Important modifications by sugammadex, a modified γ-cyclodextrin, of ion currents in differentiated NSC-34 neuronal cells
© The Author(s) 2017
Received: 22 March 2016
Accepted: 8 December 2016
Published: 3 January 2017
Sugammadex (SGX) is a modified γ-cyclodextrin used for reversal of steroidal neuromuscular blocking agents during general anesthesia. Despite its application in clinical use, whether SGX treatment exerts any effects on membrane ion currents in neurons remains largely unclear. In this study, effects of SGX treatment on ion currents, particularly on delayed-rectifier K+ current [I K(DR)], were extensively investigated in differentiated NSC-34 neuronal cells.
After cells were exposed to SGX (30 μM), there was a reduction in the amplitude of I K(DR) followed by an apparent slowing in current activation in response to membrane depolarization. The challenge of cells with SGX produced a depolarized shift by 15 mV in the activation curve of I K(DR) accompanied by increased gating charge of this current. However, the inactivation curve of I K(DR) remained unchanged following SGX treatment, as compared with that in untreated cells. According to a minimal reaction scheme, the lengthening of activation time constant of I K(DR) caused by cell treatment with different SGX concentrations was quantitatively estimated with a dissociation constant of 17.5 μM, a value that is clinically achievable. Accumulative slowing in I K(DR) activation elicited by repetitive stimuli was enhanced in SGX-treated cells. SGX treatment did not alter the amplitude of voltage-gated Na+ currents. In SGX-treated cells, dexamethasone (30 μM), a synthetic glucocorticoid, produced little or no effect on L-type Ca2+ currents, although it effectively suppressed the amplitude of this current in untreated cells.
The treatment of SGX may influence the amplitude and gating of I K(DR) and its actions could potentially contribute to functional activities of motor neurons if similar results were found in vivo.
KeywordsSugammadex Motor neuron Delayed-rectifier K+ current Activation kinetics L-type Ca2+ current Glucocorticoid
Sugammadex (SGX) is recognized as a modified γ-cyclodextrin with a lipophilic core and a hydrophilic periphery, and it has been used clinically for reversal of neuromuscular blockade caused by rocuronium or vecuronium during general anesthesia [1–5]. A previous report showed that addition of SGX could cause neuronal apoptosis in primary cultures . This compound per se was also reported to be effective at reversing neurodegenerative disorder of the lower motor neurons [7, 8]. However, interestingly, how the treatment with SGX can perturb ionic currents remains largely unexplored.
The KV3 channels, a subfamily of KV channels, are distinguished from other types of KV channels by more positively shifted voltage-dependent activation and by faster activation and deactivation rates [9, 10]. These differences enable KV3 channels to be major determinants of high-frequency firing existing in several types of central neurons [11–15]. The activity of KV3.1 channels has been recently described as playing a crucial role in controlling the amplitude of action potentials at synapses . The de novo mutations in KCNC1, which encodes KV3.1 channels, have been also found to expand phenotypic spectra of this channel to progressive myoclonus epilepsy . Therefore, these channels clearly are important targets used for investigations on electrical behaviors of central neurons including motor neurons .
The NSC-34 mouse motor neuron cell line is a hybridoma cell line derived from the fusion of mouse neuroblastoma with motor neuron-enriched spinal cord cells [18, 19]. These cells may create an easily accessible and clonally uniform motor neuron-like cell line that overcomes the problems associated with the culturing of primary spinal motor neurons. It has been demonstrated to be a suitable model for investigations on the mechanisms of neuronal development and differentiation in vitro and for studying electrophysiological properties of motor neurons in spite of being not considered as an adult motor neuron [18–20]. The biophysical properties of delayed-rectifier K+ current [I K(DR)] in NSC-34 cells were previously found to resemble the KV3.1-encoded current because of positive mRNA detection of KV3.1 (KCNC1) [21, 22]. As NSC-34 cells were differentiated, the density of I K(DR) was significantly enhanced. Previous work from our laboratory has shown that removal of membrane cholesterol by methyl-β-cyclodextrin, a cyclic oligosaccharide, could modify activation kinetics of I K(DR) in motor neuron-like cells . Additionally, although voltage-gated Na+ current (I Na) in NSC-34 neuronal cells has been previously reported , few studies have been concerned with the biophysical or pharmacological properties of Ca2+ currents in these cells.
