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.

Keywords: aminoglycosides; cancer; connexins; hemichannels; inhibitors; lens.

Figure 1

Only HeLa cells transfected with Cx46tGFP show DAPI uptake in DCFS. HeLa cells were transfected with GFP or Cx46tGFP and DAPI uptake time-lapse recordings were performed. (a) Pictures of a culture of HeLa cells expressing GFP (HeLa-GFP) revealed that almost all HeLa cells express this protein (merge panels). After 20 min of exposure to 10 μM DAPI, HeLa-GFP cells do not show DAPI uptake, either under control conditions or in DCFS (DAPI panel). (b) Representative quantification of DAPI uptake in time-lapse experiments showing that the rate of DAPI uptake in HeLa-GFP cells under control (red dots) and DCFS (blue dots) conditions is very similar. (c) HeLa cells transfected with Cx46tGFP (HeLa Cx46tGFP) do not show significant DAPI uptake in control conditions (DAPI panels). However, DAPI uptake was greatly increased when cells were placed in DCFS. (d) Representative quantification of DAPI uptake in time-lapse experiments showing a significant increase in DAPI uptake in HeLa Cx46tGFP in DCFS (blue dots) in comparison to the control condition (red dots). Each dot corresponds to the average intensity of DAPI fluorescence in the nucleus of 16 different cells. (e) Graph showing the average of n = 3 independent experiments for each condition (control and DCFS). p > 0.05 = n.s, and p < 0.001= ***.

Figure 2

KI04 acts as an inhibitor of Cx46 hemichannels. HeLa cells transfected with Cx46tGFP were placed in DCFS complemented with 10 μM DAPI, and time-lapse experiments were performed. (a) Chemical structure of aminoglycosides and KI04 that were used in this work. Dose response of (b) geneticin (n = 4), (c) amikacin (n = 5), (d) kanamycin (n = 6), and (e) KI04 (n = 7) on DAPI uptake. In each plot, the rate of DAPI uptake was normalized against the rate of DAPI uptake measured in the absence of KI04 (dot line). For each experiment, DAPI fluorescence intensity was measured in 16 cells, showing GFP or Cx46tGFP fluorescence. p < 0.01 = ***.

Figure 3

KI04 does not induce changes in Cx46 molecular weight and cellular distribution. (a) Western blot analyses show that none of the KI04 concentrations used in this study (from 0.01 to 100 μM) induced changes in Cx46 electrophoretic mobility. Similarly, (b) 10 μM KI04 did not induce evident changes in Cx46 intracellular localization. (c). Addition of 5 mM DTT shows a tendency to reduce the rate of DAPI uptake, and moreover, it did not reverse the inhibition mediated by KI04. (d) On the contrary, for Cx46tGFP cells that were transiently exposed to 10 μM KI04, the inhibitory effect was completely reversed, and moreover, there was an increase in DAPI uptake (n = 4 for each experiment, 16 cells analyzed in each case). For each experiment, DAPI fluorescence intensity was measured in 16 cells. p < 0.05 = * and no statistical differences (p > 0.05), was denoted as ns.

Figure 4

KI04 inhibits Cx46-mediated hemichannel currents. Hemichannel currents were studied in HeLa cells expressing Cx46tGFP. (A) Voltage ramps from −80 to +80 mV were applied in whole-cell patch clamp configuration. Large currents were recorded in HeLa Cx46tGFP cells that were activated above +20 mV. These currents were decreased when cells were exposed to 10 μM KI04. (B) Quantification of the maximal current at +80 mV in n = 3 independent experiments, p < 0.05 = *.

Figure 5

KI04 shows two putative binding sites with Cx46 hemichannels. Human Cx46 hemichannels were modeled in silico, and a molecular docking analysis was performed in the presence of KI04. (a) In our model, KI04 showed two putative binding sites, (b) one close to the parahelix segment and (c) another inside of the pore, close to the cytosolic entrance.