http://informahealthcare.com/ebm ISSN: 1536-8378 (print), 1536-8386 (electronic) Electromagn Biol Med, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15368378.2014.906448
ORIGINAL ARTICLE
Effects of moderate static magnetic fields on the voltage-gated sodium and calcium channel currents in trigeminal ganglion neurons Xiao-Wen Lu1,2, Li Du2, Liang Kou1,2, Ning Song1,2, Yu-Jiao Zhang1,2, Min-Ke Wu1,2, and Jie-Fei Shen1,2 State Key Laboratory of Oral Diseases and 2Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China
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Abstract
Keywords
Aim: To study the effects of static magnetic fields (SMF) on the electrophysiological properties of voltage-gated sodium and calcium channels on trigeminal ganglion (TRG) neurons. Methods: Acutely dissociated TRG neurons of neonatal SD rats were exposed to 125-mT and 12.5-mT SMF in exposure devices and whole-cell patch-clamp recordings were carried out to observe the changes of voltage-gated sodium channels (VGSC) and calcium channels (VGCC) currents, while laser scanning confocal microscopy was used to detect intracellular free Ca2+ concentration in TRG neurons, respectively. Results: (1) No obvious change of current–voltage (I–V) relationship and the peak current densities of VGSC and VGCC currents were found when TRG neurons were exposed to 125-mT and 12.5-mT SMF. However, the activation threshold, inactivation threshold and velocity of the channel currents above were significantly altered by 125-mT and 12.5-mT SMF. (2) The fluctuation of intracellular free Ca2+ concentration within TRG neurons were slowed by 125-mT and 12.5-mT SMF. When SMF was removed, the Ca2+ concentration level showed partial recovery in the TRG neurons previously exposed by 125-mT SMF, while there was a full recovery found in 12.5-mT-SMF-exposed neurons. Conclusions: Moderate-intensity SMF could affect the electrophysiological characteristics of VGCS and VGCC by altering their activation and inactivation threshold and velocity. The fluctuations of intracellular free Ca2+ caused by SMF exposure were not permanent in TRG neurons.
125-mT, 12.5-mT, calcium channel, static magnetic fields, sodium channel, trigeminal ganglion neuron, whole-cell patch-clamp
Introduction Moderate-intensity (1 mT-1T) static magnetic fields (SMF) can be produced by permanent magnets as well as other electromagnets in our daily life. Dental magnetic attachments are typical examples for the application of magnets and magnetic forces in contemporary dentistry. In their course of development from early open-circuit magnet system to nowadays close-circuit magnet system, for different type of magnets, controversial opinions persist on the biosecurity of SMF exposure. Previous studies revealed that SMF can alter the central nervous function (Rosen, 1993a; Rosen and Lubowsky, 1990), peripheral nerve conduction (Cordeiro et al., 1989), neuromuscular activity and synaptic transmission (Rosen, 1992; Satow et al., 2001). However, the mechanisms underlying such effects are not entirely clear. One notable hypothesis proposed by Rosen (1993a,b) indicated that moderate-intensity SMF up to 125-mT may induce magnetic reorientation of membrane phospholipids by diamagnetic anisotropy effects and this theory was brought to Address correspondence to Dr. Jie-Fei Shen, State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, No. 14, Section 3, Renminnan Road, Chengdu, Sichuan Province, 610041, P.R. China. Tel: +86 028 85502408. E-mail: [email protected]
History Received 22 October 2013 Revised 3 March 2014 Accepted 9 March 2014 Published online 8 April 2014
interpret a series of experiment findings about the effects of moderate-intensity SMF on the action potentials of neurons (Cavopol et al., 1995; McLean et al., 1995; Ye et al., 2004) and mechano-sensitive ion channels (Dobson et al., 2002a,b). The voltage-gated channels are important physiological regulators of membrane resting and action potentials (Catterall, 2000, 2011; Yellen, 2002). They are widely expressed in excitable tissues including sensory ganglia (Amaya et al., 2000; Novakovic et al., 1998; Rasband et al., 2001). In our previous report on trigeminal ganglion neurons (Shen et al., 2007), effects of 125-mT SMF were found on two main sub-types of voltage-gated potassium channels (VGPC), the fast-inactivating transient (IK,A) and dominant-sustained (IK,V) channels, that SMF could influence the inactivation kinetics of both VGPC currents. These findings were consistent on the research on the IK,V channel of prefrontal cortex pyramidal neurons, that even weaker SMF could also influence the amplitudes, characteristics of half-activation voltage and the k value of the VGPC activation curves (Li et al., 2010). Besides VGPC, voltage-gated sodium channels (VGSC) and voltage-gated calcium channels (VGCC) are the other two major components giving rise to the electrophysiological characteristics of neurons. As the fundamentals of the sodium inward currents, VGSC initiate the action potentials in nerve,
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muscle and other excitable cells (Catterall, 2012; Hodgkin and Huxley, 1952a, b). Malfunctions of VGSC lead to the disorders of neuronal excitability in epilepsy, chronic pain, autism, migraine, multiple sclerosis, etc. (Eijkelkamp et al., 2012). VGCC are the key signal transducers of electrical excitability, converting the electrical signal of the action potential in the cell surface membrane to an intracellular Ca2+ transients that initiate many physiological events (Catterall, 2011). The complex interaction between VGSC, VGPC and VGCC is the functional basis of profound neural electric activities. However, the effects of moderate intensity SMF on VGSC and VGCC are not thoroughly clarified. Therefore, in this study, whole-cell patch-clamp experiments were conducted on TRG neurons to investigate whether 125-mT and 12.5-mT SMF were capable of influencing the activation and inactivation characteristics of VGSC and VGCC currents.
Methods The SMF exposure devices According to our previous measurements (Shen et al., 2007), 125-mT was a common intensity of SMF around open-circuit dental magnets, while 12.5-mT was that of close-circuit ones. In this study in vitro, two exposure deices were manufactured to simulate the SMF of these two intensities, respectively. The structure and dimension of the devices were designed and adjusted by electromagnetic design software, Maxwell 3D (Ansoft Corp., Pittsburg, PA).
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Each U-shaped device has two 60 mm 60 mm square NdFeB magnets covered by polar caps and connected by a steel yoke (Figure 1A). In the course of SMF exposure, 35-mm plastic cell culture dish was placed on a base to reach the position at the vertical and horizontal center of the 50-mm-wide air gap between the two caps. Simulation results demonstrated that 125-mT and 12.5-mT SMF could be generated by these two devices of their structure and size, respectively. After the devices were assembled, precise measurement carried out by digital teslameter (TJSH-035, Fuzhou, China) revealed the consistent results and the non-homogeneity of flux density was less than 1% around the culture dish for both of the two devices (Figure 1B). The exposure device could be mounted onto an inverted microscope (IX71, Olympus, Tokyo, Japan) and patchclamp pipette was extended into the exposure area from the opening side of the device. All ferromagnetic materials on the microscope round the exposure device were replaced by acrylic or epoxy resin. Acute dissociation of TRG neurons The method obtaining TRG neurons was described in our previous report ever (Shen et al., 2007, 2012). Briefly, TRG neurons were isolated from neonatal (5–7 d) Sprague–Dawley rats anesthetized with ether. Dissected TRG neurons were washed by ice-cold Hanks’ balanced salt solution (HBSS, Sigma, St. Louis, MO), then minced into small pieces and
Figure 1. (A) The structure and dimension of 125-mT (upper) and 12.5-mT (lower) SMF exposure devices, a: magnets covered by polar caps; b: steel yoke. (B) The horizontal SMF distribution at the height where the culture dish was placed (Z ¼ 30 mm). The central point of dish bottom was designated as the horizontal coordinate origin.
Effects of SMF on TRGN Na+ and Ca2+ channels
DOI: 10.3109/15368378.2014.906448
incubated in HBSS containing 20 U/ml papain (Sigma) at 37 C for 40 min. After the digestion, the cells were washed 3 times by DMEM/F-12 culture medium (1:1 in volume, Gibco, Grand Island, NY) supplemented with 10% Fetal Bovine Serum (Gibco), and 0.5-mM glutamine (Gibco). Then, they were gently triturated by a series of fire-polished Pasteur pipettes and plated onto poly-L-lysine (Sigma) pretreated glass coverslips placed in 35-mm dishes. The dishes were maintained in a humidified atmosphere of 5% CO2 at 37 C and all TRG neurons were used within 2 and 3 h after plating. All TRG neurons could be distinguished by their relatively larger figures than other dissociated cells and identified by further Nissl substance stain with Cresyl Echt violet (Mitcheson and Sanguinetti, 1999).