Therefore, the purpose of this work was to test whether SGX treatment could exert any perturbations on ionic currents present in NSC-34 neuronal cells differentiated with low serum and retinoic acid. The biophysical and pharmacological properties of ionic currents including delayed-rectifier K+ current [I K(DR)], voltage-gated Na+ current (I Na) and L-type Ca2+ current (I Ca,L) in untreated and SGX-treated cells have been characterized and compared in this study. Interestingly, the present results indicate that SGX treatment is capable of modifying the activation kinetics of I K(DR) elicited by membrane depolarization in these cells in a concentration-, time-, and state-dependent manner.
Drugs and solutions
Composition of normal Tyrode’s solution and the pipette solution used in this study
Purpose or name
Normal Tyrode’s solution
136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.53 mM MgCl2, 5.5 mM glucose, and 5.5 mM HEPES–NaOH buffer, pH 7.4
For recordings of K+ current or membrane potential
130 mM K-aspartate, 20 mM KCl, 1 mM KH2PO4, 1 mM MgCl2, 3 mM Na2ATP, 0.1 mM Na2GTP, 0.1 mM EGTA, and 5 mM HEPES–KOH buffer, pH 7.2
For recordings of Na+ or Ca2+ currents
130 mM Cs-aspartate, 20 mM CsCl, 1 mM KH2PO4, 1 mM MgCl2, 3 mM Na2ATP, 0.1 mM Na2GTP, 0.1 mM EGTA, and 5 mM HEPES–CsOH buffer, pH 7.2
Cell preparation and differentiation
NSC-34 neuronal cells were originally produced by fusion of the motor neuron-enriched, embryonic mouse spinal cords with the mouse neuroblastoma . These cells were kindly provided by Professor Yuh-Jyh Jong (Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung City, Taiwan). They were routinely grown in 1:1 mixture of Dulbecco’s modified Eagle medium (DMEM) and Ham’s F12 medium that was supplemented with 10% (v/v) FBS and 1% penicillin–streptomycin. Cultures were incubated at 37 °C in a humidified environment of 5% CO2/95% air. The medium was replenished every 2–3 days for removal of non-adhering cells. To slow cell proliferation and enhance their maturation towards a differentiated state , before confluence, cells were grown in 1:1 DMEM plus Ham’s F12 medium supplemented with low serum (1% FBS) and 1 μM retinoic acid. The SGX-treated cells were incubated at 37 °C for 1 h in normal Tyrode’s solution containing different concentrations of this compound. The reason that the duration was set at 1 h was to ensure that SGX could exert its interaction with cell membrane.
Shortly before each experiment, cells were dissociated, and an aliquot of cell suspension was transferred to a homemade recording chamber positioned on the stage of a CKX-41 inverted microscope (Olympus, Tokyo, Japan). Cells were immersed at room temperature (20–25 °C) in normal Tyrode’s solution containing 1.8 mM CaCl2. The patch electrodes used were prepared from Kimax capillary tubes (#34500; Kimble Glass, Vineland, NJ, USA) using a vertical two-step electrode puller (PP-83 or PP-830; Narishige, Tokyo, Japan), and their tips were then fire-polished with an MF-83 micro-forge (Narishige). Experiments were performed using the whole-cell configuration of standard patch-clamp technique using either an RK-400 (Bio-Logic, Claix, France) or an Axopatch 200B (Molecular Devices, Sunnyvale, CA, USA) patch-clamp amplifier . Junctional potentials that developed when the composition of the pipette solution was different from that in the bath were nulled.
