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Human acetylcholine receptors desensitize much faster than rat acetylcholine receptors
Neuroscience Letter,~, 103 (1989) 298 302 Elsevier Scientific Publishers Ireland Lid
298
NSL 06267
Human acetylcholine receptors desensitize much faster than rat acetylcholine receptors J. Siara, J.P. R u p p e r s b e r g a n d R . Rfidel Abteilung fiir AIIgemeine Physiologie der Universitiit Ulm, Ulm. ( F.R.G.) (Received 21 March 1989; Revised version received 26 April 1989; Accepted 2 May 1989)
Key words." Acetylchotine receptor; Desensitization; Rat myoball; Human myoball; Medulloblastoma cell line; Whole-cell recording The process of acetytcholine receptor (AChR) desensitization in the presence of transmitter was evaluated for human myoballs and medullo-btastoma cells. The cell under investigation was placed in one of two parallel streams of solution ejected from a double-barelled pipette. Ultrafast ( < 50 ms) application of acetylcholine (ACh) in a specific concentration was accomplished by a shift of the cell into the other stream. ACh-induced current was measured in the whole-cell mode of patch clamping. Parameters for AChR desensitization were almost identical for the two types of human ceils. Desensitization could be described by two time constants. With an ACh concentration of 3/tM, the fast time constant, rf, was about 0.4 s and the slow time constant, rs, was about 3.6 s. When the ACh concentration was raised to l0/tM, desensitization became faster. We also measured desensitization of the AChRs in rat myoballs and compared the values to our results from human cells. With 3/tM ACh, rf in rat myoballs was 2.3 s and r~ was 12.7 s. Thus, rat AChRs desensitize more slowly than human AChRs. However, desensitization times for rat AChRs obtained in our experiments are much faster than published values. Therefore, ultrafast solution change would seem to be requisite for correct assessment of the densitization process.
The nicotinic acetylcholine receptor (AChR) in the endplate region of the muscle fiber membrane is known to desensitize [7], i.e. with prolonged exposure to the agonist the ion channel goes into a closed state even though the ligand is present. Exact determination of the decay of the channel current shows that the desensitization process may be described by two exponentials with a fast (rf) and slow (r~) time constant [3, 18]. The AChRs in various mammalian species appear to be slightly different in their amino acid sequence, and during the development of each species different forms of AChR are expressed. It is still a matter of debate whether the speed of desensitization is the same in junctional and extrajunctional AChRs [10]. The present study was designed to compare AChR desensitization in two types of human cells originating from muscular and nervous tissue, i.e., (1) human myoballs, Correspondence." R. Riidel, Abteilung fiir Atlgemeine Physiologie der Universit~it Ulm, Albert-EinsteinAllee 11, D-7900 Ulm, F.R.G. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
299
spherical muscle cell regenerates at the stage of myotubes [14], and (2) spherical cells from the medulloblastoma cell line TE 671 established by McAUister et al. [9]. The medulloblastoma cells express an AChR that resembles the extrajunctional AChR of human skeletal muscle [8]. The procedures for culturing rat [2] and human [14] myoballs have been published. Spherical cells from McAllister's medulloblastoma cell line TE 671 [9] were cultured in the usual way [15]. At the end of the culturing period cells were detached from the bottom of the culture dish by the addition of collagenase and were kept floating in a solution containing (in mM): NaCl 135, KCI 3.5, CaC12 1.5, MgSO4 1.4, HEPES 10. The pH was 7.4, the temperature 22°C. For the measurement of ACh-induced membrane current, we placed a culture dish on the stage of an inverted microscope and selected an appropriate cell with a diameter of about 60/am. A pipette with a polished opening of about 8/zm in diameter was filled with a solution containing (in mM): KC1 140, MgCl2 1.5, EGTA l0 and HEPES 10 (pH 7.4) yielding a pipette resistance of 140-240 kl'2. The pipette was gently manipulated against the cell to form a high-resistance seal (> 5 MI2 between pipette and bath). The membrane spanning the mouth of the pipette was broken by gentle suction at the pipette butt resulting in the pipette-cell configuration for whole-cell recording [5]. The membrane potential was clamped to - 8 5 mV. The low access resistance of the system ensured that even during the flow of large membrane current ( > 10 hA) the voltage clamp was good. A double-barrelled pipette was placed 400/zm from the cell so that one of the two parallel streams ejected from the pipette at 30 mm/s completely surrounded the cell. ACh Cl
a) h u m a n m y o b a l l b ) T E 671 c)rat myoball Fig. 1. Time course o f whole-cell currents induced by the fast application of 3/aM acetylcholine to (a) a human myoball, (b) a human medulloblastoma cell and (c) a rat myoball. For easier comparison o f desensitization, the current peaks were scaled to the same size.