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Whole-cell patch-clamp recordings Before patch-clamp recordings, the cell culture medium in the dishes was carefully removed and the cells were washed by external solution for 3 times. The external solution used for VGSC currents recording contained 60-mM Choline-Cl, 60-mM NaCl, 20-mM Tetraethylammonium chloride (TEA-Cl, Sigma), 5-mM KCl, 1-mM MgCl2 6H2O, 2-mM CaCl2, 0.1-mM CdCl2 5H2O, 10-mM D-glucose, 3-mM 4-aminopyridine (4-AP, Sigma), 10-mM HEPES (pH ¼ 7.4), which used for VGCC currents recording contained 110-mM Choline-Cl, 2.5-mM CsCl, 2.5-mM CaCl2, 1-mM MgCl2, 30mM tetraethylammonium (TEA, Sigma), 4-mM 4-AP, 10-mM D-glucose and 10-mM HEPES (pH ¼ 7.4). Finally, the external solution volume in the dishes was adjusted to 2 ml. About 15 min after the final wash, the dishes were transferred into the SMF exposure device on the microscope and maintained for another 15 min. Then the recordings were performed at room temperature of 25–28 C. For control groups, there was no SMF exposure to the cells. Patch-clamp pipettes were pulled from borosilicate glass and filled with internal solution composed of 135-mM CsCl, 10-mM NaCl, 2-mM MgCl2, 1-mM CaCl2, 10-mM HEPES, 10-mM EGTA (ph ¼ 7.3) for VGSC currents recording, and 120-mM CsCl, 2-mM CaCl2, 10-mM NaCl, 10-mM HEPES and 10-mM EGTA (pH ¼ 7.3) for that of VGCC, respectively. The electrodes had resistances of 2–4 M . The whole cell recordings were conducted using an Axopatch 200 B patch-clamp amplifier (Molecular Devices LLC, Sunnyvale, CA) and the output was digitized with Digidata 1440 A converter (Molecular Devices LLC). Both the capacitance and series resistance were well compensated. All data were acquired by Clampex 10.0 software (Molecular Devices LLC). Intracellular free Ca2+ imaging and fluorometry The intracellular free Ca2+ concentration ([Ca2+]i) measurements within TRG neurons were carried out by Fluo-3/AM loading and laser scanning confocal microscope (LSCM, TCS SP, Leica, Wetzlar, Germany) after the neurons were exposed under SMF for 1 h, 1.5 h, 2 h and 2.5 h. [Ca2+]i was derived by equation F¼F1-F0, in which F was actual fluorescent density, F1 and F0 were detected and background fluorescent density. All exposure procedures were kept in a humidified atmosphere of 5% CO2 at 37 C.
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Data analysis Data of patch clamp recordings were analyzed with Clampfit 10.0 software (Molecular Devices LLC). All curve fittings and statistical comparisons were performed in Origin 9.0 software (OriginLab Corp., Northampton, MA). Differences were considered to be significant at p50.05.
Results Characteristic of total VGSC and VGCC currents on rat TRG neurons In previous reports, several types of VGSC and VGCC currents which differ in their pharmacological and biophysical properties have been found in sensory neurons including TRG neurons (Amaya et al., 2000; Kim and Chung, 1999; Novakovic et al., 1998). In present study, total currents of VGSC and VGCC were the focus of our observations. In patch clamp system, the recording of activation and inactivation currents for VGSC and VGCC were based on different stimulus protocols after reaching Giga-Ohm seal between the cell and patch pipette. For the VGSC, neurons were initially held at 60 mV followed by hyperpolarization to 80 mV for 70 ms as conditioning prepulse potential. Then the inward sodium activation currents were elicited by 100 ms pulses stepping from 60 to +60 mV in 10-mV increments (Figure 2A). Their inactivation properties were studied by another stimulus protocol. The neurons being held at 80 mV were given a series of 50 ms prepulses stepping from 100 to 40 mV followed by a 50 ms test pulse depolarizing to +10 mV (Figure 3A). While, to the VGCC, they were initially held at 80 mV and activation currents were elicited by 400 ms pulses stepping from 80 mV to +100 mV in 10-mV increments (Figure 2D). For inactivation recordings, the neurons were held at 80 mV then given a series of 300-ms prepulses stepping from 80 mV to +70 mV followed by a 200 ms test pulse depolarizing to +10 mV (Figure 3D). Current densities of both channels were obtained by dividing the amplitude of the currents to their own whole-cell capacitances. Effects of SMF on the activation of VGSC and VGCC currents on TRG neurons Totally, 30 randomized selected TRG neurons were recorded for VGSC (for 125-mT SMF exposure group, n ¼ 10; for 12.5mT SMF exposure group, n ¼ 10; for control group, n ¼ 10) and another 30 randomized selected TRG neurons were used for VGCC currents recording (for 125-mT SMF exposure group, n ¼ 10; for 12.5-mT SMF exposure group, n ¼ 10; for control group, n ¼ 10). The capacitance of VGSC exposed in 125-mT SMF, 12.5-mT SMF and control groups were 32.51 ± 6.43 pF, 34.01 ± 4.81 pF and 31.15 ± 5.38 pF, while those of VGCC were 30.51 ± 5.03 pF, 33.01 ± 4.92 pF and 35.37 ± 6.02 pF (mean ± SEM). The VGSC started to be activated when the membrane potential was depolarized to about 60 mV (Figure 2B). The largest current densities were gained for all three groups of VGSC currents around 10 mV. To the VGCC, the channels started to be activated as the membrane potential was about 60 mV (Figure 2E), and the largest current densities were
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Figure 2. (A–C) Effects of 125-mT and 12.5-mT SMF on the activation of INa; (D–F) Effects of 125-mT and 12.5-mT SMF on the activation of ICa. (A,D) Typical VGSC and VGCC activation currents of TRG neurons recorded by patch clamp system and their activation patch clamp protocols; (B,E) normalized activation curves for VGSC and VGCC; (C) comparison of the fitting results of VGSC activation currents. (F) Comparison of the fitting results of VGCC activation currents. (*Difference between two SMF intensity exposure and control groups were statistically significant at p50.05.)
gained around +10 mV for all three groups. The current– voltage (I–V) relationship and largest current densities of VGSC and VGCC were not significantly altered by 125-mT and 12.5-mT SMF (data not showed). Then, channel conductance (G) at different membrane potential was obtained by equation G ¼ I/(VmVrev), in which I was the current density, Vm was the voltage command, and Vrev was the reversal potential. Normalized activation curves were plotted as G/Gmax against the voltage commands (Figure 2B and E). The curves of all groups were fitted to Boltzmann equation: G/Gmax ¼ 11/{1+exp[(VmV1/2)/k]}, in which Vm was the voltage command, V1/2 was the membrane potential at half activation and k was the slope factor. Fitting results (Figure 2C) demonstrated that, there was no significant change in V1/2 and k of VGSC between 12.5-mT SMF exposure group and control group (p40.05), while V1/2 and k of 125-mT SMF exposure group differed with both 12.5-mT exposure and control groups (p50.05). To the analysis of VGCC (Figure 2F), there was no significant change in V1/2 between two SMF exposure groups (p40.05), while V1/2 of control group differed with both of them (p50.05). For k values, no statistical difference was found among those three groups (p40.05).
