Oh S (2015), Bisphenol A and 4-tert-Octylphenol Inhibit Cx46...

XB-ART-50400

Korean J Physiol Pharmacol 2015 Jan 01;191:73-9. doi: 10.4196/kjpp.2015.19.1.73.

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Bisphenol A and 4-tert-Octylphenol Inhibit Cx46 Hemichannel Currents.

Oh S .

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Connexins (Cx) are membrane proteins and monomers for forming gap junction (GJ) channels. Cx46 and Cx50 are also known to function as conductive hemichannels. As part of an ongoing effort to find GJ-specific blocker(s), endocrine disruptors were used to examine their effect on Cx46 hemichannels expressed in Xenopus oocytes. Voltage-dependent gating of Cx46 hemichannels was characterized by slowly activating outward currents and relatively fast inward tail currents. Bisphenol A (BPA, 10 nM) reduced outward currents of Cx46 hemichannels up to ~18% of control, and its effect was reversible (n=5). 4-tert-Octylphenol (OP, 1 µM) reversibly reduced outward hemichannel currents up to ~28% (n=4). However, overall shapes of Cx46 hemichannel current traces (outward and inward currents) were not changed by these drugs. These results suggest that BPA and OP are likely to occupy the pore of Cx46 hemichannels and thus obstruct the ionic fluxes. This finding provides that BPA and OP are potential candidates for GJ channel blockers.

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Species referenced: Xenopus
Genes referenced: gja3 gja8 tert

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	Fig. 1. Voltage-dependent gating of Cx46 hemichannel. (A) When holding the oocyte expressing Cx46 hemichannels at -70 mV, currents were near zero level. When stepping to a depolarizing potential at +30 mV for 10 sec, slowly activating outward currents were observed. When stepping back to the initial holding potential at -70 mV, relatively fast inward tail currents were shown. Immediately after, currents were back to zero level. The total recording time was 40 sec. (B) Same pulse protocol used in Fig. 1A was applied to un-injected oocyte. Slowly activating outward and relatively fast inward currents as those of Cx46 cRNA injected oocytes were not observed from the un-injected oocyte. (C) Same pulse protocol used in Fig. 1A was applied to each sweep. One set of records included a total of nine sweeps (3 of each control, drug, and wash phases, respectively) and were obtained from a single oocyte expressing Cx46 hemichannels. Each sweep record was acquired immediately after the perfusion with new bath solution. Most peak currents were quite stable for the entire recording period indicated by a dotted line. The voltage-dependence of Cx46 hemichannels represented by slowly activating outward currents and relatively fast inward currents were observed without any change. Current traces for about 25 seconds before the next sweep were omitted for data illustration.
	Fig. 2. The effect of BPA (10 nM) on Cx46 hemichannel currents. (A) One set of records obtained from a single oocyte expressing Cx46 hemichannels is shown. The pulse protocol included VH=-70 mV, Vstep=+30 mV for 10 sec, and a total recording time of 40 sec was used to each sweep. In the control phase (initial three sweeps), the average peak current was 4.75±0.48 µA. The average peak current after applying 10 nM BPA (middle three sweeps) was 3.45±0.05 µA, indicating that BPA inhibited Cx46 hemichannel currents to 27.36% of control. Reversible effects of BPA were observed. In the wash phase (last three sweeps) these reduced currents were returned to control level (4.64±0.40 µA). Overall shapes for outward and inward currents were not affected by BPA. Current traces for about 20 seconds before the next sweep were omitted for better illustration. (B) Normalized peak currents of each phase determined by the ratio of average peak currents of the drug (or wash) phase to those of the control phase are shown. Normalized peak currents with BPA treatment were 0.82±0.10 (n=5). This inhibitory effect of BPA (~18% reduction) was statistically significant (*p<0.05). However, normalized peak currents in the wash phase (1.06±0.15, n=5) were not statistically different from that of control, meaning that BPA effect was reversible.
	Fig. 3. The effect of OP (1 µM) on Cx46 hemichannel currents. (A) One set of records obtained from a single oocyte expressing Cx46 hemichannels are shown. The pulse protocol included VH=-70 mV, Vstep=+30 mV for 10 sec, and a total duration of 40 sec was used to each sweep. The average peak current in the control phase (1.60±0.13 µA) was reduced to 1.06±0.02 µA by applying 1 µM OP (middle three sweeps). The reduction rate of currents for this particular record was 33.72% of control. The inhibitory effect of OP was reversible by recovering reduced current back to 1.52±0.06 uA. Overall shapes for outward and inward currents were unchanged by OP. Current traces for about 25 seconds before the next sweep were omitted for better illustration. (B) All data obtained from the four independent experiments were analyzed by normalizing peak currents of each drug (or wash) phase to those of the control phase. Normalized peak currents inhibited by OP were 0.72±0.10 (n=4) and this current reduction (~28% of control) was statistically significant (*p<0.05). Inhibitory effect of OP was also reversible (0.99±0.07, n=4).
	Fig. 4. The effects of other drugs on Cx46 hemichannel currents. (A) Representative current traces obtained from oocytes expressing Cx46 hemichannels with drug treatments are shown. The pulse protocol (VH=-70 mV, Vstep=+30 mV for 10 sec, and a total duration of 40 sec) was used for each sweep. The horizontal thick bars on the top of current traces indicate drug phases (from the top panel, NP, BP, MEHP, and DEHP, respectively). The average peak currents in control phase (initial three current traces of each record) were not changed by drug treatments indicated by dotted lines. Current traces and overall shapes for outward and inward currents of Cx46 hemichannels were not modified by these drugs. None of them appeared to block Cx46 hemichannel currents. Current traces for about 20 seconds before the next sweep were omitted for better illustration. Scale bars indicate 10 sec and 1 µA. (B) Normalized peak currents obtained from the independent experiments are shown. Normalized peak current of each phase was determined by the ratio of average peak currents of the drug (or wash) phase to those of the control phase. Normalized peak currents with drug treatments (0.99±0.05 for NP (n=3), 1.03±0.05 for BP (n=3), 0.98±0.07 for MEHP (n=4), 1.01±0.06 for DEHP (n=3), respectively) were not significantly different from those of control.

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