Korean J Physiol Pharmacol.  2024 Jul;28(4):323-333. 10.4196/kjpp.2024.28.4.323.

3,3′,4,4′-tetrachlorobiphenyl (PCB77) enhances human Kv1.3 channel currents and alters cytokine production

• Kim JH
• Hwang S
• Park SI
• Lee HJ
• Jung YJ
• Jo SH

• KMID: 2557204
• DOI: http://doi.org/10.4196/kjpp.2024.28.4.323

Abstract

Polychlorinated biphenyls (PCBs) were once used throughout various industries; however, because of their persistence in the environment, exposure remains a global threat to the environment and human health. The Kv1.3 and Kv1.5 channels have been implicated in the immunotoxicity and cardiotoxicity of PCBs, respectively. We determined whether 3,3′,4,4′-tetrachlorobiphenyl (PCB77), a dioxin-like PCB, alters human Kv1.3 and Kv1.5 currents using the Xenopus oocyte expression system. Exposure to 10 nM PCB77 for 15 min enhanced the Kv1.3 current by approximately 30.6%, whereas PCB77 did not affect the Kv1.5 current at concentrations up to 10 nM. This increase in the Kv1.3 current was associated with slower activation and inactivation kinetics as well as right-shifting of the steady-state activation curve. Pretreatment with PCB77 significantly suppressed tumor necrosis factor-α and interleukin-10 production in lipopolysaccharide-stimulated Raw264.7 macrophages. Overall, these data suggest that acute exposure to trace concentrations of PCB77 impairs immune function, possibly by enhancing Kv1.3 currents.

Keyword

Cytokines; Kv1.3 channel; Kv1.5 channel; Macrophages; PCB77

Figure

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  Fig. 1 Structure of 3,3′,4,4′-tetrachlorobiphenyl (PCB 77).

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  Fig. 2 3,3′,4,4′-tetrachlorobiphenyl (PCB77) enhanced human steady-state Kv1.3 currents expressed in Xenopus oocytes. (A) Currents were evoked by 2-s voltage pulses from a holding potential of −60 to +50 mV with an increment of 10 mV. (B) The current traces from a control oocyte and one treated with 10 nM PCB77 for 8 and 15 min. (C−F) Steady-state current–voltage (C, D) and peak current–voltage (E, F) relationships for human Kv1.3 currents in the presence of 0 (control), 3, and 10 nM PCB77 for 8 min (C, E) or 15 min (D, F). Steady-state currents were measured at the end of the 2-s pulse shown as dotted lines. Peak currents and steady-state currents were normalized to the corresponding values at +50 mV. Data are presented as the mean ± SEM (n = 4–11 oocytes per concentration group).

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  Fig. 3 3,3′,4,4′-tetrachlorobiphenyl (PCB77) does not influence human Kv1.5 channel currents. (A) Currents were evoked in oocytes using the same voltage pulse protocol as in Fig. 2. (B) Current traces from a control oocyte and one treated with 10 nM PCB77 for 8 and 15 min. (C−F) Steady-state current–voltage (C, D) and peak current–voltage (E, F) relationships for human Kv1.5 currents in the presence of 0 (control), 3, and 10 nM PCB77 for 8 min (C, E) or 15 min (D, F). Steady-state currents were measured at the end of each 2-s depolarizing pulse shown as dotted lines. Peak and steady-state current magnitudes were normalized to the corresponding values at +50 mV. Data are presented as the mean ± SEM (n = 5–11 oocytes per concentration group).

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  Fig. 4 Comparison of the effects of 3,3′,4,4′-tetrachlorobiphenyl (PCB77) on Kv1.3 and Kv1.5 currents. (A, B) Average changes in the magnitude of steady-state (Iss) and peak current (Ipeak) evoked at +50 mV by 3 and 10 nM PCB77 treatment for 8 (A) or 15 min (B) relative to the corresponding controls. Asterisks indicate significant differences between the control in the absence of PCB77 and the indicated currents in the presence of PCB77. Daggers indicate significant differences between Kv1.3 and Kv1.5 currents in the presence of PCB77. Data are presented as the mean ± SEM (n = 4–11 oocytes per concentration treatment, *, †p < 0.05).