For all the three groups of VGSC, currents began to inactivate when membrane potential was about 70 mV and reached the maximum inactivation at membrane potentials were about +40 mV (Figure 3B). As to that of VGCC (Figure 3E), both SMF exposure groups and control groups began to inactivate as membrane potential was about 60 mV. But the maximum inactivation currents appeared at different membrane potentials among the three groups, which were about 20 mV, 10 mV and 10 mV for 125-mT SMF, 12.5-mT SMF and control group, respectively. Their inactivation curves were also fitted to Boltzmann equation: I/Imax ¼ 1/{1+exp[(VmV1/2)/k]}, in which V1/2 was the membrane potential at half inactivation at this time. According to the fitting results, difference in V1/2 of VGSC was found between 125-mT SMT exposure group and both 12.5-mT SMT exposure and control groups (p50.05), while difference of 12.5-mT SMT exposure group and control group showed no significant change (p40.05). No significant changes existed in k between all the groups of VGSC (p40.05) (Figure 3C). As to VGCC, difference of V1/2 was found among all three groups (p50.05). No significant change existed in k between two SMF exposure groups (p40.05), while k of control group differed with both of the SMF exposure groups (p50.05) (Figure 3F).
Effects of SMF on the inactivation of VGSC and VGCC currents on TRG neurons
Effects of SMF on [Ca2+]i in TRG neurons
Normalized inactivation curves were plotted as the peak current density against the prepulse voltage commands.
Images of Ca2+ fluoresced TRG neurons of different exposure conditions and durations were showed as in Figure 4.
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DOI: 10.3109/15368378.2014.906448
Effects of SMF on TRGN Na+ and Ca2+ channels
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Figure 3. (A–C) Effects of 125-mT and 12.5-mT SMF on the inactivation of INa; (D–F) Effects of 125-mT and 12.5-mT SMF on the inactivation of ICa. (A,D) Typical VGSC and VGCC inactivation currents of TRG neurons recorded by patch clamp system and their activation patch clamp protocols; (B,E) normalized inactivation curves for VGSC and VGCC currents; (C) comparison of the fitting results of VGSC inactivation currents. (F) Comparison of the fitting results of VGCC inactivation currents. (*Difference between two SMF intensity exposure and control groups were statistically significant at p50.05).
It could be derived from the measurements of F value among the SMF exposure and control groups (Table 1) that at the beginning 1 h to 1.5 h, the [Ca2+]i changes were delayed in both 125-mT and 12.5-mT SMF groups followed by recovery process toward the level of the control group after 2 h of exposure. But such process differed according to different intensity of SMF loading. After 2.5 h exposure of 12.5-mT SMF, a full recovery of [Ca2+]i was met, while [Ca2+]i of the TRG neurons in 125-mT SMF group was still lower than the control.
Discussion In previous studies on the biological effects of moderateintensity SMF on the cell membrane and ion channels, 125mT and 12.5-mT were of the most discussed intensities (Rosen, 2003a; Xu et al., 2004). In this study in vitro, the field was produced by exposure devices made of permanent magnets (Figure 1A). Compared to electromagnets, no thermal changes would be generated by this device. The size was minimized by the Maxwell software while the power, scope and homogeneity of the magnetic fields were ensured. Two SMF intensity exposure devices were fully compatible with the inverted microscope and the patch clamp system. There have been various reports on the biological effect caused by electromagnets and SMF. However, the underlying mechanism, especially about the effects on the neural
activities, has not been entirely clarified. In Rosen’s hypothesis (Rosen, 2003b), the biological membrane would be deformed in SMF and the ion channels on the membrane would be influenced. The alterations on channel activity caused by SMF exposure are indirect. The primary effect of magnetic fields is to induce rotation and reorientation of the membrane lipid molecules and such reorientation could affect conformation changes of ion channels (Hughes et al., 2005). This theory has been applied to interpret experimental findings on GH3 cells including an inhibition of their sodium and calcium channel currents under moderateintensity SMF exposure (Rosen, 1996, 2003a). It provides an explanation to the effects of SMF on neurotransmitter release, the permeability of VGCC on hippocampal neurons (Wieraszko, 2000), and the change of delayed rectifier potassium channel exposed to 3-mT static magnetic field on prefrontal cortex pyramidal neurons (Li et al., 2010). VGPC, VGSC and VGCC are the major channels which establish the resting membrane potential, action potential and other electric activities of neurons and participate into the process of transducing electrical activity into intracellular biochemical signals of neurons. In our previous experiment, effects of 125-mT SMF were also found on the activation and inactivation of VGPC currents on TRG neurons. In this study, the main purpose was to observe the effects of SMF on voltage-gated sodium and calcium channels and compared to our previous findings on potassium channels. Therefore, we
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Figure 4. Intracellular free Ca2+ imaging of TRG neurons.
Table 1. Effects of 125-mT and 12.5-mT SMF on [Ca2+]i in TRG neurons (mean ± SD). SMF exposure time 1h 1.5 h 2h 2.5 h
125-mT group 179.4 ± 68.4 105.9 ± 57.3 66.0 ± 37.2 139.6 ± 53.8
(n ¼ 12)b,c,d,a (n ¼ 11)a,c,d,a (n ¼ 21)a,b,d (n ¼ 17)a,b,c,a
12.5-mT group 176.4 ± 44.0 82.6 ± 58.1 70.3 ± 45.9 175.9 ± 59.5
(n ¼ 17)b,c,a (n ¼ 30)a,d,a (n ¼ 37)a,d (n ¼ 16)b,c
Control 141.2 ± 50.8 41.4 ± 21.4 81.8 ± 61.5 188.8 ± 27.5
(n ¼ 24)b,c,d,b,g (n ¼ 19)a,c,d,b,g (n ¼ 22)a,b,d (n ¼ 9)a,b,c,g
a
p50.05 versus 1 h, bp50.05 versus 1.5 h, cp50.05 versus 2 h, dp50.05 versus 2.5 h; p50.05 versus control, bp50.05 versus 12.5-mT group, gp50.05 versus 125-mT group.
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focused on the whole-cell total currents and their overall channel kinetics, not all the subtypes of each category. As shown in the results 125-mT and 12.5-mT SMF did not exhibit effects on the peak current density of VGSC and VGCC activation, neither were the k value which indicated the activation velocity of VGCC and inactivation velocity of VGSC. But the V1/2, which was related to the activation threshold voltage, was influenced by SMF of both intensities for VGSC and VGCC (Figures 2 and 3).
