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Modulation of inward rectifier potassium channel by toosendanin, a presynaptic blocker
Neuroscience Research 40 (2001) 211– 215 www.elsevier.com/locate/neures
Modulation of inward rectifier potassium channel by toosendanin, a presynaptic blocker Zhong-Feng Wang, Yu-Liang Shi * Key Laboratory of Neurobiology, Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People’s Republic of China Received 21 November 2000; accepted 19 March 2001
Abstract The effect of toosendanin, a presynaptic blocker, on the inward rectifier potassium channel (KKir) of hippocampal CA1 pyramidal neurons of rats was studied by the single-channel patch-clamp technique. The results showed that toosendanin had an inhibitory effect on KKir in an excised inside-out patch of the neuron under a symmetrical 150 mM K+ condition. By decreasing the slower open time constant and increasing the slower close time constant, toosendanin (1 × 10 − 6 –1× 10 − 4 g/ml) significantly reduced the open probability of the channel in a concentration-dependent manner. Meanwhile, a dose-dependent reduction in unitary conductance of the channel was also detected after toosendanin application. These data offer an explanation for toosendanin-induced facilitation of neurotransmitter release and antibotulismic effect of the drug. © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Toosendanin; Presynaptic blocker; Patch-clamp; Inward rectifier potassium channel; Hippocampal pyramidal neuron
1. Introduction Toosendanin (C30H38O11, FW =574), a triterpenoid derivative (Chung et al., 1975; Shu and Liang, 1980), is an active ingredient extracted from the bark of Melia toosendan Sieb et Zucc, which is used as an anthelmintic vermifuge against ascarids in Chinese traditional medicine (Wang and Wen, 1959). Previous studies demonstrated that having no effect on the nerve impulse conduction and muscle cell membrane potential and acetylcholine sensitivity, toosendanin selectively blocks the synaptic transmission in neuromuscular junction and central synapses by interfering with the transmitter release with a facilitation at the first and blockade at the last (Shi et al., 1980, 1981a,b, 1982; Shi and Hsu, 1983; Shi and Chen, 1999; Chen et al., 1999) and might be potentially useful in medical and scientific researches (Shi et al., 1993). Moreover, toosendanin* Corresponding author. Tel.: + 86-21-64370080, ext. 154; fax: +86-21-64332445. E-mail address: [email protected] (Y.-L. Shi).
induced decrease in the number of synaptic vesicles was observed in the neuromuscular junction (Huang et al., 1980), and toosendanin-specific binding was found in the rat cerebral cortex homogenate (Shen et al., 1994). However, in spite of sharing some actions similar to those of botulinum neurotoxins (BoNTs), toosendanin was shown to have a dramatic antibotulismic effect. For example, toosendanin could prevent death in animals (mice, rats and monkeys) administered with several lethal dose of BoNT/A, B and still effective even when applied 18 h after BoNTs injection (Li et al., 1982); the tolerance to BoNT of the neuromuscular junction preparations, isolated from rats several days after a single injection of toosendanin, was enhanced significantly, and the high tolerance was associated with the toosendanin-induced facilitation (Shi and Hsu, 1983). An agent may evoke facilitation of the neurotransmitter release by inhibiting the K+ channels. It has been observed that toosendanin inhibited a subendothelial fast K+ current in mouse motor nerve terminals (Xu and Shi, 1993) and delayed rectifier K+ channel in
0168-0102/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 1 ) 0 0 2 3 3 - 4
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Z.-F. Wang, Y.-L. Shi / Neuroscience Research 40 (2001) 211–215
neuroblastoma×glioma NG108-15 cell line (Hu et al., 1997). The present work was undertaken to investigate the effect of toosendanin on inward rectifier K+ channels (KKir) in acutely isolated pyramidal neurons of the rat hippocampal CA1 region using the single-channel patch-clamp technique.
2. Materials and methods
2.1. Cell preparation A single hippocampal pyramidal neuron was isolated from the brain of Sprague– Dawley rats aged 7– 12 days using procedures previously described (Kaneda et al., 1988) with slight modifications. Briefly, after the rat was killed by decapitation, the brain was rapidly dissected out and the hippocampi were removed and sliced manually at a thickness of about 400– 600 mm. The slices were incubated in an incubation solution (in mM: NaCl 150, KCl 5, CaCl2 2, N-2-hydroxyethylpiperazine-N%-2-ethanesulfonic acid (HEPES) 10, glucose 10, the pH was adjusted to 7.4 with 2-amino-2-hydroxymethyl-propan-1,3-diol(Tris)) bubbled with 100% O2 at 37°C for 60 min and then treated with 0.5 mg/ml collagenase and 2 mg/ml pronase E for 60 min. The enzymes were washed out with the incubation solution twice. The slices were then re-incubated for 30 min. The treated slices were immersed in a standard external solution (in mM: NaCl 150, KCl 5, CaCl2 2.6, MgCl2 1.1, HEPES 10 and glucose 10, the pH was adjusted to 7.4 with Tris) containing 20% newborn calf serum, and CA1 regions were removed from the slices using small hand needles under an inverted microscope. The hippocampal cells were mechanically isolated using firepolished Pasteur pipettes with a 100– 400 mm tip diameter. All experiments conformed to the guidelines of the NIH on the ethical use of animals. All efforts were made to minimize the animal suffering and to reduce the number of animals used.
