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The inhibitory effect of propofol on Kv2.1 potassium channel in rat parietal cortical neurons
Accepted Manuscript Title: The inhibitory effect of propofol on Kv2.1 potassium channel in rat parietal cortical neurons Author: Yan-zhuo Zhang Rui Zhang Xian-zhang Zeng Chun-yu Song PII: DOI: Reference:
S0304-3940(16)30057-X http://dx.doi.org/doi:10.1016/j.neulet.2016.01.058 NSL 31816
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
17-10-2015 24-12-2015 26-1-2016
Please cite this article as: Yan-zhuo Zhang, Rui Zhang, Xian-zhang Zeng, Chun-yu Song, The inhibitory effect of propofol on Kv2.1 potassium channel in rat parietal cortical neurons, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.01.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The inhibitory effect of propofol on Kv2.1 potassium channel in rat parietal cortical neurons Yan-zhuo Zhang, Rui Zhang, Xian-zhang Zeng, Chun-yu Song* [email protected] Department of Anesthesiology, China and Heilongjiang Key Laboratory for Anesthesia and Critical Care, The Second Affiliated Hospital of Harbin Medical University, Harbin 150081, China *
Corresponding author at: Department of Anesthesiology, The Second Affiliated
Hospital of Harbin Medical University, NO 246, Xue-fu Street, Nangang District, Harbin 150081, China. Fax: +86 451 86605028. Tel: 13936130754.
Highlights Propofol had neuroprotective effect; propofol could inhibit Ik；propofol could depress the expression of Kv2.1
Abstract Excessive K+ efflux via activated voltage-gated K+ channels can deplete intracellular K+ and lead to long-lasting membrane depolarization which will promote neuronal apoptosis during ischemia/hypoxia injury. The Kv2.1 potassium channel was the major component of delayed rectifier potassium current (Ik) in pyramidal neurons in cortex and hippocampus. The neuronal protective effect of propofol has been proved. Delayed rectifier potassium current (Ik) has been shown to have close relationship with neuronal damage. The study was designed to test the inhibitory effect of propofol on Kv2.1 potassium channel in rat parietal cortical neurons. Whole-cell patch clamp recordings and Western blot analysis were used to investigate the electrophysiological function and protein expression of Kv2.1 in rat parietal cortical neurons after propofol treatment. We found that propofol concentration-dependently inhibited Ik in pyramidal neurons. Propofol also caused a downward shift of the I-V curve of Ik at 30 μM concentration. Propofol significantly inhibited the expression of Kv2.1 protein level at 30 μM, 50 μM, 100 μM concentration. In conclusion, our data showed that propofol could inhibit Ik, probably via depressing the expression of Kv2.1 protein in rat cerebral parietal cortical neurons.
Keywords: propofol; Kv2.1 potassium channel; whole-cell patch clamp; parietal cortical neuron; delayed rectifier potassium current.
1. Introduction Cerebral ischemia is the most common cause that can lead to complex chronic disability worldwide [6]. Ischemia/anoxia could induce cell membrane depolarization, and could lead to voltage gated potassium channels activation, and then potassium homeostasis imbalance. Excessive loss of intracellular potassium plays an important role in triggering cell apoptosis. Some reports have shown that potassium channel blockers could attenuate ischemia/anoxia induced cell apoptosis both in vitro and in vivo. Delayed rectifier potassium currents (Ik) are important in regulating excitability of hippocampal and cortical pyramidal neurons [2]. Kv2.1 is the main composition of IK, and has been tested widely expressed in brain, such as pyramidal neurons, hippocampus, and interneurons [12-17]. Kv2.1 plays various roles in neuronal pathophysiology, such as setting resting membrane potential, shaping action potential repolarization which regulates the excitability and process of synaptic integration in central neurons [2, 11]. Ischemia/anoxia could increase the activity of Kv2.1 by lowering the threshold of activation. This finding suggested that depression of Kv2.1 channel could provide neuroprotective effect for ischemic injuries [18]. Propofol is a commonly used anaesthetic agent that has been demonstrated to be neuroprotective. Some studies have shown that propofol had neuroprotective activities and the mechanisms were complicated, include reduction in cerebral metabolism [10], antioxidant activity [3], anti-excitotoxic properties [7], modulation of inhibitory and excitatory neurotransmitters [21], or influence on neuronal apoptosis [23]. Delayed rectifier potassium channel Kv2.1 was highly expressed on neuronal soma in the brain. Moreover, Kv2.1 was an important potassium channel subtype in regulating apoptotic signaling cascade in ischemia/anoxia injury [17]. However, the effect of propofol on delayed rectifier potassium channel Kv2.1 remains unclear. Therefore, the aim of this study is to investigate whether propofol could inhibit Ik and could block the expression of Kv2.1 protein in rat cerebral parietal cortical neurons. To test this possibility, we used whole-cell patch clamp recordings to explore the potential effects of propofol on Ik and Western blot to examine the expression of Kv2.1 in rat parietal cortical neurons.