VGSC were activated at an proximal membrane potential when SMF exposure was loaded. Though the V1/2 and k values of activation properties showed no significant changes between 12.5-mT SMF exposure group and control group, 125-mT SMF exposure made a change, which indicated that 125-mT exposure could make activation threshold voltage more positive and slow down the activation speed. As for the inactivation properties of VGSC, V1/2 values exposed under 125-mT SMF appeared some conspicuous features that
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DOI: 10.3109/15368378.2014.906448
currents began to be inactivated at more negative potentials, as 12.5-mT SMT exposure made no effect. Such results seemed to indicate that there perhaps existed a certain threshold of magnetic field intensity able to change both activation and inactivation features of sodium channel, or the effects might be intensity-depended of which 12.5-mT exposure was not enough to make an influence obviously to achieve the statistical significance. However, VGCC were activated at a more negative membrane potential without change of k values when SMF exposure was loaded. As for the inactivation properties of VGCC, both 125-mT and 12.5mT SMF could change the V1/2 and k values. The VGCC currents began to be inactivated at more negative potentials. Such effects were intensity-dependent, that the 125-mT SMF brought a stronger influence than 12.5-mT SMF. Meanwhile, both of them accelerated the inactivation speed and such effects seemed not related to their intensities. Previous studies have identified that VGSC are large multiprotein complexes with a a subunit containing four internally homologous domain (I–IV) and auxiliary b subunits. There are six a-helical transmembrane segments (S1–S6) in each of the four homologous domains. Sodium channel activation is due to a sequence of voltage induced changes in the S4 segment of the a-subunit. Depolarization of the membrane is proposed to release the S4 segments to move outward along a spiral path, initiating a conformational change that opens the pore (Catterall, 2000). The S4 segment would be vulnerable to the effects of diamagnetic reorientation of phospholipid molecules caused by SMF (Rosen, 2003a). Inactivation of the sodium channel has been discovered to involve an highly conserved intracellular loop connecting domains III and IV of the sodium channel a-subunit (Catterall, 2000). Such an intracellular mechanism may not be directly affected by physical changes within the membrane (Rosen, 2003a). VGCC on sensory neurons have a similar structure with a pore forming a1 subunit as the center piece, surrounded by auxiliary a2d, b and g subunits. The a1 subunit contains four conserved transmembrane structural domains that are linked by more variable cytoplasmic sequences (Catterall, 2011). This subunit largely determines the biophysical properties of the VGCC, and gives rise to the voltage sensing and activation characteristics of VGCC currents (Catterall et al., 2005), while the inactivation processes can occur by multiple mechanisms that requires the participation of auxiliary subunits (Dolphin, 2003). The similar effects of 125-mT and 12.5-mT SMF on the activation threshold suggested that a1 subunits of VGCC might be the indirect targets of the moderate-intensity of SMF by the diamagnetic anisotropy effects and reorientation of membrane phospholipids. However, the reason underlying the diverse changes of inactivation characteristics by 125-mT and 12.5-mT SMF exposure remained obscure. In our previous experiments on 125-mT SMF (Shen et al., 2007), similar differentiated alterations were also observed on the inactivation of two main VGPCs currents, IK,A and IK,V. N-type inactivation leading to very rapid inactivation process of IK,A is frequently described as the ‘‘ball-and-chain’’ mechanism, in which a sequence of amino acids (the inactivation domain) at the N-terminus of the VGPCs occludes the intracellular channel pore and prevents ion permeation (Hoshi et al., 1990, 1991;
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Rasmusson et al., 1998). The slower inactivation process of IK,V is more related to the C-type inactivation which involves conformational changes around the selectivity filter and extracellular entrance to the channels (Choquet and Korn, 1992). When biological membrane is deformed under SMF, the distortion of VGPCs is probably different on their inner and outer terminals. This difference might give rise to the opposite effects of 125-mT SMF on the inactivation velocity of IK,A and IK,V currents (Shen et al., 2007). It needs further studies to clarify whether 125-mT and 12.5-mT could bring such effects on VGCC currents. The maximum time of patch clamp recordings was limited to 15 min after the seal between the plasma membrane and pipette was formed, because TRG neurons could not survive a longer time after the patch break on the membrane required by whole-cell recordings. Though the time course for the influence of SMF on VGSC and VGCC was not included in our study, this issue has already been discussed in previous reports (Rosen, 2003a). It was demonstrated that the effect of SMF on the cell membrane was reversible and could persist for about 100 s after the field was turned off. In Hughes’ study (Hughes et al., 2005), ion channel activity also showed slow recovery following removal of the magnetic field. These observations indicated that after the exposure of SMF was shut off, there would be a slow reorientation process of the diamagnetic molecules on the deformed membrane and the restoration of channel functions would be delayed. This explanation could interpret our findings on the [Ca2+]i in this study (Table 1, Figure 4). It should be addressed that, in Rosen’s researches on GH3 cells, optimum effects of SMF on the ion channels were observed around 37 C (Rosen, 1996, 2003a). However, most studies of channel functions on mammal cells, especially the neurons, were carried out at room temperature (Liu and Simon, 2003; Takeda et al., 2004a,b). In normal physiological condition ranging from 23 to 37 C, no change in the current– voltage relationship of channel kinetics was found (Rosen, 2001). According to Maret and Dransfeld (1977) analysis on the joint effects of SMF and temperature on the diamagnetic anisotropic molecules, it was pointed out that within the physiologically permissible range, the effect of temperature on the interaction between SMF and those molecules was small; a 10 C difference in temperature will result in only a change less than 4%. In Hughes’ experiment (Hughes et al., 2005), membrane reorientation effects of 125-mT SMF at room temperature were found to be identical to that in Rosen’s. Wieraszko (2000) reported the similar changes in hippocampal neurons under SMF exposure at room temperature as well. In this study, our primary objective was to investigate the effects of SMF on the activation and inactivation current–voltage relationship of VGSC and VGCC. Therefore, the experiment temperature was set to the room level.
Conclusion Moderate-intensity SMF could affect electrophysiological characteristics of VGCS and VGCC by altering their activation and inactivation threshold and velocity. The fluctuation of intracellular free Ca2+ concentration within TRG neurons
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slowed by SMF could be fully or partially restorable, indicating the influence of SMF might not be permanent. The mechanism underlying those changes was believed to be related to the magnetic reorientation of membrane phospholipids caused by moderate-intensity SMF through diamagnetic anisotropy effects.
Acknowledgements We would like to thank Ms. Daqing Liao, Ms. Yanfang Chen and Ms. Xiaoyu Li for their technical assistance.
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Declaration of interest This research was funded by The National Natural Science Foundation of China for Young Scholars. Grant No. 81000456.
References Amaya, F., Decosterd, I., Samad, T. A., et al. (2000). Diversity of expression of the sensory neuron-specific TTX-resistant voltage-gated sodium ion channels SNS and SNS2. Mol. Cell. Neurosci. 15: 331–342. Catterall, W. A. (2000). From ionic currents to molecular mechanisms: The structure and function of voltage-gated sodium channels. Neuron. 26:13–25. Catterall, W. A. (2011). Voltage-gated calcium channels. Cold Spring Harb. Perspect. Biol. 3:a003947 (1–23). Catterall, W. A. (2012). Voltage-gated sodium channels at 60: Structure, function and pathophysiology. J. Physiol. 590:2577–2589. Catterall, W. A., Perez-Reyes, E., Snutch, T. P., Striessnig, J. (2005). International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels. Pharmacol. Rev. 57:411–425. Cavopol, A. V., Wamil, A. W., Holcomb, R. R., McLean, M. J. (1995). Measurement and analysis of static magnetic fields that block action potentials in cultured neurons. Bioelectromagnetics. 16:197–206. Choquet, D., Korn, H. (1992). Mechanism of 4-aminopyridine action on voltage-gated potassium channels in lymphocytes. J. Gen. Physiol. 99: 217–240. Cordeiro, P.G., Seckel, B.R., Miller, C.D., et al. (1989). Effect of a highintensity static magnetic field on sciatic nerve regeneration in the rat. Plast. Reconstr. Surg. 83:301–308. Dobson, J., Stewart, Z., Martinac, B. (2002a). Preliminary evidence for weak magnetic field effects on mechanosensitive ion channel subconducting states in E. coli. Electromagn. Biol. Med. 21:89–95. Dobson, J., Stewart, Z., Martinac, B. (2002b). Preliminary evidence for weak magnetic field effects on mechanosensitive ion channel subconducting states in E. coli-errata. Electromagn. Biol. Med. 21:309. Dolphin, A. C. (2003). Beta subunits of voltage-gated calcium channels. J. Bioenerg. Biomembr. 35:599–620. Eijkelkamp, N., Linley, J. E., Baker, M. D., et al. (2012). Neurological perspectives on voltage-gated sodium channels. Brain. 135: 2585–2612. Hodgkin, A. L., Huxley, A. F. (1952a). Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo. J. Physiol. 116:449–472. Hodgkin, A. L., Huxley, A. F. (1952b). The dual effect of membrane potential on sodium conductance in the giant axon of Loligo. J. Physiol. 116:497–506. Hoshi, T., Zagotta, W. N., Aldrich, R. W. (1990). Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science. 250:533–538. Hoshi, T., Zagotta, W. N., Aldrich, R. W. (1991). Two types of inactivation in Shaker K+ channels: Effects of alterations in the carboxy-terminal region. Neuron. 7:547–556. Hughes, S., El Haj, A. J., Dobson, J., Martinac, B. (2005). The influence of static magnetic fields on mechanosensitive ion channel activity in artificial liposomes. Eur. Biophys. J. 34:461–468.
