Introduction

Polychlorinated biphenyls (PCBs), organic chemicals, were toxic [1]. Among the environmental chemicals that may be able to disrupt the endocrine systems of animals and humans, the PCB were a chemical class of considerable concern [2]. Residue analysis of human adipose tissue, blood, and breast milk confirms that most individuals have been environmentally exposed to PCBs and contain measurable levels in the tissues [2]. Polychlorinated biphenyls consist of two six-carbon rings linked by a single carbon bond and theoretically 209 congeners can form, depending upon the number of chlorines and their location on the biphenyl rings [3].

PCB congeners are typical carcinogens [4,5], suppresses the immune system [6,7], althyroid function increase the risk of developing [8,9], and causing a decrease in the heart function such as hypertension [10-12]. PCB congeners exposure to maternal is induced fetal developmental disturbances [13]. PCB congeners having at 4 or more chlorines each have significant aryl hydrocarbon receptor (AhR)-activating potential [1,3]. Halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons like to PCB can act as AhR ligands [14]. Stimulated AhR with ligand binds to AhR nuclear translocator (ARNT) and function as a transcription factor in vivo [14]. This transcription factor binds to a specific DNA regulatory region, such as AhR-response element (Arnt), dioxin-response element and xenobiotics-response element, promoting the expression of abnormal genes, thereby induced toxicity [15]. In addition, Protein produced by stimulation of the AhR produces intermediary metabolites that have toxic, directly damage DNA, during the synthesis [16].

3,3’,4,4’,5-pentachlorobiphenyl (PCB126) (Fig. 1) was a dioxin-like PCB and a potent AhR agonist [17]. Specificity of PCB126 actions on the G protein coupled receptor pathway was shown by normal burst oxidative activation evoked by toll-like receptor 4 and protein kinase C direct activation [18]. PCB126 was a cardiovascular risk factor that increases the heart weight and serum cholesterol levels in female rats [19]. PCB126 exposure also increased nitric oxide production and nuclear factor kappa-light-chain-enhancer of activated B cells binding activity in the chondrocytes, and may be an initiator of chondrocyte apoptosis, which is closely linked to degradation of cartilage in osteoarthritis pathogenesis [20]. Also PCB126 produces thymic atrophy and immunosuppression [21].

The study identified whether cardiac and immune abnormalities with PCB126 were caused by K⁺ channels. Voltagedependent K⁺ channels (Kv) conduct K⁺ ions across the cell membrane in response to changes in the membrane voltage, thereby regulating neuronal excitability by modulating the shape and frequency of action potentials [22]. Kv channels, a superfamily comprised of 12 subfamilies (Kv1–Kv12), are involved in a number of physiological processes, were typically closed at the resting potential of the cell, but open on membrane depolarization [23]. Kv1.5 and Kv1.3, the voltage dependent K⁺ channels, are members of the Kv1 Shaker family of K⁺ channels, which are involved in tissue differentiation and cell growth [24].

Kv1.3 channel is a delayed-rectifier channel and shows slow C-type inactivation by changing the selectivity filter [25]. Kv1.3 channel is encoded by the KCNA3 gene and is specifically expressed in the central nervous system, osteoclast, and T cell, and is functional in producing human vascular smooth muscle cells [26]. Kv1.3 channel regulates membrane potential and Ca²⁺ signaling in human T cells, and its expression is increased 4- to 5-fold in activated CD4+ and CD8+ TEM/TEMRA cells [27]. The activity of the Kv1.3 channel is the K⁺ that goes out of the cell and becomes hyperpolarized [28]. When the intracellular charge falls to a negative charge, the Ca²⁺ is introduced, thereby promoting activation of Ca²⁺ dependent transcription factors and induction of T lymphocyte proliferation [28]. The Kv1.3 channel is considered a potential target for immune diseases for these reasons [29].

In heart, Kv1.5 channels determine the resting potential, shape, and length of the action potential, thus controlling cardiac performance [30]. The present work shows that arachidonic acid and some other long chain polyunsaturated fatty acids such as docosahexaenoic acid, which is abundant in fish oil, produce a direct open channel block of the major Kv1.5 cloned in cardiac cells [31]. The suppression of Kv1.5 channels inhibit delayed rectified K⁺ channels in myocardial cells and was important in processes such as long-term potentiation or depression [31]

In this study, I have analyzed the electrophysiological effects of PCB126 on human Kv1.5 and Kv1.3 channels expressed in Xenopus oocytes and a two-microelectrode voltage clamp amplifier. We consider the effects of PCB126 on Kv1.3 and Kv1.5 channel currents and determine the physiological and pharmacological meaning of these effects.