2. Materials and methods 2.1 Acute dissociation of neurons Animals used in this study were approved by and handled in accordance with the guidelines of the Animal Care and Use Committee at the Harbin Medical University, Harbin, China. Wistar rats between 10 and 14 days old of both sexes were used. After ether anesthesia, rats were decapitated and the brains were quickly removed and placed in cold artificial cerebrospinal fluid (ACSF) that contained (mM): NaCl: 126, KCl: 5, NaH2PO4: 1.25, NaHCO3: 26, CaCl2: 2, MgSO4: 2, HEPES: 10, and glucose: 10, pH: 7.2–7.4 (titrated with HCl), and were then cut into 400 µm slices. Slices were incubated for 0.5 h at 32 °C in a buffered ACSF bubbled with 95% O2 and 5% CO2. Slices were moved into buffered ACSF containing protease (0.5 mg/ml) at 32 °C for 30 min, and then rinsed three times in cold buffered ACSF. Enzyme-treated slices were mechanically dissociated with a graded series of fire-polished Pasteur pipettes with diameter 150, 300, or 500 µm. Cells were then plated into a 35 mm dish and viewed under an Olympus inverted microscope. 2.2 Electrophysiological recordings Whole-cell patch clamp recording was performed at room temperature (20–25 °C) using a PC-IIC amplifier (HUST-IBB, Wuhan, China). Patch electrodes were pulled from borosilicate glass capillaries by a PP-83 microelectrode puller (Narrishage, Japan) and had resistances of 3–5 MΩ when filled with the internal solutions that contained (mM): KCl: 140, HEPES: 10, EGTA: 10, Na2ATP: 2, and pH: 7.2–7.4 (titrated with KOH). The standard extracellular solution contained (mM): NaCl: 130, KCl: 5.4, CaCl2: 1, MgCl2: 1, glucose: 25, HEPES: 10, CdCl2: 0.2 and pH: 7.2–7.4
(titrated with NaOH). Neurons with a bright and smooth appearance were selected for recording. After whole-cell configuration was established, we waited at least 5 min for steady-state currents to stabilize. Current was filtered by a low-pass Bessel filter set at 2 kHz during data acquisition. Series resistances were compensated for 70–80%. After whole-cell configuration was established, cells were held at -80 mV. The typical IK was elicited with voltage steps with 150-ms prepulse at -40 mV to inactivate transient outward potassium currents (IA). 2.3 Drug administration The following drugs and chemicals were used: protease was purchased from Sigma Chemical (St Louis, MO, USA). Propofol (Labor Dr. Ehrenstorfer-schafers. Germany). All the agents were dissolved in extracellular fluid to make 100 × stock solutions (stored at -20 °C) and diluted in external solution just before application. To determine the effects of propofol on IK, we recorded the amplitudes of IK before and after addition of 10μM, 30μM, 50μM, 100μM, or 150 μM propofol. All drugs and chemicals were applied to bath solution. 2.3 Protein Extraction To produce protein extracts, the cerebral cortex was cut rapidly into pieces 300–400 μm wide and placed at 0–4 °C in ACSF. The samples were then treated with propofol (30µmol/L, 50µmol/L, 100µmol/L) and ventilated at 95% O2/5% CO2 for 6 h; the tissues were then washed three times using PBS buffer. Finally, the samples were placed in liquid nitrogen to isolate all the proteins.
2.4 Western blot analysis One hundred micrograms of total proteins per sample was electrophoretically separated on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and electronically transferred onto a PVDF membrane (Amersham Life Science, Buckinghamshire). The membranes were blocked by incubation for 60 min at room temperature with 5% (w/v) nonfat dry milk in TBS-T (150 mM NaCl, 10 mM Tris-HCl, 0.1% TWEEN), then incubated with mouse anti-Kv2.1 monoclonal antibody (Abcam, Cambridge, UK, 1:1000 dilution) overnight at 4 °C, washed with 1×TBS-T, and incubated for 1 h at room temperature with horseradish peroxidase-conjugated rabbit-anti-mouse IgG secondary antibody (1:5000 dilution; Amersham Life Science, Buckinghamshire). Immunoreactive proteins were visualized using an enhanced chemiluminescence system (ECL; Amersham Life Science, Buckinghamshire). Membranes were exposed for 3 to 30 sec to Kodak XOMAT AR film. Protein expression was normalized to -actin (loading control). 2.5 Quantification and statistics Currents were measured at 100 ms after the initiation of the test pulse. Percentage inhibition is indicated by (1－IK propofol / IK control) ×100%. The data were analyzed and plotted by using clampfit 8.2. Data are presented as means ± SD. A Students t-test was used for testing significance between two groups. One-way ANOVA followed by Dunnett’s t test was used in comparison of multiple groups. A p-value of less than 0.05 was considered statistically significant.