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Kim, H. C., Chung, M. K. (1999). Voltage-dependent sodium and calcium currents in acutely isolated adult rat trigeminal root ganglion neurons. J. Neurophysiol. 81:1123–1134. Li, G., Cheng, L. J., Qiao, X. Y., Lin, L. (2010). Characteristics of delayed rectifier potassium channels exposed to 3 mT static magnetic field. IEEE Trans. Magnet. 46:2635–2638. Liu, L., Simon, S. A. (2003). Modulation of IA currents by capsaicin in rat trigeminal ganglion neurons. J. Neurophysiol. 89:1387–1401. Maret, G., Dransfeld, K. (1977). Macromolecules and membranes in high magnetic fields. Physica. 86–88B:1077–1083. McLean, M. J., Holcomb, R. R., Wamil, A. W., et al. (1995). Blockade of sensory neuron action potentials by a static magnetic field in the 10 mT range. Bioelectromagnetics. 16:20–32. Mitcheson, J. S., Sanguinetti, M. C. (1999). Biophysical properties and molecular basis of cardiac rapid and slow delayed rectifier potassium channels. Cell Physiol. Biochem. 9:201–216. Novakovic, S. D., Tzoumaka, E., McGivern, J. G., et al. (1998). Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions. J. Neurosci. 18:2174–2187. Rasband, M. N., Park, E. W., Vanderah, T. W., et al. (2001). Distinct potassium channels on pain-sensing neurons. Proc. Natl. Acad. Sci. USA. 98:13373–13378. Rasmusson, R. L., Morales, M. J., Wang, S., et al. (1998). Inactivation of voltage-gated cardiac K+ channels. Circ. Res. 82:739–750. Rosen, A. D. (1992). Magnetic field influence on acetylcholine release at the neuromuscular junction. Am. J. Physiol. 262:C1418–C1422. Rosen, A. D. (1993a). Membrane response to static magnetic fields: Effect of exposure duration. Biochim. Biophys. Acta. 1148:317–320. Rosen, A. D. (1993b). A proposed mechanism for the action of strong static magnetic fields on biomembranes. Int. J. Neurosci. 73:115–119. Rosen, A. D. (1996). Inhibition of calcium channel activation in GH3 cells by static magnetic fields. Biochim. Biophys. Acta. 1282:149–155. Rosen, A. D. (2001). Nonlinear temperature modulation of sodium channel kinetics in GH(3) cells. Biochim. Biophys. Acta. 1511: 391–396. Rosen, A. D. (2003a). Effect of a 125 mT static magnetic field on the kinetics of voltage activated Na+ channels in GH3 cells. Bioelectromagnetics. 24:517–523. Rosen, A. D. (2003b). Mechanism of action of moderate-intensity static magnetic fields on biological systems. Cell Biochem. Biophys. 39: 163–173. Rosen, A. D., Lubowsky, J. (1990). Modification of spontaneous unit discharge in the lateral geniculate body by a magnetic field. Exp. Neurol. 108:261–265. Satow, Y., Matsunami, K., Kawashima, T., et al. (2001). A strong constant magnetic field affects muscle tension development in bullfrog neuromuscular preparations. Bioelectromagnetics. 22:53–59. Shen, J., Wang, H., Ma, Y., et al. (2012). Effects of intracellular osmolality changes on the voltage-gated sodium channels currents of trigeminal ganglion neuron. Hua Xi Kou Qiang Yi Xue Za Zhi. 30: 338–342. Shen, J. F., Chao, Y. L., Du, L. (2007). Effects of static magnetic fields on the voltage-gated potassium channel currents in trigeminal root ganglion neurons. Neurosci. Lett. 415:164–168. Takeda, M., Tanimoto, T., Ikeda, M., et al. (2004a). Activaton of GABAB receptor inhibits the excitability of rat small diameter trigeminal root ganglion neurons. Neuroscience. 123:491–505. Takeda, M., Tanimoto, T., Ikeda, M., et al. (2004b). Opioidergic modulation of excitability of rat trigeminal root ganglion neuron projections to the superficial layer of cervical dorsal horn. Neuroscience. 125:995–1008. Wieraszko, A. (2000). Dantrolene modulates the influence of steady magnetic fields on hippocampal evoked potentials in vitro. Bioelectromagnetics. 21:175–182. Xu, C., Chao, Y. L., Du, L., Yang, L. (2004). Measurements of the flux densities of static magnetic fields generated by two types of dental magnetic attachments and their retentive forces. Sichuan Da Xue Xue Bao Yi Xue Ban. 35:412–415. Ye, S. R., Yang, J. W., Chen, C. M. (2004). Effect of static magnetic fields on the amplitude of action potential in the lateral giant neuron of crayfish. Int. J. Radiat. Biol. 80:699–708. Yellen, G. (2002). The voltage-gated potassium channels and their relatives. Nature. 419:35–42.
ORIGINAL ARTICLE
Effects of moderate static magnetic fields on the voltage-gated sodium and calcium channel currents in trigeminal ganglion neurons Xiao-Wen Lu1,2, Li Du2, Liang Kou1,2, Ning Song1,2, Yu-Jiao Zhang1,2, Min-Ke Wu1,2, and Jie-Fei Shen1,2 State Key Laboratory of Oral Diseases and 2Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China
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Abstract
Keywords
Aim: To study the effects of static magnetic fields (SMF) on the electrophysiological properties of voltage-gated sodium and calcium channels on trigeminal ganglion (TRG) neurons. Methods: Acutely dissociated TRG neurons of neonatal SD rats were exposed to 125-mT and 12.5-mT SMF in exposure devices and whole-cell patch-clamp recordings were carried out to observe the changes of voltage-gated sodium channels (VGSC) and calcium channels (VGCC) currents, while laser scanning confocal microscopy was used to detect intracellular free Ca2+ concentration in TRG neurons, respectively. Results: (1) No obvious change of current–voltage (I–V) relationship and the peak current densities of VGSC and VGCC currents were found when TRG neurons were exposed to 125-mT and 12.5-mT SMF. However, the activation threshold, inactivation threshold and velocity of the channel currents above were significantly altered by 125-mT and 12.5-mT SMF. (2) The fluctuation of intracellular free Ca2+ concentration within TRG neurons were slowed by 125-mT and 12.5-mT SMF. When SMF was removed, the Ca2+ concentration level showed partial recovery in the TRG neurons previously exposed by 125-mT SMF, while there was a full recovery found in 12.5-mT-SMF-exposed neurons. Conclusions: Moderate-intensity SMF could affect the electrophysiological characteristics of VGCS and VGCC by altering their activation and inactivation threshold and velocity. The fluctuations of intracellular free Ca2+ caused by SMF exposure were not permanent in TRG neurons.
125-mT, 12.5-mT, calcium channel, static magnetic fields, sodium channel, trigeminal ganglion neuron, whole-cell patch-clamp
Introduction Moderate-intensity (1 mT-1T) static magnetic fields (SMF) can be produced by permanent magnets as well as other electromagnets in our daily life. Dental magnetic attachments are typical examples for the application of magnets and magnetic forces in contemporary dentistry. In their course of development from early open-circuit magnet system to nowadays close-circuit magnet system, for different type of magnets, controversial opinions persist on the biosecurity of SMF exposure. Previous studies revealed that SMF can alter the central nervous function (Rosen, 1993a; Rosen and Lubowsky, 1990), peripheral nerve conduction (Cordeiro et al., 1989), neuromuscular activity and synaptic transmission (Rosen, 1992; Satow et al., 2001). However, the mechanisms underlying such effects are not entirely clear. One notable hypothesis proposed by Rosen (1993a,b) indicated that moderate-intensity SMF up to 125-mT may induce magnetic reorientation of membrane phospholipids by diamagnetic anisotropy effects and this theory was brought to Address correspondence to Dr. Jie-Fei Shen, State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, No. 14, Section 3, Renminnan Road, Chengdu, Sichuan Province, 610041, P.R. China. Tel: +86 028 85502408. E-mail: [email protected]
History Received 22 October 2013 Revised 3 March 2014 Accepted 9 March 2014 Published online 8 April 2014
interpret a series of experiment findings about the effects of moderate-intensity SMF on the action potentials of neurons (Cavopol et al., 1995; McLean et al., 1995; Ye et al., 2004) and mechano-sensitive ion channels (Dobson et al., 2002a,b). The voltage-gated channels are important physiological regulators of membrane resting and action potentials (Catterall, 2000, 2011; Yellen, 2002). They are widely expressed in excitable tissues including sensory ganglia (Amaya et al., 2000; Novakovic et al., 1998; Rasband et al., 2001). In our previous report on trigeminal ganglion neurons (Shen et al., 2007), effects of 125-mT SMF were found on two main sub-types of voltage-gated potassium channels (VGPC), the fast-inactivating transient (IK,A) and dominant-sustained (IK,V) channels, that SMF could influence the inactivation kinetics of both VGPC currents. These findings were consistent on the research on the IK,V channel of prefrontal cortex pyramidal neurons, that even weaker SMF could also influence the amplitudes, characteristics of half-activation voltage and the k value of the VGPC activation curves (Li et al., 2010). Besides VGPC, voltage-gated sodium channels (VGSC) and voltage-gated calcium channels (VGCC) are the other two major components giving rise to the electrophysiological characteristics of neurons. As the fundamentals of the sodium inward currents, VGSC initiate the action potentials in nerve,
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muscle and other excitable cells (Catterall, 2012; Hodgkin and Huxley, 1952a, b). Malfunctions of VGSC lead to the disorders of neuronal excitability in epilepsy, chronic pain, autism, migraine, multiple sclerosis, etc. (Eijkelkamp et al., 2012). VGCC are the key signal transducers of electrical excitability, converting the electrical signal of the action potential in the cell surface membrane to an intracellular Ca2+ transients that initiate many physiological events (Catterall, 2011). The complex interaction between VGSC, VGPC and VGCC is the functional basis of profound neural electric activities. However, the effects of moderate intensity SMF on VGSC and VGCC are not thoroughly clarified. Therefore, in this study, whole-cell patch-clamp experiments were conducted on TRG neurons to investigate whether 125-mT and 12.5-mT SMF were capable of influencing the activation and inactivation characteristics of VGSC and VGCC currents.
