Electrophysiological Properties of Newborn and Adult Rat Spinal Cord

Proc. Nati. Acad. Sci. USA Vol. 91, pp. 3097-3101, April 1994 Neurobiology Electrophysiological properties of newborn and adult rat spinal cord glycine receptors expressed in Xenopus oocytes ANDRAS MORALES*, QUOC-THANG NGUYEN, AND RICARDO MILEDI Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, Irvine, CA 92717 Contributed by Ricardo Miledi, December 30, 1993

ABSTRACT The properties of glycine receptors (GlyRs) quickly frozen in liquid nitrogen and stored at -800C. New- from newborn and adult rat spinal cord were studied in born rats (postnatal days 0-3) were deeply anaesthetized with Xenopus oocytes injected with whole mRNA or the heavy (H) ether and decapitated and spinal cords were dissected as or light (L) mRNA fractions encig their respective GlyRs. above. Mean open times and conductances of channels gated by H- or mINA Isolation and Size Fractionation. mRNA preparation L-GlyRs were determined by noise analysis or voltage jumps. and size separation on sucrose gradients were as described We found that adult H- and L-GlyRs opened channels that (2). Briefly, tissues were homogenized in phenol-saturated differed in their mean open time but had the same channel homogenization buffer (200 mM Tris-HCl/50 mM NaCl/10 conductance. Both H- and L-GlyRs gated Cl currents that mM EDTA/0.5% SDS, pH 9) supplemented with heparin (1 displayed a mir strong outward rectification. Neverthe- mg/ml). The mixture was shaken for 5 min and centrifuged less, single chanls ofadult H-and L-GlyRs did not rectify and for 10 min at 15,300 x g. Nucleic acids were isolated in the their mean open times were only slightiy altered by voltage. It aqueous phase by several extractions with chloroform and follows that the outward rectification of adult GlyRs is due phenol/chloroform, 1:1 (wt/vol). Total RNA was separated mainly to a reduction in the number of open channels. in by selective precipitation in 3 M NaCl and washed three times contrast to H-GlyRs, whose characteristics seem to remain with 3 M sodium acetate (pH 6). Poly(A)+ mRNA was essentiafly unchanged with age, L-GlyRs from newborn and purified by oligo(dT) chromatography, precipitated, washed adult rats have different properties. Channels of newborn in 70% ethanol, and concentrated to 1 pg/gl in diethyl L-GlyRs have a higher conductance, longer open time, and pyrocarbonate-treated water. About 100 gg of adult or new- greater voltage dependency than those from the adult. Inter- born mRNA was size fractionated on a sucrose gradient as estingly, properties of newborn GlyRs expressed by whole described (8). A rRNA gradient was run in parallel to give the mRNA were markedly different from those encoded by new- positions of the 18S and 28S rRNAs. Thirty fractions were born or adult L or H mRNA. These results demonstrate that the collected, ethanol-precipitated, dried in vacuum, and resus- functional heterogeneity of GlyRs is developmentally regu- pended in 10-12 ;4 of diethyl pyrocarbonate-treated water. lated. Oocyte Injection and Recording. Oocytes were injected with 50 nl of whole (W) mRNA or the H- or L-GlyR fraction Glycine is the major inhibitory neurotransmitter in the spinal and kept at 15-16'C in a modified Barth's solution (with cord and acts via glycine receptors (GlyRs) that differ in their Nystatin at 50 units/ml and gentamicin at 0.1 mg/ml). Two molecular structure and functional properties and which are days after injection, oocytes were treated with collagenase developmentally regulated (1-4). These two features of (0.5 mg/ml) to remove the surrounding layers (9) and record- GlyRs, heterogeneity and developmental regulation, have ings were performed at room temperature (20-240C) 4-10 been demonstrated in the Xenopus oocyte expression sys- days after injection. tem. For example, both the adult and the neonatal spinal cord To determine the mean open time and unitary currents of contain heavy (H) and light (L) mRNAs that code for distinct channels gated by H- or L-GlyRs, responses to glycine were GlyRs, all of which gate Cl- channels. Furthermore, in