The signals consisting of voltage and current tracings were displayed and recorded online using an ASUSPRO-BU401LG computer (ASUS, Taipei City, Taiwan) equipped with a Digidata 1440A device (Molecular Devices), and the experiments were controlled by pCLAMP 10.2 software (Molecular Devices). Current signals were low-pass filtered at 3 kHz and digitized at 10 kHz. In some experiments of verifying analog-to-digital conversion, signals are digitized using a PowerLab acquisition system with LabChart 7.0 programs (AD Instruments, Gerin, Tainan City, Taiwan). The resultant data achieved during this experiment were analyzed off-line by use of various analytical tools including the LabChart 7.0 program (Gerin), Origin 8.0 (OriginLab, Northampton, MA, USA) and custom-made macro procedures run under Excel 2013 (Microsoft, Redmond, WA, USA). The voltage-step profiles digitally created from pCLAMP 10.2 were employed to evaluate current–voltage (I–V) relationships or steady-state inactivation of ionic currents [e.g., I K(DR)].
Perturbation by SGX treatment of free energy involved in the gating of I K(DR) was calculated as ΔΔG 0 = ΔG 0 SGX − ΔG 0 Ctrl = F(qV 1/2SGX − qV 1/2Ctrl) = Δ(qFV 1/2), where ΔG 0 Ctrl and ΔG 0 SGX indicate the free energy of I K(DR) activation taken from untreated cells and cells exposed to SGX respectively.
Linear or nonlinear curve-fitting to data sets presented herein was performed using either Microsoft Solver function embedded in Excel (Microsoft) or Origin 8.0 program (OriginLab). The values are provided as the mean ± standard error of the mean (SEM) with sample sizes (n) indicating the cell number from which the results were obtained. The paired or unpaired Student’s t test and a one-way analysis of variance with the least-significant difference method for multiple comparisons were used for statistical evaluation of differences among means. Statistical analyses were performed using the Statistical Package for Social Science 20 (SPSS; IBM Corp., Armonk, New York). Statistical significance was determined at a P value of <0.05.
Effect of SGX treatment on delayed-rectifier K+ currents [I K(DR)] in differentiated NSC-34 neuronal cells
The activation curve of IK(DR) obtained with or without treatment of SGX
According to the values of q and V 1/2, the free energy involved in the gating of I K(DR) at 0 mV (ΔG 0) in the absence and presence of SGX treatment was estimated to be 3.93 ± 0.12 and 10.35 ± 0.18 kJ/mol (n = 9) respectively. The perturbation by SGX treatment of free energy (ΔΔG 0) involved in I K(DR) gating was calculated to be 6.42 ± 0.15 kJ/mol. It is therefore anticipated from these data that SGX treatment can increase the free energy needed for I K(DR) activation observed in differentiated NSC-34 cells.
Kinetic evaluation of IK(DR) block by SGX
Inability of SGX to modify the steady-state inactivation curve of IK(DR)
The increase of cumulative inhibition of IK(DR) activation in SGX-treated cells
Effect of SGX treatment on voltage-gated Na+ current (INa) in differentiated NSC-34 cells
Comparison of the effect of dexamethasone (DEX) on L-type Ca2+ current (ICa,L) in SGX-treated cells
In this study, the blocking of I K(DR) by SGX treatment was noted to be not instantaneous, but to develop with time immediately after the cell became depolarized. Such treatment produces a time-dependent increase in the activation time constant of I K(DR) in response to membrane depolarization. However, the inactivation curve of I K(DR) obtained in untreated and SGX-treated cells did not differ significantly. It thus appears that, as cells are exposed to SGX, this compound has a greater affinity for the closed (resting) state in the KV channel existing on differentiated NSC-34 cells. The activated channels during SGX treatment tended to produce a lower affinity than those residing in the closed state. As a result, the transition from closed to open state became slowed during cell exposure to SGX. Based on our study, it is therefore tempting to speculate that the treatment of SGX or other structurally similar agents binds to the closed state of the channel and/or blocks a prolonged channel closing in KV3.1 channels. Alternatively, the challenge of cells with SGX or other structurally similar agents can modulate KV3.1 channels where the closed channel conformation represents the high-affinity binding site. It also needs to be noted that, according to minimal reaction scheme shown here, the dissociation constant for SGX treatment was calculated to be 17.5 μM, a value that can be clinically achievable [1, 3, 5].