3. Results 3.1 The effects of propofol on IK We tested the effects of propofol on IK in acutely dissociated rat cerebral parietal cortical neurons. After whole-cell configuration was established, cells were held at -80 mV and IK was elicited with voltage steps with 150 ms pre-pulse at -40 mV to inactivate transient outward potassium currents. Bath application of 10μM, 30μM, 50μM, 100μM, or 150 μM propofol significantly inhibited IK amplitudes by 5.6 ± 1.6%, 17.2 ± 3.8%, 34.3 ± 6.1%, 42.2 ± 6.1%, or 47.3 ± 5.5% (n = 7, p<0.001 vs control) respectively (Fig. 1A, 1B). Fig. 2A showed the representative IK traces before and after propofol (30μM ) treatment. The current was elicited with a 150 ms pre-pulse to -40 mV from a holding potential at -80 mV followed by 150 ms depolarizing pulses from -40 mV to +60 mV in 10 mV steps. The peak amplitudes of IK at the voltage of +60 mV were 2278.3 ± 601.7 pA and 1890.3 ± 529.9 pA in control and 30 μM propofol, respectively (n = 7, p<0.001vs control, Fig 2). The current-voltage relationship of IK before and after 30 μM propofol application was shown in Fig 3 (n = 7). 3.2 Effects of propofol on Kv2.1 expression Propofol dose-dependently down-regulated the protein expression of Kv2.1 in the cerebral cortex (Fig 4A). The Kv2.1 protein level was significantly lower in propofol groups (1.84±0.145, 1.73±0.178, 1.42±0.135, at 30 μM, 50 μM, and 100 μM propofol, respectively) than in control group (2.12±0.185) (Fig 4B, n=5, *p <0.05, # p <0.01).
4. Discussion In this study, we investigated the effects of propofol on Ik and the expression of Kv2.1 in acutely dissociated rat cerebral parietal cortical neurons. The results showed that propofol concentration-dependently inhibited Ik and the expression of Kv2.1 in acutely dissociated rat cerebral parental cortical neurons. Previous studies have shown that propofol could inhibit IK and suppress excess potassium current efflux during ischemia/hypoxia injury [1, 4, 5, 9, 20]. Consistently, our study showed that propofol inhibited IK in dose-dependent manner of acutely dissociated rat cerebral parietal cortical neurons. These data imply that the inhibitory effect of propofol on IK may be associated with its neuroprotective effect. In mammalian brain, a number of functionally distinct potassium channel subtypes have been identified. The delayed rectifier Kv2.1 channel is widely expressed in mammalian central nervous system. It has been demonstrated that Kv2.1 channel clustered on the soma and dendrites of both interneurons and principal neurons. Kv2.1 carries the majority of delayed rectifier potassium current (Ik). Kv2.1 plays an important role in regulating the transmission of electrical signals into and out of the neuronal soma under normal physiological conditions. Moreover, Kv2.1 might also be involved in the apoptotic signaling cascade in mammalian cortical neurons. Potassium channels play important physiological role, such as, regulation of action potentials, signal integration, and neurotransmitter release [19]. Thus, potassium channels dysfunction could cause certain pathophysiological outcomes. Ischemia/hypoxia injury could induce membrane depolarization and activate voltage-gated potassium channels and cause the loss of intracellular potassium current. Activation of potassium channels could induce excessive potassium current efflux, and lead to ischemia induced neuronal death [22]. Excessive potassium current efflux and intracellular potassium depletion are the key steps in the process of apoptotic cascade in many cells among cerebral neurons. Apoptotic injury can cause activation of IK before cells are doomed to die in the cultured cortical neurons which is consistent with the potential role of IK activation as the early mechanism of apoptosis. The close
relationship between activation of potassium channel and apoptosis is also supported by a host of studies in peripheral cells. Huang et al. showed that the potassium channel blocker, eg. tetraethylammonium (TEA) or elevated extracellular potassium current could attenuate neuronal apoptosis after global ischemia in rats [8]. The antibody that is specific for the Kv2.1 channel could block the majority of the Ik channel in hippocampal neurons. Some reports showed that depression of Kv2.1 mRNA may decrease the Ik currents of CA1 pyramidal neurons. In our study, we also found that propofol inhibited the expression of Kv2.1 in a concentration-depended manner. In this study, the cells were held at -80 mV. The typical IK was elicited with voltage steps with 150-ms pre-pulse at -40 mV to inactivate transient outward potassium currents. However, the transient potassium current was not completely blocked at initial phase (Fig B), while it faded away at later phase. The recording time used in this study was long enough to record the later phase with only IK presentation. Therefore, it seems that the transient component (which has faded away at later phase) would not influence the results. We also discovered that 150 uM propofol combined with the voltage protocol could fully block the transient current component. So, we speculate that high concentration propofol would probably inhibit the transient current component. Further study is required to test this possibility. In conclusion, we found that propofol inhibited delayed rectifier potassium channel Kv2.1 in acutely dissociated rat cerebral parietal cortical neurons. These results indicate that the brain protective effects of propofol may involve inhibition of IK. Our findings partially provided the molecular basis for the use of propofol as a neuroprotective agent. Acknowledgments This work was supported by the grant 81301668 from the State Natural Science Foundation of China. The authors have no conflicts of interest, financial or otherwise. Author contributions to the study and manuscript preparation include the following. Conception and design: Chun-yu Song. Acquisition of data and performed
experiments: Chun-yu Song and Yan-zhuo Zhang. Critically revising the article: all authors. Approved the final version of the manuscript on behalf of all authors: Chun-yu Song. Statistical analysis: Rui Zhang, Xian-zhang Zeng.