Materials and Methods

1. Expression of Kv1.3 and Kv1.5 in oocytes

Human Kv1.3 (GenBank accession no. BC035059.1) cRNA and Kv1.5 (hKv1.5, GenBank accession no. BC099665.3) cRNA were synthesized by in vitro transcription using Message Machine T7 kits (Ambion, Austin, TX, USA) and stored in nuclease- free water at –80℃. Stage V and VI oocytes were surgically eliminated from female Xenopus laevis (Nasco, Modesto, CA, USA) anesthetized with ice for 30 minutes at 10 minutes interval, and isolated from theca and follicle layers using fine forceps. After two days removal of theca and follicle, Xenopus oocytes were injected with 20 nL cRNA (0.4 μg/μL). These procedures were implemented under the Research Guidelines of Kangwon National University IACUC. Injected oocytes were maintained at 17℃ in modified Barth’s Solution, which was composed of (mM): 88 NaCl, 1 KCl, 0.4 CaCl, 0.33 Ca(NO₃)₂, 1 MgSO₄, 2.4 NaHCO₃, 10 HEPES (pH 7.4), and 50 mg/mL gentamincin sulfate. Currents were measured 4 to 5 days after injection.

2. Voltage-clamp recording from oocytes.

ND96 solution, which was composed of (mM): 96 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, and 10 HEPES (pH7.4), was used to oocytes by constant perfusion of the experimental bath chamber, and solution exchanges were concluded within 3 or 4 minutes. Currents were measured 8 minutes and 15 minutes after solution exchange at room temperature (20–23℃) with two-microelectrode voltage clamp systems (Warner Instruments, Hamden, CT, USA). Electrodes were filled with 3 M KCl with a resistance of 2.0–4.0 MΩ for voltage-recording electrodes and 2.0–2.5 MΩ for current-passing electrodes. Stimulation and data acquisition were regulated with an AD-DA converter (Axon Instruments Digidata 1200; Molecular Devices, San Jose, CA, USA) and pCLAMP software v5.1 (Molecular Devices). Stock solutions of 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 2 μM, 5 μM, and 10 μM, PCB126 were dissolved in dimethyl sulfoxide and added to the ND96 solution at suitable concentrations shortly before each experiment. PCB126 was purchased from ChemSpider (Raleigh, NC, USA) and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

3. Data analysis

Origin 8.0 (OriginLab Co., Northampton, MA, USA) software was used for data acquisition and analysis. Concentrationdependent current inhibition data were fitted to a Hill equation as follows:

IC₅₀ was the concentration at which half-maximal currents were inhibited and [D] was PCB126 concentration. Activation phase current trace was fitted with a single exponential function, respectively, which was considered the dominant time constant. Steady-state activation curves were obtained by fitting the data to a Boltzmann equation:

V was the test potential. V_1/2 was the half-activation potential (voltage at which the conductance was half-activated) and k was the slope factor. All data are expressed as mean ± SEM. Paired student’s t -tests or ANOVA were used for statistical comparisons. Differences were considered significant at p ＜ 0.05.

Results

1. Effects of PCB126 on human Kv1.3 channel currents

The effects of PCB126 on Kv1.3 channel currents were measured using Xenopus oocytes expression system. We exposed the control solution to Xenopus oocytes to normalize the peak amplitude. Fig. 2. shows peak currents and steady-state currents recordings of voltage clamps in Xenopus to control conditions exposed only to ND96 solution and after exposure to 3 nM, 10 nM, 30 nM, 100 nM, 1 μM, and 10 μM PCB126 for 8 and 15 minutes on human Kv1.3 channel currents. PCB126 did not affect either the steady-state current or peak current of Kv1.3 channel (n = 5–11) (Fig. 2).

2. Effects of PCB126 on steady-state activation of Kv1.3 channels

To obtain whether PCB126 affected the activation kinetics of Kv1.3 channel currents, two-pulse protocols were used to evoke tail currents (Fig. 3). Steady-state activation curves were obtained from normalized tail currents (Fig. 3B) and fitted by two different Boltzmann equations. In the absence of PCB126, half-activation potential (V_1/2) in the activation curve was –5.64 ± 1.09 mV, and slope value (k) were 9.03 ± 2.25 (n = 5) (Fig. 3C). Exposure to 3 nM PCB126 changed V_1/2 to –0.47 ± 2.67 mV and slope value (k) to 13.56 ± 1.55 (n = 5) (Fig. 4C). Also V_1/2 and k-values increased to 3.26 ± 3.47 mV and 13.96 ± 1.67, respectively by 10 nM PCB126 in human Kv1.3 channel currents (n = 5) (Fig. 3C). These results suggest that PCB126 right-shifted the steady-state activation curves of human Kv1.3 channels (n = 5) (Fig. 3C) (p ＜ 0.05).