2.2. Electrophysiological recordings The hippocampal cell suspension was added to a poly-L-lysine-coated glass coverslip in a recording chamber mounted on the stage of an inverted microscope. After the cells settled down, perfusion was started. The channel current was recorded at an insideout configuration (Hamill et al., 1981) using a patchclamp amplifier (Axopatch 200A, Axon Instruments, Burlingame, CA) at room temperature (20– 25°C). The current was filtered at 1 kHz (−3 dB, 4-pole Bessel filter) and stored on a computer (IBM compatible 586/DX) via an analog-to-digital interface (DigiData
1200, Axon Instruments) for further analysis. Patch pipettes were pulled from a 1.5 mm (outer diameter) capillary glass (type 95, Shanghai Institute of Physiology) using a Narishige PP-83 electrode puller (Narishige, Japan). The tip of the pipette was coated with N-trimethylsilydiethylamine and fire polished on a microforge (FP-1, Shanghai Institute of Physiology). The recording electrode was filled with the electrode solution, and had a tip resistance of 6–8 MV. The electrode solution contained (in mM): KCl 150, CaCl2 1.0, HEPES 5.0, pH 7.4. The composition of the standard internal solution perfusing the cytoplasmic face of the inside-out patch was as follows (in mM): KCl 150, CaCl2 3.0, HEPES 5, ethyleneglycol-bis(b-aminoethylether) -N,N%-tetraacetic acid (EGTA) 5.0, the pH was adjusted to 7.2 with KOH.
2.3. Current analysis and data statistics Stretches of record 30 s in length at each membrane potential were digitized at 20 kHz and the single-channel analysis was performed using the Fetchan and pStat program (pClamp 6.04, Axon Instruments). The histogram of the distribution of current amplitude was fitted by the Gaussian distribution function using the Levenberg–Marquadt non-linear least squares method. The mean current values of the peak were subsequently used to construct the current–voltage relationship and hence to determine the conductance of the channel. The channel open and close dwell time histograms were fitted by the sum of exponential probability density functions using the Maximum likelihood method. The membrane potential described in this work indicates the cytoplasmic face potential. All data were expressed as mean9 S.E.M. Statistical analysis was performed using Student’s t-test for paired data, considering P B0.05 as significant.
2.4. Drugs and reagents Toosendanin used in this work was a sample recrystallized in ethanol with a purity \ 98% (Xu and Shi, 1993; Hu et al., 1997; Shi and Chen, 1999). Toosendanin was dissolved in ethanol first and then added to the perfusion solution to obtain a final concentration of 1×10 − 6 –1× 10 − 4 g/ml ( 1.7–170 mM). The highest ethanol concentration in the perfusion solution was less than 0.1%, and the channel activity was not affected by this concentration of ethanol. Poly-L-lysine, N-trimethylsilydiethylamine, HEPES, and EGTA are all Sigma products (St Louis, MO). The newborn calf serum was purchased from HyClone & Pierce (USA). All other reagents are of analytical grade.
Z.-F. Wang, Y.-L. Shi / Neuroscience Research 40 (2001) 211–215
3. Results
3.1. Decrease in open probability of the channel The effect of toosendanin on single-channel currents
213
of KKir at inside-out patches of hippocampal pyramidal neurons was investigated. Under a 150 mM symmetrical K+ condition, the single-channel current was recorded and the current–voltage relationship showed an inward rectifier property with an unit conductance of 31.99 0.6 pS over a membrane potential range from − 90 to 0 mV (n=7). The reversal potential was close to 0 mV coinciding with the K+ equilibrium potential. Thus, the channel recorded was identified as KKir as reported previously (Oh et al., 1995; Grigg et al., 1996). Internal application of toosendanin (1× 10 − 6 –1× 10 − 4 g/ml) significantly inhibited the activity of KKir of the neuron. After perfusing the inside-out patches with a toosendanin-containing internal solution, the open probability (Po) of the channel decreased in a concentration-dependent manner (n=4–6). Fig. 1A shows a typical record before and during toosendanin (1×10 − 5 g/ml) treatment at a membrane potential of −50 mV. Toosendanin at a concentration of 1× 10 − 4 g/ml decreased the Po of the channel by 85.89 11.2% (n=5) in comparison with its control value (Fig. 1B).