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  Fig. 5 Time and voltage-dependence of steady-state Kv1.3 current modulation by 3,3′,4,4′-tetrachlorobiphenyl (PCB77). (A) Current traces elicited by 2-s depolarizations of −20, +10, and +50 mV from a holding potential of −60 mV before (control) and after exposure to 3 and 10 nM PCB77. (B–E) PCB19-induced changes in peak and steady-state Kv1.3 currents at different command potentials. For each depolarizing voltage step, the current in the presence of 3 (B, C) or 10 nM PCB77 (D, E) at 8 and 15 min were normalized to the currents obtained in the absence of PCB77. Asterisks indicate significant differences between the peak and steady-state currents at each command potential in the presence of PCB77 vs. controls. Daggers indicate significant differences compared with the response at −30 mV. Data presented as mean ± SEM (n = 4–7 oocytes per concentration, *, †p < 0.05).

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  Fig. 6 Slowing of Kv1.3 current kinetics by 3,3′,4,4′-tetrachlorobiphenyl (PCB77). The time constants of current activation and inactivation were estimated by fitting each exponential function to traces evoked by a single +50 mV pulse for a 2-s duration from a holding potential of −60 mV. (A) Representative normalized current traces of the activation phase in the absence of PCB77 (dark) and the presence of 3 and 10 nM PCB77 for 15 min (colored). Each current trace was normalized to its peak value. (B) Representative normalized current traces for the inactivation phase in the absence of PCB77 and in the presence of 3 and 10 nM PCB77 for 15 min. (C, D) Summary of the activation time constants (C) and inactivation time constants (D). Data are presented as the mean ± SEM (n = 6–11 oocytes per concentration, *p < 0.05).

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  Fig. 7 3,3′,4,4′-tetrachlorobiphenyl (PCB77) shifts the steady-state Kv1.3 current activation curve to more depolarized potentials. (A, B) Representative steady-state activation tail currents recorded at −50 mV after 100 ms of depolarizing pulses from −70 to +60 mV in the absence and presence of 3 and 10 nM PCB77. (C) Steady-state activation curves were obtained by normalizing each tail current to the traces evoked at +60 mV and by fitting the data to the Boltzmann equation. Data are presented as the mean ± SEM (n = 5–7 oocytes per treatment).

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  Fig. 8 3,3′,4,4′-tetrachlorobiphenyl (PCB77) does not affect steady-state Kv1.3 current inactivation. (A) Typical tail currents elicited by depolarizing pulses from 30 s preconditioning potentials of −80–0 to +40 mV. (B) Superimposed steady-state inactivation currents in the presence of 0 (control), 3, and 10 nM PCB77. (C) Steady-state inactivation current amplitudes were normalized to those evoked from a preconditioning potential of −60 mV. The data were fitted to the Boltzmann equation to generate the steady-state inactivation curves for each condition. Data are presented as the mean ± SEM (n = 5 oocytes per concentration).

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  Fig. 9 Potentiating effects of 3,3′,4,4′-tetrachlorobiphenyl (PCB77) on Kv1.3 current are irreversible. Currents were evoked by a 200 ms depolarizing pulse to +60 mV from a holding potential of −60 mV at 10 s intervals before exposure to PCB77 (baseline), during the application of 10 nM PCB77, and washout. Steady-state currents were normalized to the baseline values. Normalized currents during PCB77 washout were compared with currents evoked in untreated oocytes after the same recording duration (n = 3 oocytes per treatment).

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  Fig. 10 3,3′,4,4′-tetrachlorobiphenyl (PCB77) inhibits lipopolysaccharide-induced production of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in mouse macrophages. (A) Raw264.7 cells were treated with PCB77 (10 μM) for 12 or 24 h. The release rates of TNF-α and IL-10 into the culture supernatant were measured using an enzyme-linked immunosorbent assay (ELISA). (B) Raw264.7 cells were pretreated with PCB77 for 24 h and then stimulated with lipopolysaccharide (LPS) for 12 or 24 h. The release rates of TNF-α and IL-10 into the culture supernatant were measured using an ELISA. All experiments were performed in triplicate and compared with a one-way analysis of variance followed by Tukey’s post-hoc test for multiple comparisons; **p < 0.01; ***p < 0.001, and not significant (ns) (p > 0.05).

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