Figueroa VA, Retamal MA, Cea LA, Salas JD, Vargas AA, Verdugo CA, Jara O, Martínez AD, Sáez JC.

	FIGURE 1. Gentamicin blocks the ethidium uptake induced by a divalent cation-free solution (DCFS) in HeLa-Cx26 cells. The ethidium (Etd) uptake was evaluated in HeLa-Parental or -Cx26 cells bathed with divalent cation (Ca2+/Mg2+) or DCFS solution in time-lapse measurement experiments. (A) Representative fluorescent fields showing Etd florescence of HeLa-Parental or -rCx26 cells incubated in saline solution containing 5 μM Etd, under control conditions (Ca2+/Mg2+, 5 min), after exposure to DCFS (10 min) or after 5 min exposure to DCFS containing 200 μM gentamicin. Scale bar, 40 μm. (B) Representative time-lapse experiments showing Etd uptake in HeLa-Cx26 and -Parental cells under control conditions (Ca2+/Mg2+, first 5 min), after exposure to DCFS solution followed by the application of 200 μM gentamicin (last 5 min). m1, m2, m3 = average slope. Measurements were taken every 30 s as fluorescence emission intensity of Etd bound to DNA and referred as fluorescence intensity expressed in arbitrary units (AUs). Each value corresponds to the mean ± SEM of at least 30 cells. (C) Etd uptake rate of Hela-Parental or -rCx26 cells measured under control conditions and after exposure to DCFS with or without 200 μM La3+ or 200 μM gentamicin. Data are presented as means ± SEM, the digit within each bar corresponds to the number of independent experiments under that condition. ***P < 0.001.
	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.
	FIGURE 3. Gentamicin inhibits the membrane current mediated by human connexin hemichannels. The membrane current of Xenopus laevis oocytes injected with connexin38 antisense oligonucleotide and human connexin (Cx26) cRNA was evaluated in whole cell modality using two electrodes. (A) Currents induced by depolarization from -60 to +40 mV (10 mV steps, for 15 s) under control condition (upper panel) or in the presence of 300 or 600 μM gentamicin (middle panel) and 300 μM gentamicin plus 200 μM La3+ (lower panel). (B) Currents induced by depolarization from -60 to +40 mV in oocytes non-injected with Cx26 cRNA under control conditions (upper panel) or in the presence of 300 or 600 μM gentamicin (middle panel) and 300 μM gentamicin plus 200 μM La3+ (lower panel). (C) Average maximal tail currents from 5 oocytes in the presence or absence of 300 or 600 μM gentamicin and 300 μM gentamicin plus 200 μM La3+. ***P < 0.005 and n.s: not significant.
	FIGURE 4. Gentamicin inhibits the membrane current mediated by human connexin26 hemichannels in nominal Ca2+-free solution. The membrane current of Xenopus laevis oocytes injected with connexin38 antisense oligonucleotide and human connexin26 cRNA was evaluated in whole cell modality using two electrodes in ND96 without Ca2+ and Mg2+. (A) Currents induced by depolarization from -60 to +40 mV (10 mV steps, for 15 s) under control conditions (upper panel) or in the presence of 300 μM gentamicin (lower panel). (B) Average maximal currents at +40 mV from 5 oocytes in the presence or absence of 300 μM gentamicin. *P < 0.01.
	FIGURE 5. Gentamicin and hemichannel blockers reduce the ATP-evoked Ca2+ signal in HeLa-rCx26GFP cells. HeLa-Parental cells or HeLa cells stably transfected with rCx26-GFP were loaded with Fura-2 and then stimulated with bath application of 10 μM ATP. (A) Time course of ATP-evoked intracellular Ca2+ signal under control conditions in HeLa-Parental cells. (B) Time course of ATP-evoked intracellular Ca2+ signal in HeLa-Parental cells pre-incubated for 5 min with 200 μM gentamicin. Five representative cells are shown in each case. (C) Time course of ATP-evoked intracellular Ca2+ signal under control conditions in HeLa-rC26-GFP cells. (D) Time course of ATP-evoked intracellular Ca2+ signal in HeLa-rC26-GFP cells pre-incubated for 5 min with 200 μM gentamicin. (E) Compiled data showing the amplitude of Ca2+ signal changes evoked by extracellular ATP (mean ± SEM) in HeLa parental and HeLa-rCx26GFP, measured as area under curve expressed as AU in control conditions or after 5 min preincubation with 200 μM gentamicin, 10 min with 100 μM carbenoxolone (CBX) or 20 min with 200 μM GAP-26. (F) Duration (time), from the first Ca2+ signal increase until the return to baseline (mean ± SEM) in HeLa-Parental and HeLa-rCx26GFP in control conditions or after 5 min preincubation with 200 μM gentamicin, 10 min with 100 μM CBX or 20 min with 200 μM GAP-26. For calculations of the average traces (as shown in A) 40 cells per experiment were included. The number of independent experiments is indicated in each bar. **P < 0.01, ***P < 0.001.