3.2. Shortening of open dwell time of the channel by decreasing the slower open time constant The distributions of open and close times of the channel could be best fitted by the two-exponential function. For example, at a −50 mV membrane potential, the fast open time constant (~fast) and slow open time constant (~slow) were 0.489 0.10 ms and 15.569 1.19 ms (n= 7), and the fast and slow close time constants were 0.329 0.05 ms and 18.7691.06 ms (n= 7), respectively. It was found that toosendanin at a concentration of 1×10 − 6 –1× 10 − 4 g/ml significantly decreased ~slow in a dose-dependent manner and had no effect on ~fast at each tested membrane potential (Fig. 1C). Moreover, the slow close time constant was markedly increased after toosendanin application. For example, at a − 50 mV membrane potential, toosendanin at concentrations of 1×10 − 6, 1×10 − 5 and 1× 10 − 4 g/ml, the slow close time constants increased to 32.93910.47 (n= 6), 59.759 13.89 (n=5) and 112.329 25.16 ms (n= 5) from the control value of
Fig. 1.
Fig. 1. Inhibition of the single-channel inward rectifier K+ current by toosendanin in a symmetrical 150 mM K+ solution; inside-out patch of rat hippocampal CA1 pyramidal neuron. (A) Original recordings before and during internal application of toosendanin (1 × 10 − 5 g/ml) at a membrane potential of −50 mV in the same patch. (B) Relationship between the concentration of toosendanin and the open probability of the channel. The membrane potential was held at − 50 mV. (C) Relationship between the concentration of toosendanin and the open time constant. The membrane potential was held at − 50 mV. The distribution of the open dwell time was best fitted with the two-exponential function, and the fast (~fast) and slow open time constants (~slow) were obtained. *PB0.05, **PB 0.01 vs. control. n =4 – 6.
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Z.-F. Wang, Y.-L. Shi / Neuroscience Research 40 (2001) 211–215
18.7691.06 ms (n= 7), respectively (P B 0.05 or PB 0.01), whereas no change was detected in the fast close time constant.
3.3. Decrease in unitary current amplitude of the channel The effect of toosendanin on the single-channel conductance of KKir was also studied. A significant decrease in the single-channel current amplitude of the channel was detected after perfusing with toosendanin at a concentration of 1× 10 − 5 g/ml (n =5) as shown in Fig. 2A as a typical record. Toosendanin at concentrations of 1× 10 − 6, 1 ×10 − 5 and 1×10 − 4 g/ml reduced the single-channel conductance to 25.49 1.2 (n= 6), 21.09 0.7 (n =5) and 15.49 0.9 pS (n= 5) from the control value of 31.990.6 pS (n = 7), re-
Fig. 2. Effect of toosendanin on unitary conductance of KKir in a symmetrical 150 mM K+ solution. (A) Original recordings before and during toosendanin application. The membrane potentials and closed current levels are shown on the left and right rows, respectively. (B) Current – voltage relationships. The conductances of the channel before and during toosendanin (1 ×10 − 5 g/ml) application were 31.9 9 0.6 and 21.0 9 0.7 pS, respectively. n=4–6.
spectively (PB 0.01). Fig. 2B shows the I–V curves before and during toosendanin application (1× 10 − 5 g/ml).