References [1] Baum VC, Distinctive effects of three intravenous anesthetics on the inward rectifier (IK1) and the delayed rectifier (IK) potassium currents in myocardium: implications for the mechanism of action. Anesth Analg 76 (1993) 18-23. [2] Bekkers JM, Distribution and and activation of voltage-gated potassium channels in cell-attached and outside-out patches from large layer 5 cortical pyramidal neurons of the rat. J Physiol 525 (2000) 611-20. [3] Ergun R, Akdemir G, Sen S, Tasci A, Ergungor F, Neuroprotective effects of propofol following global cerebral ischemia in rats. Neurosurg Rev 25 (2002) 95-98. [4] Friederich P, Benzenberg D, Urban BW, Ketamine and propofol differentially inhibit human neuronal K (+) channels. Eur J Anaesthesiol 18 (2001) 177-183. [5] Friederich P, Urban BW, Interaction of intravenous anesthetics with human neuronal potassium currents in relation to clinical concentrations. Anesthesiology 91(1999) 1853-60. [6] Flynn RW, MacWalte r RS, Doney AS, The cost of cerebral ischaemia. Neuropharmacology 55 (2008) 250-256. [7] Hans P, Bonhomme V, Collette J, Albert A, Moonen G, Propofol protects cultured rat hippocampal neurons against N-methyl-D-aspartate receptor-mediated glutamate toxicity. J Neurosurg Anesthesiol. 6 (1994) 249-253. [8] Huang H, Gao TM, Gong L, Zhuang Z, Li X, Potassium channel blocker TEA prevents CA1 hippocampal injury following transient forebrain ischemia in adult rats. Neurosci Lett 305 (2001) 83-86. [9] Kawano T, Oshita S, Takahashi A, Tsutsumi Y, Tomiyama Y, Kitahata H, Kuroda Y, Nakaya Y, Molecular mechanisms of the inhibitory effects of propofol and thiamylal on sarcolemmal adenosine triphosphate-sensitive potassium channels. Anesthesiology 100 (2004) 338-346. [10] Kochs E, Hoffman WE, Werner C, Thomas C, Albrecht RF, Schulte am Esch J, The effects of propofol on brain electrical activity, neurologic outcome, and neuronal damage following incomplete ischemia in rats. Anesthesiology. 76 (1992) 245-252. [11] Korngreen A, Sakmann B, Voltage-gated K+ channels in layer 5 neocortical pyramidal neurons from young rats: subtypes and gradients. J Physiol 525 (2000) 621-639.