The KV3.1-encoded currents were reported to be the major molecular components of I K(DR) in these cells [20, 21]. Importantly, our study demonstrated that the activation kinetics of I K(DR) (i.e., KV3.1-encoded current) in SGX-treated cells virtually became slowed in a time- and state-dependent manner. The results were in contrast with inhibitory effects of midazolam or aconitine on I K(DR) [12, 21]. These two compounds exerted inhibitory effects via a mechanism through binding to the open state of the channel followed by increased rate of I K(DR) inactivation. During repetitive stimuli, the perturbation by SGX treatment of I K(DR) activation was potentiated.
By virtue of computational analysis, previous work has shown that changes in activation kinetics of I K(DR) might lead to generation of action potentials with spike-frequency adaptation . The ability of KV3.1-encoded currents to control the waveforms of action potentials at synapses has recently been reported . Indeed, different de novo mutations in KCNC1 have been reported to display a wide variety of progressive myoclonus epilepsy . However, SGX treatment had little or no effect on the peak amplitude of I Na. Therefore, the present results showing any changes in the amplitude and gating by SGX treatment of I K(DR) can be of pharmacological and clinical relevance.
Following SGX treatment, I K(DR) enriched in differentiated NSC-34 cells became activated at more depolarized voltages in comparison with that from untreated cells. Moreover, the steepness of activation curve for I K(DR) became significantly greater in cells exposed to SGX, indicating that the effective number of elementary charges during channel activation in SGX-treated cells was significantly raised. These results are important because they led us to estimate that energy change (ΔG 0 SGX ) for generation of I K(DR) was a value of 10.35 kJ/mol. This value was found to be significantly greater than that ΔG 0 Ctrl (i.e., 3.93 kJ/mol) in untreated cells. SGX treatment apparently is involved in voltage-sensitive gating functions of I K(DR), despite no clear change in inactivation curve of I K(DR) between the two groups of cells. The results lead us to propose that following SGX treatment, the energy barrier for activation of KV3.1 channels became elevated.
In our experimental conditions, supplementation of the medium with retinoic acid resulted in changes in cell morphology and an increase in mRNA expression of the KV3.1 subunit in differentiated NSC-34 neuronal cells [20, 21]. However, the modification of I K(DR) kinetics by SGX presented here did not appear to occur by the gene regulation of these channels, because significant changes in this current in differentiated NSC-34 cells generally occurred with a short time course. Moreover, no changes in I K(DR) density after treatment with SGX were observed, suggesting that such maneuver did not alter the main parts of ion channel permeation pathway (i.e., the S5 and S6 regions). It is thus possible that SGX treatment can regulate the gating kinetics of I K(DR) with no apparent change in the number of functional channels on plasma membrane.
Consistent with previous studies , we clearly demonstrated that addition of DEX suppressed the peak amplitude of I Ca,L in differentiated NSC-34 neuronal cells. It is important to note, however, that the inhibition by DEX of I Ca,L did not occur in SGX-treated cells, despite the ability of MgSO4 to suppress I Ca,L amplitude in both untreated and SGX-treated cells (data not shown). Previous observations have shown that DEX did not increase the activity of large-conductance Ca2+-activated K+ (BKCa) channels in pituitary cells treated with methyl-β-cyclodextrin , suggesting that the binding of DEX to the protein(s) of BKCa channels relies on membrane cholesterol.
Whether DEX produces any significant effect on I Na or I K(DR) in NSC-34 cells or primary motor neurons needs to be further investigated. Whether the presence of SGX alters the cholesterol content in surface membranes and influences the DEX effect on I Ca,L also remains to be explored. Nonetheless, our experimental results are consistent with earlier work showing that DEX is effective at exerting inhibitory effects on SGX reversal of rocuronium-induced neuromuscular block . Alternatively, removal by SGX of DEX-induced block of I Ca,L could be due mostly to the possibility that, similar to a mechanism by which it can reverse muscle relaxing effects by rocuronium, the SGX molecule can effectively encapsulate the DEX molecule [1, 3–5].