the subjected to noise analysis (5) or voltage jumps (10). Cells adult spinal cord the preponderant GlyR mRNA is of the H were voltage clamped with a conventional two-electrode type, whereas in the neonatal spinal cord the main GlyR voltage-clamp amplifier and exposed to glycine at a concen- mRNA is of the L type (2-4). The present study was tration (100-200 ;LM) well below the EC50 (2). Currents were undertaken, several years ago, to determine the character- ac-filtered (high-pass filter, one-pole, -3 decibels; fre- istics of the single channels opened by H- or L-GlyRs and to quency, 2 Hz or 0.015 Hz) and the resulting fluctuations, as see whether those characteristics were developmentally reg- well as the dc current, were recorded separately on an FM ulated. For that purpose we chose to use noise analysis (5) tape recorder or on a digital videotape recorder, for off-line rather than the patch-clamp method because some of the analysis. Current relaxations were obtained by stepping the responses were rather small due to the small amounts of holding potential from -60 mV to the desired voltage before mRNAs in some fractions (see ref. 2). Preliminary results and during the application of glycine (100-200 ,AM). Re- have been published elsewhere (6, 7). sponses to voltage jumps were recorded on a digital oscillo- scope and stored on floppy disks for further analysis. Cur- METHODS rent-voltage relations were obtained by applying 2-s pulses to different potentials before and during glycine application. Tissue Collection. Adult rats were anaesthetized with Nem- Data Analysis. Mean open times and elementary currents butal and decapitated. Spinal cords were immediately dis- were determined from current fluctuations by using a pro- sected out from the upper cervical to the lower lumbar gram written Rico Miledi or the SPAN segments. During the operation, the cords were kept moist by program (J. Demp- with a physiological solution. After removal, the tissues were Abbreviations: GlyR, glycine receptor; W-, H-, or L-GlyRs, glycine receptors expressed by whole (W), heavy (H), or light (L) mRNA The publication costs of this article were defrayed in part by page charge fractions. payment. This article must therefore be hereby marked "advertisement" *Present address: Departamento de Fisiologfa, Facultad de Medic- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ina, Universidad de Alicante, Alicante, Spain.

3097 Downloaded by guest on September 24, 2021 3098 Neurobiology: Morales et al. Proc. NatL Acad. Sci. USA 91 (1994) ster, University of Strathclyde, Glasgow, U.K.). Prior to potential in oocytes (11). As shown by the graph in Fig. 1, the sampling, ac currents were low-pass filtered in an anti- power spectra of current fluctuations induced by glycine aliasing filter (Butterworth, four poles) set to half the sam- acting on both types of GlyRs could be well fitted by a single plingfrequency. After sampling ac and dc currents, variances Lorentzian curve. However, the half-power frequencies of and power spectra were calculated. Mean open times (X) were the spectra derived from H- and L-GlyRs were significantly derived from the corner frequency (fj) of the power spectra different, indicating a difference in the mean open time ofthe by the formula X = 1/(2irf,). Elementary currents were corresponding membrane channels. At -60 mV the mean determined from the total variance of fluctuations and, as a open time (X) for H-GlyRs was 7.2 ± 1.3 ms (n = 22), while control, they were also calculated from the power spectrum. for L-GlyRs it was 18.1 ± 4.3 ms (n = 18); P < 0.01. Values given by the two methods were in good agreement. We also studied glycine current fluctuations in oocytes Fitting of current relaxations was performed by another injected with the W mRNA from adult rat spinal cord. As program written by Rico Miledi. Responses to voltagejumps expected, these GlyRs showed elementary currents similar to before glycine application were subtracted from those re- H- and L-GlyRs (-1.51 ± 0.46 pA; n = 13). The power sponses during drug application. The resulting