4. Discussion The present study showed that toosendanin inhibited the activity of KKir in hippocampal CA1 pyramidal neurons. After application of toosendanin to the intracellular face in the inside-out patch of hippocampal neuron, the activity of KKir was inhibited in a concentration-dependent manner. The inhibition was observed as a reduction in Po and unitary current amplitude, and the reduction of Po resulted from a shrinkage of the open dwell time of the channel by mainly decreasing the slower open time constant. The previous study suggested that liposoluble toosendanin might diffuse into the interior of the cell and act intracellularly (Hu et al., 1997). Therefore, we examined its action on KKir in an inside-out patch configuration in this work. KKir distributed widely throughout the central nervous system allows a much larger K+ influx than efflux, controlling the membrane potential and diverse cellular functions without causing massive loss of K+ ions (Hille, 1992; Jan and Jan, 1994). In hippocampal pyramidal neurons, KKir could be modulated by neurotransmitters via G-protein coupled receptors (Thompson et al., 1992; Okuhara and Beck, 1994; Sodickson and Bean, 1998). The present work did not clarify the mechanism by which toosendanin induced the reduction in unitary conductance and slower open time constant of KKir. Toosendanin might act directly on the channel protein, but not through signal transduction pathways because the effect was detected in cell-free patches. Interestingly, we found that toosendanin inhibited several types of K+ channels but had no effect on Na+ channels (Xu and Shi, 1993). It is suggested that the action of toosendanin on these K+ channels may share a similar mechanism and there could be a common structural component in these channel proteins on which toosendanin acts. The phasic release of neurotransmitters is triggered by [Ca2 + ]i elevation regulated by Ca2 + and K+ channels, and the open K+ channels stabilize the membrane potential, indicating a negative regulation on cell excitability. Therefore, inhibition of K+ channels will enhance Ca2 + entry into cell and facilitate transmitter release. Previous studies demonstrated that toosendanin decreased the subendothelial K+ current in mouse motor nerve terminals (Xu and Shi, 1993) and delayed rectifier K+ current in NG 108-15 cells (Hu et al., 1997). The inhibition of K+ channels including KKir should be the underlying mechanism for toosendanin-
Z.-F. Wang, Y.-L. Shi / Neuroscience Research 40 (2001) 211–215
induced facilitatory phase of transmitter release and antibotulismic effect of the drug. Clarification of the mechanisms of toosendanin action and its antibotulismic effect may provide new clues to the molecular mechanism of neurotransmitter release. In conclusion, this study demonstrates that toosendanin inhibits KKir in a concentration-dependent manner. The inhibitory effect is observed as a decrease in the open probability and unitary conductance of the channel. These results, together with those of our previous study, agree with the assumption that the toosendanin-induced facilitatory phase of neurotransmitter release results from the inhibition of K+ channels and the concomitant increase in Ca2 + inflow.
Acknowledgements This work was supported by the National Basic Research Program (G1999054000) of China, National Natural Science Foundation of China (39870249) and Shanghai Metropolitan Fund for Research and Development (00JC 140407) to Y.L. Shi, and K.C. Wong Education Foundation and Chinese Postdoctoral Science Foundation to Z.F. Wang.
References Chen, W.Y., Yin, P.B., Ye, W.L., Shi, Y.L., 1999. Toosendanin-induced change of dopamine level detected by microdialysis in vivo at rat striatum. Chin. Sci. Bull. 44, 1106 –1110. Chung, C.C., Hsie, T.H., Liang, H.T., 1975. The structure of chuanliansu. Acta Chem. Sin. 33, 35 –47. Grigg, J.J., Kozasa, T., Nakajima, Y., Nakajima, S., 1996. Singlechannel properties of a G-protein-coupled inward rectifier potassium channel in brain neurons. J. Neurophysiol. 75, 318 – 328. Hamill, O.P., Marty, A., Neher, E., Sakamann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflu¨ gers Arch. 391, 85 – 100. Hille, B., 1992. G protein-coupled mechanisms and nervous signaling. Neuron 9, 187 – 195. Hu, Q., Huang, F.S., Shi, Y.L., 1997. Inhibition of toosendanin on the delayed rectifier potassium current in neuroblastoma × glioma NG 108-15 cells. Brain Res. 751, 47 –53. Huang, S.K., Song, X.E., Shi, Y.L., 1980. Effects of toosendanin on the fine structure of the neuromuscular junction. Acta Physiol. Sin. 32, 224 – 230.