A recent report showed that methyl-β-cyclodextrin, a cholesterol-depleting agent, could induce activation of matrix metalloproteinase-2 (MMP-2) . However, the reduction by SGX treatment of I K(DR) activation rate observed in differentiated NSC-34 cells was unable to be reversed by isobavachalcone (10 μM) known to be an inhibitor of MMP-2 activity . Therefore, alterations by SGX treatment of activation kinetics of I K(DR) observed in differentiated NSC-34 cells are not closely associated with a mechanism linked to MMP-2 activation.
It should be noted that the pipette solution used in this study contained 3 mM ATP, which can adequately suppress the activity of ATP-sensitive K+ (KATP) channels. The activity of KATP channels did not differ between the untreated and SGX-treated cells (data not shown). Changes by SGX treatment of I K(DR) amplitude and gating observed in differentiated NSC-34 cells is unlikely to arise from inhibition of KATP channels.
The observed block of I K(DR) caused by SGX treatment actually provides an intriguing mechanism for its inhibition that relies on the closed (resting) state of the KV3.1-encoded channels. The KV3.1-encoded currents were enriched in many central neurons including hippocampal pyramidal neurons, auditory neurons, and Purkinje cells [9, 11, 13, 14]. The activity of these KV channels is recognized as participating in electrical behaviors of fast-spiking neurons [9, 10, 15, 16]. Challenging cells with SGX reduced the amplitude of I K(DR) and slowed the activation time course of this current recorded from differentiated NSC-34 cells as well. The present observations would clearly initiate further studies to understand the SGX effects on electrical activity of motor neurons. Whether SGX-induced reversal of rocuronium-induced neuromuscular blockade is due partly to its blocking of I K(DR) in motor neurons in vivo remains to be further investigated. Some adverse effect such as movement of a limb or the body may be partly explained by its inhibitory effect on I K(DR).
It is noted that neostigmine, an inhibitor of acetylcholinesterase activity, is a typical drug used in anesthesia for reversing the effect of rocuronium-induced neuromuscular blockade. In our study, neostigmine (1 μM) did not exert any effect on the amplitude and gating of I K(DR) in differentiated NSC-34 cells (Additional file 1: Fig. S1). Findings from our study might explain previous observations showing that SGX could reverse more rapidly rocuronium-induced neuromuscular blockade  or that the treatment with SGX was associated with less frequent dry mouth than that of neostigmine . Therefore, it remains to be further delineated whether SGX might exert differential actions when it is used with patients who have been administrated with DEX or other glucocorticoids , if similar findings presented here occur in vivo. Nonetheless, as motor neurons are exposed to SGX, the amplitude and gating of I K(DR) could be modified and these actions might significantly contribute to functional activities of motor neurons.
The SGX treatment may influence the amplitude and gating of I K(DR) and its actions could contribute to functional activities of motor neurons if similar findings occurred in vivo.
- I K(DR) :
delayed-rectifier K+ current
- I Na :
voltage-gated Na+ current
- I Ca,L :
L-type Ca2+ current
fetal bovine serum
Dulbecco’s modified Eagle medium
- I–V :
current versus voltage
- ΔG 0 :
the free energy involved in the gating of I K(DR)
standard error of the mean
- ΔΔG 0 :
the perturbation by SGX treatment of free energy
- BKCa :
large-conductance Ca2+-activated K+
- KATP channel:
ATP-sensitive K+ channel
H-TH performed the research and analyzed the data; H-TH, Y-CL and S-NW designed the research study and wrote the paper; Yi-CL, Y-MH, Y-TT, and S-NW contributed essential reagents and tools. All authors read and approved the final manuscript.
We thank Ming-Chun Hsu, Yan-Ming Huang and Huei-Jen Chen for their assistance in cell preparations. We also appreciated the English editing by Steve Tredrea.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
Funding was provided by Kaohsiung Medical University (Award Number: kmtth-104-051).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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