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A single amino acid difference between ether-a-go-go- related gene channel subtypes determines differential sensitivity to a small molecule activator.

ABSTRACT: Activators of human ether-a-go-go-related gene 1 (hERG1) channels, such as (3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid (RPR260243), reverse the effect of hERG1 blockers and shorten the duration of cardiac action potentials. RPR260243 (RPR) slows the rate of deactivation and shifts the voltage dependence of channel inactivation to more positive potentials. We recently mapped the binding site for RPR to several residues located near the cytoplasmic ends of the S5 and S6 helices of the hERG1 subunit. These residues are conserved in the highly homologous ether-a-go-go-related gene 3 (ERG3) subunit; however, RPR blocks ERG3 channels. Here, we compare hERG1 and rat ERG3 (rERG3) channels to explore the molecular basis for differential channel sensitivity to RPR. Channels were heterologously expressed in Xenopus laevis oocytes, and currents were recorded using the two-electrode voltage-clamp technique. Site-directed mutagenesis was used to swap the two residues within the putative binding domain that differed between hERG1 and rERG3. The differential sensitivity of hERG1 and rERG3 channels to the agonist effect of RPR could be accounted for by a single S5 residue (Thr556 in hERG1, Ile558 in rERG3). A Thr in this position favors agonist activity, whereas an Ile reveals a secondary blocking effect of RPR.

SUBMITTER: Perry M

PROVIDER: S-EPMC2517227 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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A single amino acid difference between ether-a-go-go- related gene channel subtypes determines differential sensitivity to a small molecule activator.

Perry Matthew M Sanguinetti Michael C MC

Molecular pharmacology 20071227 4

Activators of human ether-a-go-go-related gene 1 (hERG1) channels, such as (3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid (RPR260243), reverse the effect of hERG1 blockers and shorten the duration of cardiac action potentials. RPR260243 (RPR) slows the rate of deactivation and shifts the voltage dependence of channel inactivation to more positive potentials. We recently mapped the binding site for RPR to several residu ...[more]

PMID: 18162604

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Project description:The human ether-à-go-go-1 (h-eag1) K(+) channel is expressed in a variety of cell lines derived from human malignant tumors and in clinical samples of several different cancers, but is otherwise absent in normal tissues. It was found to be necessary for cell cycle progression and tumorigenesis. Specific inhibition of h-eag1 expression leads to inhibition of tumor cell proliferation. We report here that h-eag1 expression is controlled by the p53-miR-34-E2F1 pathway through a negative feed-forward mechanism. We first established E2F1 as a transactivator of h-eag1 gene through characterizing its promoter region. We then revealed that miR-34, a known transcriptional target of p53, is an important negative regulator of h-eag1 through dual mechanisms by directly repressing h-eag1 at the post-transcriptional level and indirectly silencing h-eag1 at the transcriptional level via repressing E2F1. There is a strong inverse relationship between the expression levels of miR-34 and h-eag1 protein. H-eag1antisense antagonized the growth-stimulating effects and the upregulation of h-eag1 expression in SHSY5Y cells, induced by knockdown of miR-34, E2F1 overexpression, or inhibition of p53 activity. Therefore, p53 negatively regulates h-eag1 expression by a negative feed-forward mechanism through the p53-miR-34-E2F1 pathway. Inactivation of p53 activity, as is the case in many cancers, can thus cause oncogenic overexpression of h-eag1 by relieving the negative feed-forward regulation. These findings not only help us understand the molecular mechanisms for oncogenic overexpression of h-eag1 in tumorigenesis but also uncover the cell-cycle regulation through the p53-miR-34-E2F1-h-eag1 pathway. Moreover, these findings place h-eag1 in the p53-miR-34-E2F1-h-eag1 pathway with h-eag as a terminal effecter component and with miR-34 (and E2F1) as a linker between p53 and h-eag1. Our study therefore fills the gap between p53 pathway and its cellular function mediated by h-eag1.

Dataset Information

A single amino acid difference between ether-a-go-go- related gene channel subtypes determines differential sensitivity to a small molecule activator.

Publications

A single amino acid difference between ether-a-go-go- related gene channel subtypes determines differential sensitivity to a small molecule activator.

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