[12] Malin SA, Nerbonne JM, Delayed rectifier K+ currents, Ik currents, Ik, are encoded by Kv2.1 alpha-subunits and regulate tonic firing in mammalian sympathetic neurons. J Neurosci 22 (2002) 10094-10105. [13] Mingna Liu, Bo Gong, Zhi Qi, Comparison of the endogenous Ik currents in rat hippocampal neurons and cloned Kv2.1 channels in CHO cells. Cell Biology International 32 (2008) 1514-1520. [14] Misonou H, Mohapatra DP, Menegola M, Trimmer JS, Calcium- and metabolic state-dependent modulation of the voltage-dependent Kv2.1 channel regulates neuronal excitability in response to ischemia. J Neurosci 25 (2005) 11184-11193. [15] Misonou H, Mohapatra DP, Park EW, Leung V, Zhen D, Misonou K, Anderson AE, Trimmer JS, Regulation of ion channel localization and phosphorylation by neuronal activity. Nat Neurosci 7(2004) 711-718. [16] Misonou H, Trimmer JS, Determinants of voltage-gated potassium channel surface expression and localization in Mammalian neurons. Crit Rev Biochem Mol Biol 39 (2004) 125-145. [17] Pal S, Hartnett KA, Nerbonne JM, Levitan ES, Aizenmen E, Mediation of neuronal apoptosis by Kv2.1-encoded potassium channels. J Neurosci 23 (2003) 4798-4802. [18] Park KS, Mohapatra DP, Misonou H, Trimmer JS, Graded regulation of the Kv2.1 potassium channel by variable phosphorylation. Science 313 (2006) 976-9. [19] Qu L, Li Y, Tian H, Wang Z, Cui L, Jin H, Wang W, Yang L, Effects of PKC on inhibition of delayed rectifier potassium currents by N/OFQ. Biochem Biophys Res Commun 356 (2007) 582-586. [20] Song CY, Xi HJ, Yang L, Qu LH, Yue ZY, Zhou J, Cui XG, Gao W, Wang N, Pan ZW, li WZ, Propofol inhibited the delayed rectifier potassium current (Ik) via activation of protein kinase C epsilon in rat parietal cortical neurons. European Journal of Pharmacology 653 (2011) 16-20. [21] Wang J, Yang X, Camporesi CV, Yang Z, Bosco G, Chen C, Camporesi EM, Propofol reduces infarct size and striatal dopamine accumulation following transient middle cerebral artery occlusion: a microdialysis study. Eur J Pharmacol 452 (2002) 303-8. [22] Wei L, Yu SP, Gottron F, Snider BJ, Zipfel GJ, Choi DW, Potassium Channel Blockers Attenuate Hypoxia- and Ischemia-Induced Neuronal Death In Vitro and
In Vivo. Stroke 34 (2003) 1281-1286. [23] Xi HJ, Zhang TH, Tao T, Song CY*, Lu SJ, Cui XG, Yue ZY, Propofol improved neurobehavioral outcome of cerebral ischemia-reperfusion rats by regulating Bcl-2 and Bax expression. Brain Res 1410 (2011) 24-32.
Figures Captions
Fig. 1 Dose-response relationship for inhibition of IK by propofol in acutely dissociated rat parietal cortical neurons. A. The percentage inhibition of different doses of propofol on IK at +60 mV (n = 7, * p<0.001 versus control group). B. The representative current traces of IK at +60 mV selected from a neuron before and after treatment with propofol.
A percent inhibition
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Fig. 2 Effects of 30 μM propofol on IK in acutely dissociated rat parietal cortical neurons. A. The representative current traces of IK selected from a neuron before and after treatment with 30 μM propofol. B. The IK values of control, 30 μM propofol at the voltage of +60 mV (n = 7, * p<0.001).
Fig. 3 Current-voltage relationships of IK before and after application of 30 μM propofol in acutely dissociated rat parietal cortical neurons (n = 7).
Fig. 4 Effects of propofol on Kv2.1 expression. A. Propofol enhances the expression of Kv2.1. B. The effect of propofol on Kv2.1 expression (n = 6, *p<0.05, #p <0.01).
S0304-3940(16)30057-X http://dx.doi.org/doi:10.1016/j.neulet.2016.01.058 NSL 31816
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
17-10-2015 24-12-2015 26-1-2016
Please cite this article as: Yan-zhuo Zhang, Rui Zhang, Xian-zhang Zeng, Chun-yu Song, The inhibitory effect of propofol on Kv2.1 potassium channel in rat parietal cortical neurons, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.01.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The inhibitory effect of propofol on Kv2.1 potassium channel in rat parietal cortical neurons Yan-zhuo Zhang, Rui Zhang, Xian-zhang Zeng, Chun-yu Song* [email protected] Department of Anesthesiology, China and Heilongjiang Key Laboratory for Anesthesia and Critical Care, The Second Affiliated Hospital of Harbin Medical University, Harbin 150081, China *
Corresponding author at: Department of Anesthesiology, The Second Affiliated
Hospital of Harbin Medical University, NO 246, Xue-fu Street, Nangang District, Harbin 150081, China. Fax: +86 451 86605028. Tel: 13936130754.
Highlights Propofol had neuroprotective effect; propofol could inhibit Ik；propofol could depress the expression of Kv2.1
Abstract Excessive K+ efflux via activated voltage-gated K+ channels can deplete intracellular K+ and lead to long-lasting membrane depolarization which will promote neuronal apoptosis during ischemia/hypoxia injury. The Kv2.1 potassium channel was the major component of delayed rectifier potassium current (Ik) in pyramidal neurons in cortex and hippocampus. The neuronal protective effect of propofol has been proved. Delayed rectifier potassium current (Ik) has been shown to have close relationship with neuronal damage. The study was designed to test the inhibitory effect of propofol on Kv2.1 potassium channel in rat parietal cortical neurons. Whole-cell patch clamp recordings and Western blot analysis were used to investigate the electrophysiological function and protein expression of Kv2.1 in rat parietal cortical neurons after propofol treatment. We found that propofol concentration-dependently inhibited Ik in pyramidal neurons. Propofol also caused a downward shift of the I-V curve of Ik at 30 μM concentration. Propofol significantly inhibited the expression of Kv2.1 protein level at 30 μM, 50 μM, 100 μM concentration. In conclusion, our data showed that propofol could inhibit Ik, probably via depressing the expression of Kv2.1 protein in rat cerebral parietal cortical neurons.