300 The composition of the fluid in the two barrels was identical to the solution in the culture dish, except that the solution in one barrel also contained 3 or 10/~M ACh. Application and washout of ACh was achieved by a quick shift of the cell from one stream into the other. The shifting was performed manually by injecting fluid into a hydraulic manipulator. The change of the solution bathing the cell occurred in < 50 ms, as judged from the delay of the peak of the ACh-induced current. Upon fast application of 3 / t M ACh to human myoballs and medulloblastoma cells, an inward current instantaneously surged to values of ~ l0 nA, decayed due to desensitization and stabilized after 20 s at about 5%peak value (Fig. 1). Recovery was complete: upon fast washout, the sensitivity of the A C h R s returned to 100% in < 30 s. The current transients were stored on an AT computer. The sum of two exponentials was fitted to the falling phase of the current with Marquardt's algorithm, yielding the time constants of the fast and slow components of desensitization, rr and rs. They proved to be faster than any desensitization value reported so far for an AChR. The values were almost identical for the two types of human cells. Also, the relative intensity of the fast and slow desensitization components, Ir/(If+ Is), was the same in both cell types (Table I). When the ACh concentration was increased from 3 to l 0 / t M , the peak currents were about 3 times larger and rr and rs became smaller. The relative intensity, If/ (If+Is), was also increased (Table I). Again, no statistically significant difference existed between the values obtained with human myoballs and medulloblastoma cells. We consider this to be corroborating evidence for the contention [8] that medulloblastoma cells express muscle AChRs. Finally, we repeated these experiments with rat myoballs. The experimental conditions were exactly the same as for the human cells except that the rat myoballs had
TABLE I TIME CONSTANTS OF FAST AND SLOW DESENSITIZATION,rf AND rs, AND RELATIVE INTENSITYOF THE TWO COMPONENTSOF DESENSITIZATION,Ir/(lr+lO, OF THE ACETYLCHOLINE RECEPTOR, DETERMINED AT 22°C IN THREE DIFFERENT CELL TYPES WITH TWO DIFFERENT ACh CONCENTRATIONS Figures are means+ S.D. of the results obtained from n different cells. ACh (3/~M) n
rr (S)
ACh (10/~M) "cs (S)
If
n
z'f (S)
~'s (S)
if+& Human myoball
11
Human medulloblastoma cell Rat myoball
36 10
0.38 ___0.16 0.49 +__0.11 2.33 4-0.35
3.6 +0.9 3.9 ___1.1 12.7 +2.4
0.64 _+0.11 0.60 __+0.12 0.43 +_0.10
If
l,,+& 6 15 10
0.23 _0.10 0.19 +0.05 0.35 +0,19
1.42 ___0.43 1.46 +0.55 1.97 +0.72
0.84 +0.08 0.85 __+0.09 0.71 _+0.14
301 diameters of only 30/zm. The p e a k of the current induced by application of ACh was about the same as in human myoballs of 60 gm diameter. As with the human cells, two components of desensitization could be distinguished (Fig. 1) but the • values were much larger and the relative intensity, Ill(If+ Is), was smaller. Mean values are given in Table I. Our results thus confirm that desensitization of the A C h R has a fast and a slow component as previously reported for frog [3] and rat [18] muscle. In the frog, desensitization is so slow (zf=2.3, s, zs=67 s with 100 gM ACh) that a solution change at 15 ml/min for a 1-ml chamber may be sufficient [3]. In mammalian muscle, desensitization of the AChRs is so fast that very fast solution changes are necessary for a correct measurement, in particular of the fast component. In an early study on rat myoballs [6], the times of changing solution were in the range of seconds. This is certainly much too slow. In a more recent study on rat myoballs, Wagoner [18] achieved changing times of 0.2q).4 s and found desensitization time constants that were about ten times larger than ours. This shows that ultrafast solution change is really a necessity for the correct assessment of desensitization of mammalian AChRs. The fastest method for application of ACh on muscle AChRs is iontophoresis from fine micropipettes positioned under microscopic control [13]. One disadvantage of this approach is that the ACh concentration at the receptor site changes during application. As shown here, the time constants of desensitization of the AChR are concentration-dependent. The double-jet stream method combines the advantages of fast application and stable concentration. Comparison of the properties of the extrajunctional AChRs in different species shows that the single channel conductance is nearly identical (human myotubes 40-45 pS [1]; torpedo AChR implanted into toad oocytes 40 + 2 pS [11]; medulloblastoma cells 44-45 pS [8] and 40_+ 1.0 pS [17]; bovine AChR implanted into toad oocytes 40_+ 1 pS [12]; rat myotubes 37-+08 pS [4]; rat muscle 42-+3 pS [19]) while the desensitization time constants vary between species. Comparison of the amino acid sequence of the AChRs from different species shows that some regions are highly conserved throughout evolution while others are not [8]. Perhaps conserved regions are responsible for ion conductance, while non-conserved regions are responsible for desensitization. We are grateful to Ms. G. Hack for culturing the cells and to Drs. Ch. Franke, H. Lorkovic, T. Pr6bstle and W. Spittelmeister for valuable comments. Supported by the Deutsche Forschungsgemeinschaft (Ru 138-15). 1 Adams, D.J. and Bevan, S., Some properties of acetylcholinereceptors in human cultured myotubes, Proc. R. Soc. Lond., B224(1985) 183-196. 2 Boldin,S., J/i.ger,U., Ruppersberg,J.P., Pentz, S. and Riidel, R., Cultivationmorphologyand electrophysiologyof contractilerat myoballs,PflfigersArch., 409 (1987) 462-467. 3 Feltz, A. and Trautmann, A., Desensitizationat the frog neuromuscularjunction: a biphasic process, J. Physiol.(Lond.), 322 (1982) 257-272. 4 Grassi, F., Monaco, L. and Eusebi, F., Acetylcholinereceptor channel properties in rat myotubes exposed to forskolin,Biochem.Biophys.Res. Commun., 147 (1987) 1000-1007.