Methods The SMF exposure devices According to our previous measurements (Shen et al., 2007), 125-mT was a common intensity of SMF around open-circuit dental magnets, while 12.5-mT was that of close-circuit ones. In this study in vitro, two exposure deices were manufactured to simulate the SMF of these two intensities, respectively. The structure and dimension of the devices were designed and adjusted by electromagnetic design software, Maxwell 3D (Ansoft Corp., Pittsburg, PA).
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Each U-shaped device has two 60 mm 60 mm square NdFeB magnets covered by polar caps and connected by a steel yoke (Figure 1A). In the course of SMF exposure, 35-mm plastic cell culture dish was placed on a base to reach the position at the vertical and horizontal center of the 50-mm-wide air gap between the two caps. Simulation results demonstrated that 125-mT and 12.5-mT SMF could be generated by these two devices of their structure and size, respectively. After the devices were assembled, precise measurement carried out by digital teslameter (TJSH-035, Fuzhou, China) revealed the consistent results and the non-homogeneity of flux density was less than 1% around the culture dish for both of the two devices (Figure 1B). The exposure device could be mounted onto an inverted microscope (IX71, Olympus, Tokyo, Japan) and patchclamp pipette was extended into the exposure area from the opening side of the device. All ferromagnetic materials on the microscope round the exposure device were replaced by acrylic or epoxy resin. Acute dissociation of TRG neurons The method obtaining TRG neurons was described in our previous report ever (Shen et al., 2007, 2012). Briefly, TRG neurons were isolated from neonatal (5–7 d) Sprague–Dawley rats anesthetized with ether. Dissected TRG neurons were washed by ice-cold Hanks’ balanced salt solution (HBSS, Sigma, St. Louis, MO), then minced into small pieces and
Figure 1. (A) The structure and dimension of 125-mT (upper) and 12.5-mT (lower) SMF exposure devices, a: magnets covered by polar caps; b: steel yoke. (B) The horizontal SMF distribution at the height where the culture dish was placed (Z ¼ 30 mm). The central point of dish bottom was designated as the horizontal coordinate origin.
Effects of SMF on TRGN Na+ and Ca2+ channels
DOI: 10.3109/15368378.2014.906448
incubated in HBSS containing 20 U/ml papain (Sigma) at 37 C for 40 min. After the digestion, the cells were washed 3 times by DMEM/F-12 culture medium (1:1 in volume, Gibco, Grand Island, NY) supplemented with 10% Fetal Bovine Serum (Gibco), and 0.5-mM glutamine (Gibco). Then, they were gently triturated by a series of fire-polished Pasteur pipettes and plated onto poly-L-lysine (Sigma) pretreated glass coverslips placed in 35-mm dishes. The dishes were maintained in a humidified atmosphere of 5% CO2 at 37 C and all TRG neurons were used within 2 and 3 h after plating. All TRG neurons could be distinguished by their relatively larger figures than other dissociated cells and identified by further Nissl substance stain with Cresyl Echt violet (Mitcheson and Sanguinetti, 1999).
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Whole-cell patch-clamp recordings Before patch-clamp recordings, the cell culture medium in the dishes was carefully removed and the cells were washed by external solution for 3 times. The external solution used for VGSC currents recording contained 60-mM Choline-Cl, 60-mM NaCl, 20-mM Tetraethylammonium chloride (TEA-Cl, Sigma), 5-mM KCl, 1-mM MgCl2 6H2O, 2-mM CaCl2, 0.1-mM CdCl2 5H2O, 10-mM D-glucose, 3-mM 4-aminopyridine (4-AP, Sigma), 10-mM HEPES (pH ¼ 7.4), which used for VGCC currents recording contained 110-mM Choline-Cl, 2.5-mM CsCl, 2.5-mM CaCl2, 1-mM MgCl2, 30mM tetraethylammonium (TEA, Sigma), 4-mM 4-AP, 10-mM D-glucose and 10-mM HEPES (pH ¼ 7.4). Finally, the external solution volume in the dishes was adjusted to 2 ml. About 15 min after the final wash, the dishes were transferred into the SMF exposure device on the microscope and maintained for another 15 min. Then the recordings were performed at room temperature of 25–28 C. For control groups, there was no SMF exposure to the cells. Patch-clamp pipettes were pulled from borosilicate glass and filled with internal solution composed of 135-mM CsCl, 10-mM NaCl, 2-mM MgCl2, 1-mM CaCl2, 10-mM HEPES, 10-mM EGTA (ph ¼ 7.3) for VGSC currents recording, and 120-mM CsCl, 2-mM CaCl2, 10-mM NaCl, 10-mM HEPES and 10-mM EGTA (pH ¼ 7.3) for that of VGCC, respectively. The electrodes had resistances of 2–4 M . The whole cell recordings were conducted using an Axopatch 200 B patch-clamp amplifier (Molecular Devices LLC, Sunnyvale, CA) and the output was digitized with Digidata 1440 A converter (Molecular Devices LLC). Both the capacitance and series resistance were well compensated. All data were acquired by Clampex 10.0 software (Molecular Devices LLC). Intracellular free Ca2+ imaging and fluorometry The intracellular free Ca2+ concentration ([Ca2+]i) measurements within TRG neurons were carried out by Fluo-3/AM loading and laser scanning confocal microscope (LSCM, TCS SP, Leica, Wetzlar, Germany) after the neurons were exposed under SMF for 1 h, 1.5 h, 2 h and 2.5 h. [Ca2+]i was derived by equation F¼F1-F0, in which F was actual fluorescent density, F1 and F0 were detected and background fluorescent density. All exposure procedures were kept in a humidified atmosphere of 5% CO2 at 37 C.
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Data analysis Data of patch clamp recordings were analyzed with Clampfit 10.0 software (Molecular Devices LLC). All curve fittings and statistical comparisons were performed in Origin 9.0 software (OriginLab Corp., Northampton, MA). Differences were considered to be significant at p50.05.