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Jan, L.Y., Jan, Y.N., 1994. Potassium channels and their evolving gates. Nature 371, 119 – 122. Kaneda, M., Nakamura, H., Akaike, N., 1988. Mechanical and enzymatic isolation of mammalian CNS neurons. Neurosci. Res. 5, 299 – 315. Li, P.Z., Zhou, J., Miao, W.Y., Ding, F.H., Meng, J.Y., Jia, G.R., Li, J.F., Ye, H.J., He, X.Y., Chen, M.Y., Huang, Z.M., 1982. Therapeutic effect of toosendanin on animal botulism. Chin. Tradit. Herb. Drugs 13, 28 – 30. Oh, U., Ho, Y.K., Kim, D., 1995. Modulation of serotonin-activated K+ channel by G protein subunits and nucleotides in hippocampal neurons. J. Membr. Biol. 147, 241 – 253. Okuhara, D.Y., Beck, S.J., 1994. 5-HT1A receptor linked to inwardrectifying potassium current in hippocampal CA3 pyramidal cells. J. Neurophysiol. 71, 2161 – 2167. Shen, G.G., Zhuo, X.L., Shi, Y.L., 1994. The binding site of 3Htoosendanin in rat cerebral cortex homogenate. Acta Physiol. Sin. 46, 546 – 552. Shi, Y.L., Hsu, K., 1983. Anti-botulismic effect of toosendanin and its facilitatory action on miniature end-plate potentials. Jpn. J. Physiol. 33, 677 – 680. Shi, Y.L., Chen, W.Y., 1999. Effect of toosendanin on acetylcholine level of rat brain, a microdialysis study. Brain Res. 850, 173 –178. Shi, Y.L., Wei, N.S., Yang, Y.Q., Wang, Z.X., 1980. Toosendanin — a presynaptic neuromuscular blocking agent. Acta Physiol. Sin. 32, 293 – 297. Shi, Y.L., Wang, W.P., Hsu, K., 1981a. Electrophysiological analysis on the presynaptic blocking effects of toosendanin on neuromuscular transmission. Acta Physiol. Sin. 33, 259 – 265. Shi, Y.L., Yang, Y.Q., Wang, W.P., Hsu, K., 1981b. Influence of stimulus frequency, temperature and Ca2 + on onset of toosendanin paralysis of isolated neuromuscular preparations. Acta Physiol. Sin. 33, 141 – 146. Shi, Y.L., Wang, W.P., Yan, S.C., Hsu, K., 1982. Effects of calcium ion and nerve impulses on changes in miniature end-plate potential frequency produced by toosendanin. Acta Physiol. Sin. 34, 304 – 309. Shi, Y.L., Furuya, K., Wang, W.P., Terakawa, S., Xu, K., Yamagishi, S., 1993. Toosendanin induced Ca2 + conductance increase in neuroblastoma ×glioma hybrid cells. Chin. Sci. Bull. 38, 1489 – 1493. Shu, G.X., Liang, X.T., 1980. A correction of structure of chuanliansu. Acta Chem. Sin. 38, 196 – 198. Sodickson, D.L., Bean, B.P., 1998. Neurotransmitter activation of inwardly rectifying potassium current in dissociated hippocampal CA3 neurons: interactions among multiple receptors. J. Neurosci. 18, 8153 – 8162. Thompson, S.M., Haas, H.L., Gahwiler, B.H., 1992. Comparison of the action of adenosine at pre- and postsynaptic receptors in the rat hippocampus in vitro. J. Physiol. (Lond.) 451, 347 – 363. Wang, Y.B., Wen, Y.X., 1959. A comprehensive report on clinical ascaris anthelmintic therapeutic effect of toosendanin pills. J. Tradit. Chin. Med. 262, 46 – 49. Xu, Y.F., Shi, Y.L., 1993. Action of toosendanin on the membrane current of mouse motor nerve terminals. Brain Res. 631, 46 –50.
Modulation of inward rectifier potassium channel by toosendanin, a presynaptic blocker Zhong-Feng Wang, Yu-Liang Shi * Key Laboratory of Neurobiology, Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People’s Republic of China Received 21 November 2000; accepted 19 March 2001
Abstract The effect of toosendanin, a presynaptic blocker, on the inward rectifier potassium channel (KKir) of hippocampal CA1 pyramidal neurons of rats was studied by the single-channel patch-clamp technique. The results showed that toosendanin had an inhibitory effect on KKir in an excised inside-out patch of the neuron under a symmetrical 150 mM K+ condition. By decreasing the slower open time constant and increasing the slower close time constant, toosendanin (1 × 10 − 6 –1× 10 − 4 g/ml) significantly reduced the open probability of the channel in a concentration-dependent manner. Meanwhile, a dose-dependent reduction in unitary conductance of the channel was also detected after toosendanin application. These data offer an explanation for toosendanin-induced facilitation of neurotransmitter release and antibotulismic effect of the drug. © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Toosendanin; Presynaptic blocker; Patch-clamp; Inward rectifier potassium channel; Hippocampal pyramidal neuron
1. Introduction Toosendanin (C30H38O11, FW =574), a triterpenoid derivative (Chung et al., 1975; Shu and Liang, 1980), is an active ingredient extracted from the bark of Melia toosendan Sieb et Zucc, which is used as an anthelmintic vermifuge against ascarids in Chinese traditional medicine (Wang and Wen, 1959). Previous studies demonstrated that having no effect on the nerve impulse conduction and muscle cell membrane potential and acetylcholine sensitivity, toosendanin selectively blocks the synaptic transmission in neuromuscular junction and central synapses by interfering with the transmitter release with a facilitation at the first and blockade at the last (Shi et al., 1980, 1981a,b, 1982; Shi and Hsu, 1983; Shi and Chen, 1999; Chen et al., 1999) and might be potentially useful in medical and scientific researches (Shi et al., 1993). Moreover, toosendanin* Corresponding author. Tel.: + 86-21-64370080, ext. 154; fax: +86-21-64332445. E-mail address: [email protected] (Y.-L. Shi).