302 5 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch.. 391 (1981) 85--100. 6 Horn, R. and Brodwick, M.S., Acetylcholine-induced current in perfused rat myoballs, J. Gen. Physiol., 75 (1980) 297-321. 7 Katz, B. and Thesleff, S., A study of the desensitization produced by acetylcholine at the neuromuscular junction, J. Physiol. (Lond.), 138 (1957) 63-~-80. 8 Luther, M.A., Schoepfer, R., Whiting, P., Casey, B., Blatt, Y., Montal, M.S., Montal, M. and Lindstrom, J., A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE 671, J. Neurosci., in press. 9 McAllister, R.M., Isaacs, H., Rongey, R., Peer, M., Au, W., Soukup, S.W. and Gardner, M.B., Establishment of a human medulloblastoma cell line, Int. J. Cancer, 20 (1977) 206-212. 10 McArdle, J.J., Molecular aspects of the trophic influence of nerve on muscle, Progr. Neurobiol.. 21 (1983) 135 198. 1l Methfessel, C., Witzemann, W., Takahashi, T., Mishina, M., Numa, S. and Sakmann, B., Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels, Pfliigers Arch., 407 (1986) 577-588. 12 Mishina, N., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S., Methfesset, C. and Sakmann, B., Molecular distinction between fetal and adult forms of muscle acetylcholine receptor, Nature (Lond.), 321 (1986) 406-411. 13 Peper, K., Bradley, R.J. and Dreyer, F., The acetylcholine receptor at the neuromuscular junction, Physiol. Rev., 62 (1982) 1271-1340. 14 Pr6bstle, T., Rfidel, R. and Ruppersberg, J.P., Hodgkin-Huxley parameters of the sodium channels in human myoballs, Pfliigers Arch., 412 (1988) 264-269. 15 Ruppersberg, J.P., Spittelmeister, W., Marx, A., Siara, J., Fakler, B. and Riidel, R., Ion channels in human medulloblastoma cells, Pfliigers Arch., 412 (1988) R 16. 16 Schoepfer, R., Luther, M. and Lindstrom, J., The human medulloblastoma celt line TE 671 expresses a muscle-like acetylcholine receptor: cloning of the ~- subunit eDNA, FEBS Lett., 226 (1988)235--240. 17 Siara, J., Ruppersberg, J.P. and Riidel, R., Human acetylcholine receptor: desensitization and the influence of second messengers, Pfliigers Arch., submitted. 18 Wagoner, P.K. and Pallotta, B.S., Modulation of acetylcholine receptor desensitization by forskolin is independent of cAMP, Science, 240 (1988) 1655 1657. 19 Witzemann, V., Barg, B., Nishikawa, Y., Sakmann, B. and Numa, S., Differential regulation of muscle acetylcholine receptor 7- and e-subunit mRNAs, FEBS Lett., 223 (1987) 104-112.
298
NSL 06267
Human acetylcholine receptors desensitize much faster than rat acetylcholine receptors J. Siara, J.P. R u p p e r s b e r g a n d R . Rfidel Abteilung fiir AIIgemeine Physiologie der Universitiit Ulm, Ulm. ( F.R.G.) (Received 21 March 1989; Revised version received 26 April 1989; Accepted 2 May 1989)
Key words." Acetylchotine receptor; Desensitization; Rat myoball; Human myoball; Medulloblastoma cell line; Whole-cell recording The process of acetytcholine receptor (AChR) desensitization in the presence of transmitter was evaluated for human myoballs and medullo-btastoma cells. The cell under investigation was placed in one of two parallel streams of solution ejected from a double-barelled pipette. Ultrafast ( < 50 ms) application of acetylcholine (ACh) in a specific concentration was accomplished by a shift of the cell into the other stream. ACh-induced current was measured in the whole-cell mode of patch clamping. Parameters for AChR desensitization were almost identical for the two types of human ceils. Desensitization could be described by two time constants. With an ACh concentration of 3/tM, the fast time constant, rf, was about 0.4 s and the slow time constant, rs, was about 3.6 s. When the ACh concentration was raised to l0/tM, desensitization became faster. We also measured desensitization of the AChRs in rat myoballs and compared the values to our results from human cells. With 3/tM ACh, rf in rat myoballs was 2.3 s and r~ was 12.7 s. Thus, rat AChRs desensitize more slowly than human AChRs. However, desensitization times for rat AChRs obtained in our experiments are much faster than published values. Therefore, ultrafast solution change would seem to be requisite for correct assessment of the densitization process.