	FIGURE 6. Concentration-dependent effects of phospholipase C and intracellular Ca2+ stores inhibitors on the ATP-induced Ca2+ signal. The effect of different concentrations of (A) U73122, a phospholipase C (PLC) inhibitor, (B) CPA, an endoplasmic reticulum Ca2+-ATPase pump inhibitor and (C) CCCP, a protonophore, was tested on the Ca2+ signal induced by 10 μM ATP. Representative average traces (including 40 cells per trace) measured in Fura-2 loaded HeLa-rCx26-GFP cells are shown. Cells were preincubated with each blocker during 10 min prior to stimulation with ATP. (D) Compiled data showing the amplitude of the ATP-evoked Ca2+ signals (mean ± SEM) measured as area under the curve and expressed as AU (mean ± SEM). Each trace represents the average from 40 cells per experiment. The number of independent experiments is indicated within each bar. *P < 0.05, ***P < 0.001.
	FIGURE 7. Detection of P2Y2, P2Y4, and P2Y6 receptors in HeLa-Parental and HeLa-Cx26 cells. Total homogenates of HeLa-Parental and HeLa cells expressing rCx26 or rCx26-GFP were resolved in sodium dodecyl sulfate polyacrylamide gel electrophoresis (12% SDS-PAGE). Membranes were probed with (A) anti-P2Y2R and anti-P2Y4R polyclonal or (B) anti-P2Y6R antibodies. Protein loading was controlled with anti-α-tubulin monoclonal antibody. Bands were detected with secondary anti-rabbit or anti-mouse antiserum coupled to horseradish peroxidase by electrogenerated chemiluminescence (ECL). MB, mouse brain (tissue lysate); Lane 1, HeLa-Parental; Lane 2, HeLa-rCx26; Lane 3, HeLa-rCx26-GFP.
	FIGURE 8. Gentamicin reduces the ATP release induced by DCFS or UTP in HeLa-rCx26 cells. ATP release from HeLa-rCx26 cells in response DCFS and purinergic receptor stimulation by UTP. ATP released to the extracellular solution was determined with an ATP bioluminescence assay kit 5 min after stimulation. (A) ATP released by HeLa-rCx26 cells exposed to DCFS, DCFS + 200 μM gentamicin (DCFS + Gen), DCFS + 200 μM CBX (DCFS + CBX) or with Ca2+/Mg2+ containing solution (control). (B) ATP released by HeLa-rCx26 stimulated with 100 μM UTP, 100 μM UTP + 200 μM gentamicin (UTP + Gen), 100 μM UTP +100 μM CBX (UTP + CBX) in Ca2+/Mg2+ solution (control). The data are shown as means [ATP] ± SEM of three independent experiments. ***P < 0.001.
	FIGURE 9. Gentamicin does not inhibit the intercellular transfer of ethidium or Lucifer yellow between HeLa-rCx26 –mCx43 cells. Gap junctional communication, expressed as dye coupling index, was evaluated in confluent cultures of HeLa-rCx26 or –mCx43 cells. A single cell was microinjected with ethidium (75 mM Etd in water) or Lucifer yellow (5% LY w/v in 150 mM LiCl) and the number of cells to which Etd or LY was transferred in 2 min was scored. Gentamicin was applied extracellularly or was included in the pipette at a final concentration of 200 μM. (A) Photomicrographs showing a fluorescent field of HeLa-xCx26 cells microinjected with Etd or LY and transfer to adjacent cells under control conditions and in the presence of 200 μM gentamicin applied extracellularly 5 min before the assay. Scale bar, 20 μm. (B) Quantitative analysis of dye coupling between HeLa-rCx26 and HeLa-mCx43 cells under control conditions (control) or treated with 200 μM gentamicin in the extracellular solution or in the pipette, as indicated. Bar graphs summarize the results of three independent experiments for each condition (the number of microinjections is indicated within each bar). Values correspond to the mean ± SEM of fluorescent cells.

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