induced decrease in the number of synaptic vesicles was observed in the neuromuscular junction (Huang et al., 1980), and toosendanin-specific binding was found in the rat cerebral cortex homogenate (Shen et al., 1994). However, in spite of sharing some actions similar to those of botulinum neurotoxins (BoNTs), toosendanin was shown to have a dramatic antibotulismic effect. For example, toosendanin could prevent death in animals (mice, rats and monkeys) administered with several lethal dose of BoNT/A, B and still effective even when applied 18 h after BoNTs injection (Li et al., 1982); the tolerance to BoNT of the neuromuscular junction preparations, isolated from rats several days after a single injection of toosendanin, was enhanced significantly, and the high tolerance was associated with the toosendanin-induced facilitation (Shi and Hsu, 1983). An agent may evoke facilitation of the neurotransmitter release by inhibiting the K+ channels. It has been observed that toosendanin inhibited a subendothelial fast K+ current in mouse motor nerve terminals (Xu and Shi, 1993) and delayed rectifier K+ channel in
0168-0102/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 1 ) 0 0 2 3 3 - 4
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Z.-F. Wang, Y.-L. Shi / Neuroscience Research 40 (2001) 211–215
neuroblastoma×glioma NG108-15 cell line (Hu et al., 1997). The present work was undertaken to investigate the effect of toosendanin on inward rectifier K+ channels (KKir) in acutely isolated pyramidal neurons of the rat hippocampal CA1 region using the single-channel patch-clamp technique.
2. Materials and methods
2.1. Cell preparation A single hippocampal pyramidal neuron was isolated from the brain of Sprague– Dawley rats aged 7– 12 days using procedures previously described (Kaneda et al., 1988) with slight modifications. Briefly, after the rat was killed by decapitation, the brain was rapidly dissected out and the hippocampi were removed and sliced manually at a thickness of about 400– 600 mm. The slices were incubated in an incubation solution (in mM: NaCl 150, KCl 5, CaCl2 2, N-2-hydroxyethylpiperazine-N%-2-ethanesulfonic acid (HEPES) 10, glucose 10, the pH was adjusted to 7.4 with 2-amino-2-hydroxymethyl-propan-1,3-diol(Tris)) bubbled with 100% O2 at 37°C for 60 min and then treated with 0.5 mg/ml collagenase and 2 mg/ml pronase E for 60 min. The enzymes were washed out with the incubation solution twice. The slices were then re-incubated for 30 min. The treated slices were immersed in a standard external solution (in mM: NaCl 150, KCl 5, CaCl2 2.6, MgCl2 1.1, HEPES 10 and glucose 10, the pH was adjusted to 7.4 with Tris) containing 20% newborn calf serum, and CA1 regions were removed from the slices using small hand needles under an inverted microscope. The hippocampal cells were mechanically isolated using firepolished Pasteur pipettes with a 100– 400 mm tip diameter. All experiments conformed to the guidelines of the NIH on the ethical use of animals. All efforts were made to minimize the animal suffering and to reduce the number of animals used.
2.2. Electrophysiological recordings The hippocampal cell suspension was added to a poly-L-lysine-coated glass coverslip in a recording chamber mounted on the stage of an inverted microscope. After the cells settled down, perfusion was started. The channel current was recorded at an insideout configuration (Hamill et al., 1981) using a patchclamp amplifier (Axopatch 200A, Axon Instruments, Burlingame, CA) at room temperature (20– 25°C). The current was filtered at 1 kHz (−3 dB, 4-pole Bessel filter) and stored on a computer (IBM compatible 586/DX) via an analog-to-digital interface (DigiData
1200, Axon Instruments) for further analysis. Patch pipettes were pulled from a 1.5 mm (outer diameter) capillary glass (type 95, Shanghai Institute of Physiology) using a Narishige PP-83 electrode puller (Narishige, Japan). The tip of the pipette was coated with N-trimethylsilydiethylamine and fire polished on a microforge (FP-1, Shanghai Institute of Physiology). The recording electrode was filled with the electrode solution, and had a tip resistance of 6–8 MV. The electrode solution contained (in mM): KCl 150, CaCl2 1.0, HEPES 5.0, pH 7.4. The composition of the standard internal solution perfusing the cytoplasmic face of the inside-out patch was as follows (in mM): KCl 150, CaCl2 3.0, HEPES 5, ethyleneglycol-bis(b-aminoethylether) -N,N%-tetraacetic acid (EGTA) 5.0, the pH was adjusted to 7.2 with KOH.