The nicotinic acetylcholine receptor (AChR) in the endplate region of the muscle fiber membrane is known to desensitize [7], i.e. with prolonged exposure to the agonist the ion channel goes into a closed state even though the ligand is present. Exact determination of the decay of the channel current shows that the desensitization process may be described by two exponentials with a fast (rf) and slow (r~) time constant [3, 18]. The AChRs in various mammalian species appear to be slightly different in their amino acid sequence, and during the development of each species different forms of AChR are expressed. It is still a matter of debate whether the speed of desensitization is the same in junctional and extrajunctional AChRs [10]. The present study was designed to compare AChR desensitization in two types of human cells originating from muscular and nervous tissue, i.e., (1) human myoballs, Correspondence." R. Riidel, Abteilung fiir Atlgemeine Physiologie der Universit~it Ulm, Albert-EinsteinAllee 11, D-7900 Ulm, F.R.G. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
299
spherical muscle cell regenerates at the stage of myotubes [14], and (2) spherical cells from the medulloblastoma cell line TE 671 established by McAUister et al. [9]. The medulloblastoma cells express an AChR that resembles the extrajunctional AChR of human skeletal muscle [8]. The procedures for culturing rat [2] and human [14] myoballs have been published. Spherical cells from McAllister's medulloblastoma cell line TE 671 [9] were cultured in the usual way [15]. At the end of the culturing period cells were detached from the bottom of the culture dish by the addition of collagenase and were kept floating in a solution containing (in mM): NaCl 135, KCI 3.5, CaC12 1.5, MgSO4 1.4, HEPES 10. The pH was 7.4, the temperature 22°C. For the measurement of ACh-induced membrane current, we placed a culture dish on the stage of an inverted microscope and selected an appropriate cell with a diameter of about 60/am. A pipette with a polished opening of about 8/zm in diameter was filled with a solution containing (in mM): KC1 140, MgCl2 1.5, EGTA l0 and HEPES 10 (pH 7.4) yielding a pipette resistance of 140-240 kl'2. The pipette was gently manipulated against the cell to form a high-resistance seal (> 5 MI2 between pipette and bath). The membrane spanning the mouth of the pipette was broken by gentle suction at the pipette butt resulting in the pipette-cell configuration for whole-cell recording [5]. The membrane potential was clamped to - 8 5 mV. The low access resistance of the system ensured that even during the flow of large membrane current ( > 10 hA) the voltage clamp was good. A double-barrelled pipette was placed 400/zm from the cell so that one of the two parallel streams ejected from the pipette at 30 mm/s completely surrounded the cell. ACh Cl
a) h u m a n m y o b a l l b ) T E 671 c)rat myoball Fig. 1. Time course o f whole-cell currents induced by the fast application of 3/aM acetylcholine to (a) a human myoball, (b) a human medulloblastoma cell and (c) a rat myoball. For easier comparison o f desensitization, the current peaks were scaled to the same size.