Results Characteristic of total VGSC and VGCC currents on rat TRG neurons In previous reports, several types of VGSC and VGCC currents which differ in their pharmacological and biophysical properties have been found in sensory neurons including TRG neurons (Amaya et al., 2000; Kim and Chung, 1999; Novakovic et al., 1998). In present study, total currents of VGSC and VGCC were the focus of our observations. In patch clamp system, the recording of activation and inactivation currents for VGSC and VGCC were based on different stimulus protocols after reaching Giga-Ohm seal between the cell and patch pipette. For the VGSC, neurons were initially held at 60 mV followed by hyperpolarization to 80 mV for 70 ms as conditioning prepulse potential. Then the inward sodium activation currents were elicited by 100 ms pulses stepping from 60 to +60 mV in 10-mV increments (Figure 2A). Their inactivation properties were studied by another stimulus protocol. The neurons being held at 80 mV were given a series of 50 ms prepulses stepping from 100 to 40 mV followed by a 50 ms test pulse depolarizing to +10 mV (Figure 3A). While, to the VGCC, they were initially held at 80 mV and activation currents were elicited by 400 ms pulses stepping from 80 mV to +100 mV in 10-mV increments (Figure 2D). For inactivation recordings, the neurons were held at 80 mV then given a series of 300-ms prepulses stepping from 80 mV to +70 mV followed by a 200 ms test pulse depolarizing to +10 mV (Figure 3D). Current densities of both channels were obtained by dividing the amplitude of the currents to their own whole-cell capacitances. Effects of SMF on the activation of VGSC and VGCC currents on TRG neurons Totally, 30 randomized selected TRG neurons were recorded for VGSC (for 125-mT SMF exposure group, n ¼ 10; for 12.5mT SMF exposure group, n ¼ 10; for control group, n ¼ 10) and another 30 randomized selected TRG neurons were used for VGCC currents recording (for 125-mT SMF exposure group, n ¼ 10; for 12.5-mT SMF exposure group, n ¼ 10; for control group, n ¼ 10). The capacitance of VGSC exposed in 125-mT SMF, 12.5-mT SMF and control groups were 32.51 ± 6.43 pF, 34.01 ± 4.81 pF and 31.15 ± 5.38 pF, while those of VGCC were 30.51 ± 5.03 pF, 33.01 ± 4.92 pF and 35.37 ± 6.02 pF (mean ± SEM). The VGSC started to be activated when the membrane potential was depolarized to about 60 mV (Figure 2B). The largest current densities were gained for all three groups of VGSC currents around 10 mV. To the VGCC, the channels started to be activated as the membrane potential was about 60 mV (Figure 2E), and the largest current densities were
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Figure 2. (A–C) Effects of 125-mT and 12.5-mT SMF on the activation of INa; (D–F) Effects of 125-mT and 12.5-mT SMF on the activation of ICa. (A,D) Typical VGSC and VGCC activation currents of TRG neurons recorded by patch clamp system and their activation patch clamp protocols; (B,E) normalized activation curves for VGSC and VGCC; (C) comparison of the fitting results of VGSC activation currents. (F) Comparison of the fitting results of VGCC activation currents. (*Difference between two SMF intensity exposure and control groups were statistically significant at p50.05.)
gained around +10 mV for all three groups. The current– voltage (I–V) relationship and largest current densities of VGSC and VGCC were not significantly altered by 125-mT and 12.5-mT SMF (data not showed). Then, channel conductance (G) at different membrane potential was obtained by equation G ¼ I/(VmVrev), in which I was the current density, Vm was the voltage command, and Vrev was the reversal potential. Normalized activation curves were plotted as G/Gmax against the voltage commands (Figure 2B and E). The curves of all groups were fitted to Boltzmann equation: G/Gmax ¼ 11/{1+exp[(VmV1/2)/k]}, in which Vm was the voltage command, V1/2 was the membrane potential at half activation and k was the slope factor. Fitting results (Figure 2C) demonstrated that, there was no significant change in V1/2 and k of VGSC between 12.5-mT SMF exposure group and control group (p40.05), while V1/2 and k of 125-mT SMF exposure group differed with both 12.5-mT exposure and control groups (p50.05). To the analysis of VGCC (Figure 2F), there was no significant change in V1/2 between two SMF exposure groups (p40.05), while V1/2 of control group differed with both of them (p50.05). For k values, no statistical difference was found among those three groups (p40.05).
For all the three groups of VGSC, currents began to inactivate when membrane potential was about 70 mV and reached the maximum inactivation at membrane potentials were about +40 mV (Figure 3B). As to that of VGCC (Figure 3E), both SMF exposure groups and control groups began to inactivate as membrane potential was about 60 mV. But the maximum inactivation currents appeared at different membrane potentials among the three groups, which were about 20 mV, 10 mV and 10 mV for 125-mT SMF, 12.5-mT SMF and control group, respectively. Their inactivation curves were also fitted to Boltzmann equation: I/Imax ¼ 1/{1+exp[(VmV1/2)/k]}, in which V1/2 was the membrane potential at half inactivation at this time. According to the fitting results, difference in V1/2 of VGSC was found between 125-mT SMT exposure group and both 12.5-mT SMT exposure and control groups (p50.05), while difference of 12.5-mT SMT exposure group and control group showed no significant change (p40.05). No significant changes existed in k between all the groups of VGSC (p40.05) (Figure 3C). As to VGCC, difference of V1/2 was found among all three groups (p50.05). No significant change existed in k between two SMF exposure groups (p40.05), while k of control group differed with both of the SMF exposure groups (p50.05) (Figure 3F).
Effects of SMF on the inactivation of VGSC and VGCC currents on TRG neurons
Effects of SMF on [Ca2+]i in TRG neurons
Normalized inactivation curves were plotted as the peak current density against the prepulse voltage commands.
Images of Ca2+ fluoresced TRG neurons of different exposure conditions and durations were showed as in Figure 4.
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DOI: 10.3109/15368378.2014.906448
Effects of SMF on TRGN Na+ and Ca2+ channels
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Figure 3. (A–C) Effects of 125-mT and 12.5-mT SMF on the inactivation of INa; (D–F) Effects of 125-mT and 12.5-mT SMF on the inactivation of ICa. (A,D) Typical VGSC and VGCC inactivation currents of TRG neurons recorded by patch clamp system and their activation patch clamp protocols; (B,E) normalized inactivation curves for VGSC and VGCC currents; (C) comparison of the fitting results of VGSC inactivation currents. (F) Comparison of the fitting results of VGCC inactivation currents. (*Difference between two SMF intensity exposure and control groups were statistically significant at p50.05).
It could be derived from the measurements of F value among the SMF exposure and control groups (Table 1) that at the beginning 1 h to 1.5 h, the [Ca2+]i changes were delayed in both 125-mT and 12.5-mT SMF groups followed by recovery process toward the level of the control group after 2 h of exposure. But such process differed according to different intensity of SMF loading. After 2.5 h exposure of 12.5-mT SMF, a full recovery of [Ca2+]i was met, while [Ca2+]i of the TRG neurons in 125-mT SMF group was still lower than the control.
Discussion In previous studies on the biological effects of moderateintensity SMF on the cell membrane and ion channels, 125mT and 12.5-mT were of the most discussed intensities (Rosen, 2003a; Xu et al., 2004). In this study in vitro, the field was produced by exposure devices made of permanent magnets (Figure 1A). Compared to electromagnets, no thermal changes would be generated by this device. The size was minimized by the Maxwell software while the power, scope and homogeneity of the magnetic fields were ensured. Two SMF intensity exposure devices were fully compatible with the inverted microscope and the patch clamp system. There have been various reports on the biological effect caused by electromagnets and SMF. However, the underlying mechanism, especially about the effects on the neural
activities, has not been entirely clarified. In Rosen’s hypothesis (Rosen, 2003b), the biological membrane would be deformed in SMF and the ion channels on the membrane would be influenced. The alterations on channel activity caused by SMF exposure are indirect. The primary effect of magnetic fields is to induce rotation and reorientation of the membrane lipid molecules and such reorientation could affect conformation changes of ion channels (Hughes et al., 2005). This theory has been applied to interpret experimental findings on GH3 cells including an inhibition of their sodium and calcium channel currents under moderateintensity SMF exposure (Rosen, 1996, 2003a). It provides an explanation to the effects of SMF on neurotransmitter release, the permeability of VGCC on hippocampal neurons (Wieraszko, 2000), and the change of delayed rectifier potassium channel exposed to 3-mT static magnetic field on prefrontal cortex pyramidal neurons (Li et al., 2010). VGPC, VGSC and VGCC are the major channels which establish the resting membrane potential, action potential and other electric activities of neurons and participate into the process of transducing electrical activity into intracellular biochemical signals of neurons. In our previous experiment, effects of 125-mT SMF were also found on the activation and inactivation of VGPC currents on TRG neurons. In this study, the main purpose was to observe the effects of SMF on voltage-gated sodium and calcium channels and compared to our previous findings on potassium channels. Therefore, we
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Figure 4. Intracellular free Ca2+ imaging of TRG neurons.