Keywords: propofol; Kv2.1 potassium channel; whole-cell patch clamp; parietal cortical neuron; delayed rectifier potassium current.
1. Introduction Cerebral ischemia is the most common cause that can lead to complex chronic disability worldwide [6]. Ischemia/anoxia could induce cell membrane depolarization, and could lead to voltage gated potassium channels activation, and then potassium homeostasis imbalance. Excessive loss of intracellular potassium plays an important role in triggering cell apoptosis. Some reports have shown that potassium channel blockers could attenuate ischemia/anoxia induced cell apoptosis both in vitro and in vivo. Delayed rectifier potassium currents (Ik) are important in regulating excitability of hippocampal and cortical pyramidal neurons [2]. Kv2.1 is the main composition of IK, and has been tested widely expressed in brain, such as pyramidal neurons, hippocampus, and interneurons [12-17]. Kv2.1 plays various roles in neuronal pathophysiology, such as setting resting membrane potential, shaping action potential repolarization which regulates the excitability and process of synaptic integration in central neurons [2, 11]. Ischemia/anoxia could increase the activity of Kv2.1 by lowering the threshold of activation. This finding suggested that depression of Kv2.1 channel could provide neuroprotective effect for ischemic injuries [18]. Propofol is a commonly used anaesthetic agent that has been demonstrated to be neuroprotective. Some studies have shown that propofol had neuroprotective activities and the mechanisms were complicated, include reduction in cerebral metabolism [10], antioxidant activity [3], anti-excitotoxic properties [7], modulation of inhibitory and excitatory neurotransmitters [21], or influence on neuronal apoptosis [23]. Delayed rectifier potassium channel Kv2.1 was highly expressed on neuronal soma in the brain. Moreover, Kv2.1 was an important potassium channel subtype in regulating apoptotic signaling cascade in ischemia/anoxia injury [17]. However, the effect of propofol on delayed rectifier potassium channel Kv2.1 remains unclear. Therefore, the aim of this study is to investigate whether propofol could inhibit Ik and could block the expression of Kv2.1 protein in rat cerebral parietal cortical neurons. To test this possibility, we used whole-cell patch clamp recordings to explore the potential effects of propofol on Ik and Western blot to examine the expression of Kv2.1 in rat parietal cortical neurons.
2. Materials and methods 2.1 Acute dissociation of neurons Animals used in this study were approved by and handled in accordance with the guidelines of the Animal Care and Use Committee at the Harbin Medical University, Harbin, China. Wistar rats between 10 and 14 days old of both sexes were used. After ether anesthesia, rats were decapitated and the brains were quickly removed and placed in cold artificial cerebrospinal fluid (ACSF) that contained (mM): NaCl: 126, KCl: 5, NaH2PO4: 1.25, NaHCO3: 26, CaCl2: 2, MgSO4: 2, HEPES: 10, and glucose: 10, pH: 7.2–7.4 (titrated with HCl), and were then cut into 400 µm slices. Slices were incubated for 0.5 h at 32 °C in a buffered ACSF bubbled with 95% O2 and 5% CO2. Slices were moved into buffered ACSF containing protease (0.5 mg/ml) at 32 °C for 30 min, and then rinsed three times in cold buffered ACSF. Enzyme-treated slices were mechanically dissociated with a graded series of fire-polished Pasteur pipettes with diameter 150, 300, or 500 µm. Cells were then plated into a 35 mm dish and viewed under an Olympus inverted microscope. 2.2 Electrophysiological recordings Whole-cell patch clamp recording was performed at room temperature (20–25 °C) using a PC-IIC amplifier (HUST-IBB, Wuhan, China). Patch electrodes were pulled from borosilicate glass capillaries by a PP-83 microelectrode puller (Narrishage, Japan) and had resistances of 3–5 MΩ when filled with the internal solutions that contained (mM): KCl: 140, HEPES: 10, EGTA: 10, Na2ATP: 2, and pH: 7.2–7.4 (titrated with KOH). The standard extracellular solution contained (mM): NaCl: 130, KCl: 5.4, CaCl2: 1, MgCl2: 1, glucose: 25, HEPES: 10, CdCl2: 0.2 and pH: 7.2–7.4
(titrated with NaOH). Neurons with a bright and smooth appearance were selected for recording. After whole-cell configuration was established, we waited at least 5 min for steady-state currents to stabilize. Current was filtered by a low-pass Bessel filter set at 2 kHz during data acquisition. Series resistances were compensated for 70–80%. After whole-cell configuration was established, cells were held at -80 mV. The typical IK was elicited with voltage steps with 150-ms prepulse at -40 mV to inactivate transient outward potassium currents (IA). 2.3 Drug administration The following drugs and chemicals were used: protease was purchased from Sigma Chemical (St Louis, MO, USA). Propofol (Labor Dr. Ehrenstorfer-schafers. Germany). All the agents were dissolved in extracellular fluid to make 100 × stock solutions (stored at -20 °C) and diluted in external solution just before application. To determine the effects of propofol on IK, we recorded the amplitudes of IK before and after addition of 10μM, 30μM, 50μM, 100μM, or 150 μM propofol. All drugs and chemicals were applied to bath solution. 2.3 Protein Extraction To produce protein extracts, the cerebral cortex was cut rapidly into pieces 300–400 μm wide and placed at 0–4 °C in ACSF. The samples were then treated with propofol (30µmol/L, 50µmol/L, 100µmol/L) and ventilated at 95% O2/5% CO2 for 6 h; the tissues were then washed three times using PBS buffer. Finally, the samples were placed in liquid nitrogen to isolate all the proteins.