300 The composition of the fluid in the two barrels was identical to the solution in the culture dish, except that the solution in one barrel also contained 3 or 10/~M ACh. Application and washout of ACh was achieved by a quick shift of the cell from one stream into the other. The shifting was performed manually by injecting fluid into a hydraulic manipulator. The change of the solution bathing the cell occurred in < 50 ms, as judged from the delay of the peak of the ACh-induced current. Upon fast application of 3 / t M ACh to human myoballs and medulloblastoma cells, an inward current instantaneously surged to values of ~ l0 nA, decayed due to desensitization and stabilized after 20 s at about 5%peak value (Fig. 1). Recovery was complete: upon fast washout, the sensitivity of the A C h R s returned to 100% in < 30 s. The current transients were stored on an AT computer. The sum of two exponentials was fitted to the falling phase of the current with Marquardt's algorithm, yielding the time constants of the fast and slow components of desensitization, rr and rs. They proved to be faster than any desensitization value reported so far for an AChR. The values were almost identical for the two types of human cells. Also, the relative intensity of the fast and slow desensitization components, Ir/(If+ Is), was the same in both cell types (Table I). When the ACh concentration was increased from 3 to l 0 / t M , the peak currents were about 3 times larger and rr and rs became smaller. The relative intensity, If/ (If+Is), was also increased (Table I). Again, no statistically significant difference existed between the values obtained with human myoballs and medulloblastoma cells. We consider this to be corroborating evidence for the contention [8] that medulloblastoma cells express muscle AChRs. Finally, we repeated these experiments with rat myoballs. The experimental conditions were exactly the same as for the human cells except that the rat myoballs had
TABLE I TIME CONSTANTS OF FAST AND SLOW DESENSITIZATION,rf AND rs, AND RELATIVE INTENSITYOF THE TWO COMPONENTSOF DESENSITIZATION,Ir/(lr+lO, OF THE ACETYLCHOLINE RECEPTOR, DETERMINED AT 22°C IN THREE DIFFERENT CELL TYPES WITH TWO DIFFERENT ACh CONCENTRATIONS Figures are means+ S.D. of the results obtained from n different cells. ACh (3/~M) n
rr (S)
ACh (10/~M) "cs (S)
If
n
z'f (S)
~'s (S)
if+& Human myoball
11
Human medulloblastoma cell Rat myoball
36 10
0.38 ___0.16 0.49 +__0.11 2.33 4-0.35
3.6 +0.9 3.9 ___1.1 12.7 +2.4
0.64 _+0.11 0.60 __+0.12 0.43 +_0.10
If
l,,+& 6 15 10
0.23 _0.10 0.19 +0.05 0.35 +0,19
1.42 ___0.43 1.46 +0.55 1.97 +0.72
0.84 +0.08 0.85 __+0.09 0.71 _+0.14