Table 1. Effects of 125-mT and 12.5-mT SMF on [Ca2+]i in TRG neurons (mean ± SD). SMF exposure time 1h 1.5 h 2h 2.5 h
125-mT group 179.4 ± 68.4 105.9 ± 57.3 66.0 ± 37.2 139.6 ± 53.8
(n ¼ 12)b,c,d,a (n ¼ 11)a,c,d,a (n ¼ 21)a,b,d (n ¼ 17)a,b,c,a
12.5-mT group 176.4 ± 44.0 82.6 ± 58.1 70.3 ± 45.9 175.9 ± 59.5
(n ¼ 17)b,c,a (n ¼ 30)a,d,a (n ¼ 37)a,d (n ¼ 16)b,c
Control 141.2 ± 50.8 41.4 ± 21.4 81.8 ± 61.5 188.8 ± 27.5
(n ¼ 24)b,c,d,b,g (n ¼ 19)a,c,d,b,g (n ¼ 22)a,b,d (n ¼ 9)a,b,c,g
a
p50.05 versus 1 h, bp50.05 versus 1.5 h, cp50.05 versus 2 h, dp50.05 versus 2.5 h; p50.05 versus control, bp50.05 versus 12.5-mT group, gp50.05 versus 125-mT group.
a
focused on the whole-cell total currents and their overall channel kinetics, not all the subtypes of each category. As shown in the results 125-mT and 12.5-mT SMF did not exhibit effects on the peak current density of VGSC and VGCC activation, neither were the k value which indicated the activation velocity of VGCC and inactivation velocity of VGSC. But the V1/2, which was related to the activation threshold voltage, was influenced by SMF of both intensities for VGSC and VGCC (Figures 2 and 3).
VGSC were activated at an proximal membrane potential when SMF exposure was loaded. Though the V1/2 and k values of activation properties showed no significant changes between 12.5-mT SMF exposure group and control group, 125-mT SMF exposure made a change, which indicated that 125-mT exposure could make activation threshold voltage more positive and slow down the activation speed. As for the inactivation properties of VGSC, V1/2 values exposed under 125-mT SMF appeared some conspicuous features that
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DOI: 10.3109/15368378.2014.906448
currents began to be inactivated at more negative potentials, as 12.5-mT SMT exposure made no effect. Such results seemed to indicate that there perhaps existed a certain threshold of magnetic field intensity able to change both activation and inactivation features of sodium channel, or the effects might be intensity-depended of which 12.5-mT exposure was not enough to make an influence obviously to achieve the statistical significance. However, VGCC were activated at a more negative membrane potential without change of k values when SMF exposure was loaded. As for the inactivation properties of VGCC, both 125-mT and 12.5mT SMF could change the V1/2 and k values. The VGCC currents began to be inactivated at more negative potentials. Such effects were intensity-dependent, that the 125-mT SMF brought a stronger influence than 12.5-mT SMF. Meanwhile, both of them accelerated the inactivation speed and such effects seemed not related to their intensities. Previous studies have identified that VGSC are large multiprotein complexes with a a subunit containing four internally homologous domain (I–IV) and auxiliary b subunits. There are six a-helical transmembrane segments (S1–S6) in each of the four homologous domains. Sodium channel activation is due to a sequence of voltage induced changes in the S4 segment of the a-subunit. Depolarization of the membrane is proposed to release the S4 segments to move outward along a spiral path, initiating a conformational change that opens the pore (Catterall, 2000). The S4 segment would be vulnerable to the effects of diamagnetic reorientation of phospholipid molecules caused by SMF (Rosen, 2003a). Inactivation of the sodium channel has been discovered to involve an highly conserved intracellular loop connecting domains III and IV of the sodium channel a-subunit (Catterall, 2000). Such an intracellular mechanism may not be directly affected by physical changes within the membrane (Rosen, 2003a). VGCC on sensory neurons have a similar structure with a pore forming a1 subunit as the center piece, surrounded by auxiliary a2d, b and g subunits. The a1 subunit contains four conserved transmembrane structural domains that are linked by more variable cytoplasmic sequences (Catterall, 2011). This subunit largely determines the biophysical properties of the VGCC, and gives rise to the voltage sensing and activation characteristics of VGCC currents (Catterall et al., 2005), while the inactivation processes can occur by multiple mechanisms that requires the participation of auxiliary subunits (Dolphin, 2003). The similar effects of 125-mT and 12.5-mT SMF on the activation threshold suggested that a1 subunits of VGCC might be the indirect targets of the moderate-intensity of SMF by the diamagnetic anisotropy effects and reorientation of membrane phospholipids. However, the reason underlying the diverse changes of inactivation characteristics by 125-mT and 12.5-mT SMF exposure remained obscure. In our previous experiments on 125-mT SMF (Shen et al., 2007), similar differentiated alterations were also observed on the inactivation of two main VGPCs currents, IK,A and IK,V. N-type inactivation leading to very rapid inactivation process of IK,A is frequently described as the ‘‘ball-and-chain’’ mechanism, in which a sequence of amino acids (the inactivation domain) at the N-terminus of the VGPCs occludes the intracellular channel pore and prevents ion permeation (Hoshi et al., 1990, 1991;
Effects of SMF on TRGN Na+ and Ca2+ channels
7
Rasmusson et al., 1998). The slower inactivation process of IK,V is more related to the C-type inactivation which involves conformational changes around the selectivity filter and extracellular entrance to the channels (Choquet and Korn, 1992). When biological membrane is deformed under SMF, the distortion of VGPCs is probably different on their inner and outer terminals. This difference might give rise to the opposite effects of 125-mT SMF on the inactivation velocity of IK,A and IK,V currents (Shen et al., 2007). It needs further studies to clarify whether 125-mT and 12.5-mT could bring such effects on VGCC currents. The maximum time of patch clamp recordings was limited to 15 min after the seal between the plasma membrane and pipette was formed, because TRG neurons could not survive a longer time after the patch break on the membrane required by whole-cell recordings. Though the time course for the influence of SMF on VGSC and VGCC was not included in our study, this issue has already been discussed in previous reports (Rosen, 2003a). It was demonstrated that the effect of SMF on the cell membrane was reversible and could persist for about 100 s after the field was turned off. In Hughes’ study (Hughes et al., 2005), ion channel activity also showed slow recovery following removal of the magnetic field. These observations indicated that after the exposure of SMF was shut off, there would be a slow reorientation process of the diamagnetic molecules on the deformed membrane and the restoration of channel functions would be delayed. This explanation could interpret our findings on the [Ca2+]i in this study (Table 1, Figure 4). It should be addressed that, in Rosen’s researches on GH3 cells, optimum effects of SMF on the ion channels were observed around 37 C (Rosen, 1996, 2003a). However, most studies of channel functions on mammal cells, especially the neurons, were carried out at room temperature (Liu and Simon, 2003; Takeda et al., 2004a,b). In normal physiological condition ranging from 23 to 37 C, no change in the current– voltage relationship of channel kinetics was found (Rosen, 2001). According to Maret and Dransfeld (1977) analysis on the joint effects of SMF and temperature on the diamagnetic anisotropic molecules, it was pointed out that within the physiologically permissible range, the effect of temperature on the interaction between SMF and those molecules was small; a 10 C difference in temperature will result in only a change less than 4%. In Hughes’ experiment (Hughes et al., 2005), membrane reorientation effects of 125-mT SMF at room temperature were found to be identical to that in Rosen’s. Wieraszko (2000) reported the similar changes in hippocampal neurons under SMF exposure at room temperature as well. In this study, our primary objective was to investigate the effects of SMF on the activation and inactivation current–voltage relationship of VGSC and VGCC. Therefore, the experiment temperature was set to the room level.
Conclusion Moderate-intensity SMF could affect electrophysiological characteristics of VGCS and VGCC by altering their activation and inactivation threshold and velocity. The fluctuation of intracellular free Ca2+ concentration within TRG neurons
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X.-W. Lu et al.
slowed by SMF could be fully or partially restorable, indicating the influence of SMF might not be permanent. The mechanism underlying those changes was believed to be related to the magnetic reorientation of membrane phospholipids caused by moderate-intensity SMF through diamagnetic anisotropy effects.
Acknowledgements We would like to thank Ms. Daqing Liao, Ms. Yanfang Chen and Ms. Xiaoyu Li for their technical assistance.
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Declaration of interest This research was funded by The National Natural Science Foundation of China for Young Scholars. Grant No. 81000456.
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