2.3. Current analysis and data statistics Stretches of record 30 s in length at each membrane potential were digitized at 20 kHz and the single-channel analysis was performed using the Fetchan and pStat program (pClamp 6.04, Axon Instruments). The histogram of the distribution of current amplitude was fitted by the Gaussian distribution function using the Levenberg–Marquadt non-linear least squares method. The mean current values of the peak were subsequently used to construct the current–voltage relationship and hence to determine the conductance of the channel. The channel open and close dwell time histograms were fitted by the sum of exponential probability density functions using the Maximum likelihood method. The membrane potential described in this work indicates the cytoplasmic face potential. All data were expressed as mean9 S.E.M. Statistical analysis was performed using Student’s t-test for paired data, considering P B0.05 as significant.
2.4. Drugs and reagents Toosendanin used in this work was a sample recrystallized in ethanol with a purity \ 98% (Xu and Shi, 1993; Hu et al., 1997; Shi and Chen, 1999). Toosendanin was dissolved in ethanol first and then added to the perfusion solution to obtain a final concentration of 1×10 − 6 –1× 10 − 4 g/ml ( 1.7–170 mM). The highest ethanol concentration in the perfusion solution was less than 0.1%, and the channel activity was not affected by this concentration of ethanol. Poly-L-lysine, N-trimethylsilydiethylamine, HEPES, and EGTA are all Sigma products (St Louis, MO). The newborn calf serum was purchased from HyClone & Pierce (USA). All other reagents are of analytical grade.
Z.-F. Wang, Y.-L. Shi / Neuroscience Research 40 (2001) 211–215
3. Results
3.1. Decrease in open probability of the channel The effect of toosendanin on single-channel currents
213
of KKir at inside-out patches of hippocampal pyramidal neurons was investigated. Under a 150 mM symmetrical K+ condition, the single-channel current was recorded and the current–voltage relationship showed an inward rectifier property with an unit conductance of 31.99 0.6 pS over a membrane potential range from − 90 to 0 mV (n=7). The reversal potential was close to 0 mV coinciding with the K+ equilibrium potential. Thus, the channel recorded was identified as KKir as reported previously (Oh et al., 1995; Grigg et al., 1996). Internal application of toosendanin (1× 10 − 6 –1× 10 − 4 g/ml) significantly inhibited the activity of KKir of the neuron. After perfusing the inside-out patches with a toosendanin-containing internal solution, the open probability (Po) of the channel decreased in a concentration-dependent manner (n=4–6). Fig. 1A shows a typical record before and during toosendanin (1×10 − 5 g/ml) treatment at a membrane potential of −50 mV. Toosendanin at a concentration of 1× 10 − 4 g/ml decreased the Po of the channel by 85.89 11.2% (n=5) in comparison with its control value (Fig. 1B).
3.2. Shortening of open dwell time of the channel by decreasing the slower open time constant The distributions of open and close times of the channel could be best fitted by the two-exponential function. For example, at a −50 mV membrane potential, the fast open time constant (~fast) and slow open time constant (~slow) were 0.489 0.10 ms and 15.569 1.19 ms (n= 7), and the fast and slow close time constants were 0.329 0.05 ms and 18.7691.06 ms (n= 7), respectively. It was found that toosendanin at a concentration of 1×10 − 6 –1× 10 − 4 g/ml significantly decreased ~slow in a dose-dependent manner and had no effect on ~fast at each tested membrane potential (Fig. 1C). Moreover, the slow close time constant was markedly increased after toosendanin application. For example, at a − 50 mV membrane potential, toosendanin at concentrations of 1×10 − 6, 1×10 − 5 and 1× 10 − 4 g/ml, the slow close time constants increased to 32.93910.47 (n= 6), 59.759 13.89 (n=5) and 112.329 25.16 ms (n= 5) from the control value of
Fig. 1.
Fig. 1. Inhibition of the single-channel inward rectifier K+ current by toosendanin in a symmetrical 150 mM K+ solution; inside-out patch of rat hippocampal CA1 pyramidal neuron. (A) Original recordings before and during internal application of toosendanin (1 × 10 − 5 g/ml) at a membrane potential of −50 mV in the same patch. (B) Relationship between the concentration of toosendanin and the open probability of the channel. The membrane potential was held at − 50 mV. (C) Relationship between the concentration of toosendanin and the open time constant. The membrane potential was held at − 50 mV. The distribution of the open dwell time was best fitted with the two-exponential function, and the fast (~fast) and slow open time constants (~slow) were obtained. *PB0.05, **PB 0.01 vs. control. n =4 – 6.