301 diameters of only 30/zm. The p e a k of the current induced by application of ACh was about the same as in human myoballs of 60 gm diameter. As with the human cells, two components of desensitization could be distinguished (Fig. 1) but the • values were much larger and the relative intensity, Ill(If+ Is), was smaller. Mean values are given in Table I. Our results thus confirm that desensitization of the A C h R has a fast and a slow component as previously reported for frog [3] and rat [18] muscle. In the frog, desensitization is so slow (zf=2.3, s, zs=67 s with 100 gM ACh) that a solution change at 15 ml/min for a 1-ml chamber may be sufficient [3]. In mammalian muscle, desensitization of the AChRs is so fast that very fast solution changes are necessary for a correct measurement, in particular of the fast component. In an early study on rat myoballs [6], the times of changing solution were in the range of seconds. This is certainly much too slow. In a more recent study on rat myoballs, Wagoner [18] achieved changing times of 0.2q).4 s and found desensitization time constants that were about ten times larger than ours. This shows that ultrafast solution change is really a necessity for the correct assessment of desensitization of mammalian AChRs. The fastest method for application of ACh on muscle AChRs is iontophoresis from fine micropipettes positioned under microscopic control [13]. One disadvantage of this approach is that the ACh concentration at the receptor site changes during application. As shown here, the time constants of desensitization of the AChR are concentration-dependent. The double-jet stream method combines the advantages of fast application and stable concentration. Comparison of the properties of the extrajunctional AChRs in different species shows that the single channel conductance is nearly identical (human myotubes 40-45 pS [1]; torpedo AChR implanted into toad oocytes 40 + 2 pS [11]; medulloblastoma cells 44-45 pS [8] and 40_+ 1.0 pS [17]; bovine AChR implanted into toad oocytes 40_+ 1 pS [12]; rat myotubes 37-+08 pS [4]; rat muscle 42-+3 pS [19]) while the desensitization time constants vary between species. Comparison of the amino acid sequence of the AChRs from different species shows that some regions are highly conserved throughout evolution while others are not [8]. Perhaps conserved regions are responsible for ion conductance, while non-conserved regions are responsible for desensitization. We are grateful to Ms. G. Hack for culturing the cells and to Drs. Ch. Franke, H. Lorkovic, T. Pr6bstle and W. Spittelmeister for valuable comments. Supported by the Deutsche Forschungsgemeinschaft (Ru 138-15). 1 Adams, D.J. and Bevan, S., Some properties of acetylcholinereceptors in human cultured myotubes, Proc. R. Soc. Lond., B224(1985) 183-196. 2 Boldin,S., J/i.ger,U., Ruppersberg,J.P., Pentz, S. and Riidel, R., Cultivationmorphologyand electrophysiologyof contractilerat myoballs,PflfigersArch., 409 (1987) 462-467. 3 Feltz, A. and Trautmann, A., Desensitizationat the frog neuromuscularjunction: a biphasic process, J. Physiol.(Lond.), 322 (1982) 257-272. 4 Grassi, F., Monaco, L. and Eusebi, F., Acetylcholinereceptor channel properties in rat myotubes exposed to forskolin,Biochem.Biophys.Res. Commun., 147 (1987) 1000-1007.
