FIGURE 2.

Gentamicin blocks connexin26 hemichannels in a reversible- and concentration-dependent manner and also blocks connexin43 and connexin45 hemichanels. (A) Representative time-lapse experiment showing ethidium (Etd) uptake in cells transfected with rat connexin26 (HeLa-rCx26 cells) under control conditions (Ca²⁺/Mg²⁺), after exposure to DCFS, followed by 5 min exposure to DCFS containing 200 μM gentamicin and washout of gentamicin with DCFS (last 10 min), m₁, m₂, m₃, m₄ = average slope. (B) Average Etd uptake rate (experiments as shown in A); data were normalized to the control Etd uptake level (dotted line). (C) Concentration-response curve showing the inhibition of DCFS-induced Etd uptake prompted by different gentamicin concentrations in HeLa-rCx26 cells. Each point represents the mean ± SEM (n = 3). Values were normalized to the maximal response in the absence of gentamicin and were included in the Hill equation, IC₅₀ = 133.4 ± 1.1 μM, R² = 0.95. (D) Etd uptake rates of HeLa cells expressing rat Cx26 (rCx26), mouse Cx26 (mCx26), rat Cx26-GFP (rCx26-GFP), mouse Cx43 (mCx43) or mouse Cx45 (mCx45), after exposure to DCFS followed by 5 min exposure to DCFS containing 200 μM gentamicin. Data were normalized to the Etd uptake value measured under control conditions (Ca²⁺/Mg²⁺ solution, dotted line) and are presented as mean ± SEM (n = 3). (E) Representative time-lapse curve showing Etd uptake in HeLa-rCx26 cells in control conditions and after exposure to DCFS followed by 200 μM streptomycin, m₁, m₂, m₃ = average slope. (F) DCFS-induced Etd uptake normalized to control uptake in absence (gray bar) or presence (white bar) of 200 μM streptomycin of HeLa cells expressing rCx26, mCx43, or mCx45. Values recorded in at least 30 cells per experiment were included (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.