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18.7691.06 ms (n= 7), respectively (P B 0.05 or PB 0.01), whereas no change was detected in the fast close time constant.
3.3. Decrease in unitary current amplitude of the channel The effect of toosendanin on the single-channel conductance of KKir was also studied. A significant decrease in the single-channel current amplitude of the channel was detected after perfusing with toosendanin at a concentration of 1× 10 − 5 g/ml (n =5) as shown in Fig. 2A as a typical record. Toosendanin at concentrations of 1× 10 − 6, 1 ×10 − 5 and 1×10 − 4 g/ml reduced the single-channel conductance to 25.49 1.2 (n= 6), 21.09 0.7 (n =5) and 15.49 0.9 pS (n= 5) from the control value of 31.990.6 pS (n = 7), re-
Fig. 2. Effect of toosendanin on unitary conductance of KKir in a symmetrical 150 mM K+ solution. (A) Original recordings before and during toosendanin application. The membrane potentials and closed current levels are shown on the left and right rows, respectively. (B) Current – voltage relationships. The conductances of the channel before and during toosendanin (1 ×10 − 5 g/ml) application were 31.9 9 0.6 and 21.0 9 0.7 pS, respectively. n=4–6.
spectively (PB 0.01). Fig. 2B shows the I–V curves before and during toosendanin application (1× 10 − 5 g/ml).
4. Discussion The present study showed that toosendanin inhibited the activity of KKir in hippocampal CA1 pyramidal neurons. After application of toosendanin to the intracellular face in the inside-out patch of hippocampal neuron, the activity of KKir was inhibited in a concentration-dependent manner. The inhibition was observed as a reduction in Po and unitary current amplitude, and the reduction of Po resulted from a shrinkage of the open dwell time of the channel by mainly decreasing the slower open time constant. The previous study suggested that liposoluble toosendanin might diffuse into the interior of the cell and act intracellularly (Hu et al., 1997). Therefore, we examined its action on KKir in an inside-out patch configuration in this work. KKir distributed widely throughout the central nervous system allows a much larger K+ influx than efflux, controlling the membrane potential and diverse cellular functions without causing massive loss of K+ ions (Hille, 1992; Jan and Jan, 1994). In hippocampal pyramidal neurons, KKir could be modulated by neurotransmitters via G-protein coupled receptors (Thompson et al., 1992; Okuhara and Beck, 1994; Sodickson and Bean, 1998). The present work did not clarify the mechanism by which toosendanin induced the reduction in unitary conductance and slower open time constant of KKir. Toosendanin might act directly on the channel protein, but not through signal transduction pathways because the effect was detected in cell-free patches. Interestingly, we found that toosendanin inhibited several types of K+ channels but had no effect on Na+ channels (Xu and Shi, 1993). It is suggested that the action of toosendanin on these K+ channels may share a similar mechanism and there could be a common structural component in these channel proteins on which toosendanin acts. The phasic release of neurotransmitters is triggered by [Ca2 + ]i elevation regulated by Ca2 + and K+ channels, and the open K+ channels stabilize the membrane potential, indicating a negative regulation on cell excitability. Therefore, inhibition of K+ channels will enhance Ca2 + entry into cell and facilitate transmitter release. Previous studies demonstrated that toosendanin decreased the subendothelial K+ current in mouse motor nerve terminals (Xu and Shi, 1993) and delayed rectifier K+ current in NG 108-15 cells (Hu et al., 1997). The inhibition of K+ channels including KKir should be the underlying mechanism for toosendanin-
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induced facilitatory phase of transmitter release and antibotulismic effect of the drug. Clarification of the mechanisms of toosendanin action and its antibotulismic effect may provide new clues to the molecular mechanism of neurotransmitter release. In conclusion, this study demonstrates that toosendanin inhibits KKir in a concentration-dependent manner. The inhibitory effect is observed as a decrease in the open probability and unitary conductance of the channel. These results, together with those of our previous study, agree with the assumption that the toosendanin-induced facilitatory phase of neurotransmitter release results from the inhibition of K+ channels and the concomitant increase in Ca2 + inflow.
Acknowledgements This work was supported by the National Basic Research Program (G1999054000) of China, National Natural Science Foundation of China (39870249) and Shanghai Metropolitan Fund for Research and Development (00JC 140407) to Y.L. Shi, and K.C. Wong Education Foundation and Chinese Postdoctoral Science Foundation to Z.F. Wang.
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