302 5 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch.. 391 (1981) 85--100. 6 Horn, R. and Brodwick, M.S., Acetylcholine-induced current in perfused rat myoballs, J. Gen. Physiol., 75 (1980) 297-321. 7 Katz, B. and Thesleff, S., A study of the desensitization produced by acetylcholine at the neuromuscular junction, J. Physiol. (Lond.), 138 (1957) 63-~-80. 8 Luther, M.A., Schoepfer, R., Whiting, P., Casey, B., Blatt, Y., Montal, M.S., Montal, M. and Lindstrom, J., A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE 671, J. Neurosci., in press. 9 McAllister, R.M., Isaacs, H., Rongey, R., Peer, M., Au, W., Soukup, S.W. and Gardner, M.B., Establishment of a human medulloblastoma cell line, Int. J. Cancer, 20 (1977) 206-212. 10 McArdle, J.J., Molecular aspects of the trophic influence of nerve on muscle, Progr. Neurobiol.. 21 (1983) 135 198. 1l Methfessel, C., Witzemann, W., Takahashi, T., Mishina, M., Numa, S. and Sakmann, B., Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels, Pfliigers Arch., 407 (1986) 577-588. 12 Mishina, N., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S., Methfesset, C. and Sakmann, B., Molecular distinction between fetal and adult forms of muscle acetylcholine receptor, Nature (Lond.), 321 (1986) 406-411. 13 Peper, K., Bradley, R.J. and Dreyer, F., The acetylcholine receptor at the neuromuscular junction, Physiol. Rev., 62 (1982) 1271-1340. 14 Pr6bstle, T., Rfidel, R. and Ruppersberg, J.P., Hodgkin-Huxley parameters of the sodium channels in human myoballs, Pfliigers Arch., 412 (1988) 264-269. 15 Ruppersberg, J.P., Spittelmeister, W., Marx, A., Siara, J., Fakler, B. and Riidel, R., Ion channels in human medulloblastoma cells, Pfliigers Arch., 412 (1988) R 16. 16 Schoepfer, R., Luther, M. and Lindstrom, J., The human medulloblastoma celt line TE 671 expresses a muscle-like acetylcholine receptor: cloning of the ~- subunit eDNA, FEBS Lett., 226 (1988)235--240. 17 Siara, J., Ruppersberg, J.P. and Riidel, R., Human acetylcholine receptor: desensitization and the influence of second messengers, Pfliigers Arch., submitted. 18 Wagoner, P.K. and Pallotta, B.S., Modulation of acetylcholine receptor desensitization by forskolin is independent of cAMP, Science, 240 (1988) 1655 1657. 19 Witzemann, V., Barg, B., Nishikawa, Y., Sakmann, B. and Numa, S., Differential regulation of muscle acetylcholine receptor 7- and e-subunit mRNAs, FEBS Lett., 223 (1987) 104-112.