2.4 Western blot analysis One hundred micrograms of total proteins per sample was electrophoretically separated on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and electronically transferred onto a PVDF membrane (Amersham Life Science, Buckinghamshire). The membranes were blocked by incubation for 60 min at room temperature with 5% (w/v) nonfat dry milk in TBS-T (150 mM NaCl, 10 mM Tris-HCl, 0.1% TWEEN), then incubated with mouse anti-Kv2.1 monoclonal antibody (Abcam, Cambridge, UK, 1:1000 dilution) overnight at 4 °C, washed with 1×TBS-T, and incubated for 1 h at room temperature with horseradish peroxidase-conjugated rabbit-anti-mouse IgG secondary antibody (1:5000 dilution; Amersham Life Science, Buckinghamshire). Immunoreactive proteins were visualized using an enhanced chemiluminescence system (ECL; Amersham Life Science, Buckinghamshire). Membranes were exposed for 3 to 30 sec to Kodak XOMAT AR film. Protein expression was normalized to -actin (loading control). 2.5 Quantification and statistics Currents were measured at 100 ms after the initiation of the test pulse. Percentage inhibition is indicated by (1－IK propofol / IK control) ×100%. The data were analyzed and plotted by using clampfit 8.2. Data are presented as means ± SD. A Students t-test was used for testing significance between two groups. One-way ANOVA followed by Dunnett’s t test was used in comparison of multiple groups. A p-value of less than 0.05 was considered statistically significant.
3. Results 3.1 The effects of propofol on IK We tested the effects of propofol on IK in acutely dissociated rat cerebral parietal cortical neurons. After whole-cell configuration was established, cells were held at -80 mV and IK was elicited with voltage steps with 150 ms pre-pulse at -40 mV to inactivate transient outward potassium currents. Bath application of 10μM, 30μM, 50μM, 100μM, or 150 μM propofol significantly inhibited IK amplitudes by 5.6 ± 1.6%, 17.2 ± 3.8%, 34.3 ± 6.1%, 42.2 ± 6.1%, or 47.3 ± 5.5% (n = 7, p<0.001 vs control) respectively (Fig. 1A, 1B). Fig. 2A showed the representative IK traces before and after propofol (30μM ) treatment. The current was elicited with a 150 ms pre-pulse to -40 mV from a holding potential at -80 mV followed by 150 ms depolarizing pulses from -40 mV to +60 mV in 10 mV steps. The peak amplitudes of IK at the voltage of +60 mV were 2278.3 ± 601.7 pA and 1890.3 ± 529.9 pA in control and 30 μM propofol, respectively (n = 7, p<0.001vs control, Fig 2). The current-voltage relationship of IK before and after 30 μM propofol application was shown in Fig 3 (n = 7). 3.2 Effects of propofol on Kv2.1 expression Propofol dose-dependently down-regulated the protein expression of Kv2.1 in the cerebral cortex (Fig 4A). The Kv2.1 protein level was significantly lower in propofol groups (1.84±0.145, 1.73±0.178, 1.42±0.135, at 30 μM, 50 μM, and 100 μM propofol, respectively) than in control group (2.12±0.185) (Fig 4B, n=5, *p <0.05, # p <0.01).
