footprinting and molecular modeling.

Publications

Probing the outer mouth structure of the HERG channel with peptide toxin footprinting and molecular modeling.

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