Dataset Information

KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells.

ABSTRACT:

Background

Connexins (Cxs) are proteins that help cells to communicate with the extracellular media and with the cytoplasm of neighboring cells. Despite their importance in several human physiological and pathological conditions, their pharmacology is very poor. In the last decade, some molecules derived from aminoglycosides have been developed as inhibitors of Cxs hemichannels. However, these studies have been performed in E. coli, which is a very simple model. Therefore, our main goal is to test whether these molecules have similar effects in mammalian cells.

Methods

We transfected HeLa cells with the human Cx46tGFP and characterized the effect of a kanamycin-derived molecule (KI04) on Cx46 hemichannel activity by time-lapse recordings, changes in phosphorylation by Western blot, localization by epifluorescence, and possible binding sites by molecular dynamics (MD).

Results

We observed that kanamycin and KI04 were the most potent inhibitors of Cx46 hemichannels among several aminoglycosides, presenting an IC₅₀ close to 10 μM. The inhibitory effect was not associated with changes in Cx46 electrophoretic mobility or its intracellular localization. Interestingly, 5 mM DTT did not reverse KI04 inhibition, but the KI04 effect completely disappeared after washing out KI04 from the recording media. MD analysis revealed two putative binding sites of KI04 in the Cx46 hemichannel.

Results

These results demonstrate that KI04 could be used as a Cx46 inhibitor and could help to develop future selective Cx46 inhibitors.

SUBMITTER: Chang CT

PROVIDER: S-EPMC10046693 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Publications

KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells.

Chang Cheng-Wei T CT Poudyal Naveena N Verdugo Daniel A DA Peña Francisca F Stehberg Jimmy J Retamal Mauricio A MA

Biomolecules 20230222 3

<h4>Background</h4>Connexins (Cxs) are proteins that help cells to communicate with the extracellular media and with the cytoplasm of neighboring cells. Despite their importance in several human physiological and pathological conditions, their pharmacology is very poor. In the last decade, some molecules derived from aminoglycosides have been developed as inhibitors of Cxs hemichannels. However, these studies have been performed in <i>E. coli</i>, which is a very simple model. Therefore, our mai ...[more]

PMID: 36979346

Similar Datasets

Project description:Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca2+. Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs under physiological and pathological conditions in cells that express them.

Dataset Information

KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells.

Background

Methods

Results

Results

Publications

KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells.

Similar Datasets

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