4. Discussion In this study, we investigated the effects of propofol on Ik and the expression of Kv2.1 in acutely dissociated rat cerebral parietal cortical neurons. The results showed that propofol concentration-dependently inhibited Ik and the expression of Kv2.1 in acutely dissociated rat cerebral parental cortical neurons. Previous studies have shown that propofol could inhibit IK and suppress excess potassium current efflux during ischemia/hypoxia injury [1, 4, 5, 9, 20]. Consistently, our study showed that propofol inhibited IK in dose-dependent manner of acutely dissociated rat cerebral parietal cortical neurons. These data imply that the inhibitory effect of propofol on IK may be associated with its neuroprotective effect. In mammalian brain, a number of functionally distinct potassium channel subtypes have been identified. The delayed rectifier Kv2.1 channel is widely expressed in mammalian central nervous system. It has been demonstrated that Kv2.1 channel clustered on the soma and dendrites of both interneurons and principal neurons. Kv2.1 carries the majority of delayed rectifier potassium current (Ik). Kv2.1 plays an important role in regulating the transmission of electrical signals into and out of the neuronal soma under normal physiological conditions. Moreover, Kv2.1 might also be involved in the apoptotic signaling cascade in mammalian cortical neurons. Potassium channels play important physiological role, such as, regulation of action potentials, signal integration, and neurotransmitter release [19]. Thus, potassium channels dysfunction could cause certain pathophysiological outcomes. Ischemia/hypoxia injury could induce membrane depolarization and activate voltage-gated potassium channels and cause the loss of intracellular potassium current. Activation of potassium channels could induce excessive potassium current efflux, and lead to ischemia induced neuronal death [22]. Excessive potassium current efflux and intracellular potassium depletion are the key steps in the process of apoptotic cascade in many cells among cerebral neurons. Apoptotic injury can cause activation of IK before cells are doomed to die in the cultured cortical neurons which is consistent with the potential role of IK activation as the early mechanism of apoptosis. The close
relationship between activation of potassium channel and apoptosis is also supported by a host of studies in peripheral cells. Huang et al. showed that the potassium channel blocker, eg. tetraethylammonium (TEA) or elevated extracellular potassium current could attenuate neuronal apoptosis after global ischemia in rats [8]. The antibody that is specific for the Kv2.1 channel could block the majority of the Ik channel in hippocampal neurons. Some reports showed that depression of Kv2.1 mRNA may decrease the Ik currents of CA1 pyramidal neurons. In our study, we also found that propofol inhibited the expression of Kv2.1 in a concentration-depended manner. In this study, the cells were held at -80 mV. The typical IK was elicited with voltage steps with 150-ms pre-pulse at -40 mV to inactivate transient outward potassium currents. However, the transient potassium current was not completely blocked at initial phase (Fig B), while it faded away at later phase. The recording time used in this study was long enough to record the later phase with only IK presentation. Therefore, it seems that the transient component (which has faded away at later phase) would not influence the results. We also discovered that 150 uM propofol combined with the voltage protocol could fully block the transient current component. So, we speculate that high concentration propofol would probably inhibit the transient current component. Further study is required to test this possibility. In conclusion, we found that propofol inhibited delayed rectifier potassium channel Kv2.1 in acutely dissociated rat cerebral parietal cortical neurons. These results indicate that the brain protective effects of propofol may involve inhibition of IK. Our findings partially provided the molecular basis for the use of propofol as a neuroprotective agent. Acknowledgments This work was supported by the grant 81301668 from the State Natural Science Foundation of China. The authors have no conflicts of interest, financial or otherwise. Author contributions to the study and manuscript preparation include the following. Conception and design: Chun-yu Song. Acquisition of data and performed
experiments: Chun-yu Song and Yan-zhuo Zhang. Critically revising the article: all authors. Approved the final version of the manuscript on behalf of all authors: Chun-yu Song. Statistical analysis: Rui Zhang, Xian-zhang Zeng.
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Figures Captions
Fig. 1 Dose-response relationship for inhibition of IK by propofol in acutely dissociated rat parietal cortical neurons. A. The percentage inhibition of different doses of propofol on IK at +60 mV (n = 7, * p<0.001 versus control group). B. The representative current traces of IK at +60 mV selected from a neuron before and after treatment with propofol.
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Fig. 2 Effects of 30 μM propofol on IK in acutely dissociated rat parietal cortical neurons. A. The representative current traces of IK selected from a neuron before and after treatment with 30 μM propofol. B. The IK values of control, 30 μM propofol at the voltage of +60 mV (n = 7, * p<0.001).
Fig. 3 Current-voltage relationships of IK before and after application of 30 μM propofol in acutely dissociated rat parietal cortical neurons (n = 7).
Fig. 4 Effects of propofol on Kv2.1 expression. A. Propofol enhances the expression of Kv2.1. B. The effect of propofol on Kv2.1 expression (n = 6, *p<0.05, #p <0.01).