3. Effects of PCB126 on human Kv1.5 channel currents

The effects of PCB126 on Kv1.5 channel currents were measured using Xenopus oocytes expression system. We exposed the ND96 solution to Xenopus oocytes to normalize the peak amplitude. Fig. 4. shows peak currents and steady-state currents recordings of voltage clamps in Xenopus to control conditions exposed only to ND 96 solution and after exposure to 30 nM, 100 nM, 300 nM, 1 μM, 2 μM, 5 μM, and 10 μM PCB126 for 8 (Fig. 4 C and 4E) and 15 minutes (Fig. 4D and 4F) on human Kv1.5 channel currents. PCB126 did not affect either the steady-state current or peak current of Kv1.5 channel (n = 5–11) (Fig. 4).

Discussion

PCBs are persistent environmental pollutants that elicit a number of adverse health effects including teratogenesis, neurotoxicity, immunotoxicity, reproductive toxicity, endocrine disruption, and carcinogenesis [32]. PCB126 supposed one of the most potent dioxin-like and AhR agonist [17]. PCB126 and AhR activation disrupt actin/myosin interaction to cause the prototypical cardiotoxicity [33].

Fig. 2. shows peak currents and steady-state currents recordings of voltage clamps in Xenopus to control conditions exposed only to ND96 solution and after exposure PCB126 on human Kv1.3 and Kv1.5 channel currents. PCB126 did not affect either the steady-state current or peak current of Kv1.3 and Kv1.5 channel (Fig. 2). V_1/2 of the activation curve was –5.64 ± 1.09 mV. k-value was 9.03 ± 2.25 in the absence of PCB126. Exposure to 3 nM PCB126 changed V_1/2 to –0.47 ± 2.67 mV and k-value to 13.56 ± 1.55 (n = 5) (Fig. 3C). Also, V_1/2 and k-value increased to 3.26 ± 3.47 mV and 13.96 ± 1.67 by 10 nM PCB126, respectively (n = 5) (Fig. 3C). PCB126 at 3–10 nM increased the V_1/2 compared with control, indicating right-shift of the steady-state activation curves by PCB126 (n = 5) (Fig. 3C) (p ＜ 0.05). However, the increase in PCB126 concentration from 3 nM to 10 nM did not change the V_1/2 of steady-state activation curves (n = 5) (Fig. 3C) (p ＞ 0.05). These results suggest that PCB126 could make activation of Kv1.3 channel difficult.

Kv channels are regulated by a variety of stimuli of membrane voltage, protein phosphorylation, intracellular Ca²⁺ and accessory proteins [34,35]. Phosphatidylinositol 4,5-bisphosphate (PIP₂) a phospholipid, is also known to be a regulator of the Kv channel [36]. PIP₂ can directly regulate the gating of the Kv channel by directly binding to the channel structure, and generates a second messenger such as diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃) by cleavage of PIP₂ by phospholipase C [36]. DAG and IP₃ activate enzymes such as protein kinase C to increase intracellular Ca²⁺ ions [37]. Studies have shown that PIP₂ directly regulates some Kv channels [38]. In particular, the addition of PIP₂ to the cytoplasm significantly increases the amplitude of the current due to almost no inactivation of the Kv1.3 and Kv1.5 channels [25]. PIP₂ eliminated Kvβ1.3-induced N-type fast inactivation of Kv1.5 measured in inside-out macro-patches from Xenopus oocytes [25]. PIP₂ antagonized Kv1.5 inactivation by Kvβ1.3 in a concentrationdependent manner [25]. We confirmed the activation curve right-shift of the Kv1.3 channel, however, PIP₂ was shifted the activation curve to the negative side [25]. In addition, PCB126 did not affect the amplitude of Kv1.3 and Kv1.5 channels in our study. On the contrary, PIP₂ significantly affected the amplitude of Kv1.3 and Kv1.5 channel currents [25]. Therefore, we could exclude the hypothesis that PCB126 could block Kv1.3 channels by stimulating PIP₂.

Kv1.3 channel promote the sustained Ca²⁺ influx necessary for complete T cell activation, and are highly expressed in TEM cells and regulate their activity [39]. Kv1.3 channel is functional in generating human vascular smooth muscle cells. Kv1.3 was one of the first voltage-gated potassium channels reported to be modulated during apoptosis and was shown to contribute to the increased K⁺ efflux underlying the late phase of lymphocyte apoptosis. Lack or down-regulation of expression of Kv1.3 in lymphocytes conferred resistance to apoptosis [40]. Kv1.3 channels are recognized as therapeutic targets for autoimmune diseases, but blockage of Kv1.3 may cause various diseases such as immune diseases, and is also closely related to the activation of other channels, which may lead to a combination of different problems.

The presence of PCBs in the environment presents various toxic effects in vivo and in vitro. The exposure of PCB126 right-shifted the steady-state activation curves of human Kv1.3 channels. These results suggest that PCBs could affect the heart in a way that did not block a voltage-dependent potassium channel, Kv1.3 channel directly. We need more study related to the pharmacology and physiological mechanisms by which PCB126 blocks Kv1.3 channels.