decays were spectrum of these current fluctuations was well fitted by a usually well fitted by one or two exponentials. Unless oth- single Lorentzian, with a T of 8.36 ± 1.74 ms (n = 13). This values in the text correspond to the value is very similar to that obtained for receptors expressed erwise specified, given by H-GlyR mRNA (7.21 ± 1.25 ms, n = 22), which is by far mean and standard deviation. Means were compared by the predominant type in the adult rat spinal cord (2-4). Student's t test. Voltage Dependence of Adult W-, H-, and L-GIyR Unitary Conductance and Mean Open Time. As already described for RESULTS various GlyRs (12-16), the current-voltage relation of channels gated by H- or L-GlyRs showed a marked rectification Unitary Conductances and Mean Open Times of Adult W-, at hyperpolarizing potentials (Fig. 2). To identify the mech- H-, and L-GlyRs. Oocytes were injected with W, H, or L anism underlying this outward rectification, we determined mRNA from the spinal cord of the adult rat and only those the mean elementary currents and open times of GlyR oocytes in which glycine (100-200 p;M) generated currents channels at various membrane potentials. Fig. 3A shows an over 50 nA at a holding potential of -60 mV were included example of the membrane current fluctuations elicited by for noise analysis. Fig. 1 Inset shows examples of current glycine applied at various potentials in an oocyte injected fluctuations induced by glycine in oocytes injected with H or with H-GlyR mRNA. As seen in Fig. 3B, the channels gated L mRNA. At -60 mV, the elementary current amplitude (i) by H- or L-GlyRs had a reversal potential close to -20 mV. of channels associated with the two types of GlyRs were Furthermore, all showed an ohmic behavior-i.e., the ele- similar. The mean value for H-GlyRs (22 oocytes from five mentary current increased fairly linearly with the membrane donors) was -1.23 ± 0.45 pA, while for L-GlyRs it was -1.13 voltage. This implies that the channel conductance remained ± 0.43 pA (18 oocytes from the same donors). These i values constant, at about 30 pS, at all voltages tested. correspond to a single-channel conductance ('y) of about 30 The mean channel open times of H-GlyRs, derived from pS, since the reversal potential of the currents induced by power spectra, were weakly voltage-dependent and de- activation of both types of GlyRs was close to -20 mV (see creased by only 36% when the membrane potential was below), in good agreement with the estimated Cl- equilibrium changed from 0 to -120 mV. In contrast, L-GlyRs from the adult cord did not show a clear voltage dependency (Fig. 4). In two oocytes injected with adult L-GlyR mRNA and one 1.00 injected with H-GlyR mRNA, the membrane potential was stepped to various values before and during glycine application to determine the relaxation time constants ofthe glycine response. The channel open times obtained in this way were O - L 1 nearly identical to those derived from noise analysis. 1-1 co 200 _0.10 O :H 0 :L A :W 0.) 100 Membrane Potential (mV) Xj 1150WL AAvx -1 40 -120 -100 -80 -60 -40 -2 4+20 1509 nA 0.01 I I I I C)c 1 10 100 -100- 0

Frequency (Hz) N FiG. 1. (Inset) Examples of glycine-induced membrane currents E0 in oocytes injected with adult H-GlyR (left) or L-GlyR (right) -200 - mRNA. Upper traces show the dc-coupled record ofglycine-induced z currents at a holding potential of -60 mV; downward deflections denote inward currents. Lower traces are simultaneous high-gain FIG. 2. Voltage dependence of glycine (100-200 ;L)induced ac-coupled records to show the current noise. Glycine application is currents from adult W-, H-, and L-GlyRs expressed in oocytes. Note indicated by the bars. The graph shows the corresponding power the marked rectification at potentials more negative than -40 mV for spectra ofglycine-induced currentfluctuations from H-GlyRs (A) and all groups. Data were obtained from fourXenopus, 12-15 oocytes for L-GlyRs (o). The H-GlyR spectrum has a corner frequency of 31.4 each. Currents were normalized by taking the value at -60 mV as Hz, while that for L-GlyRs is 11.5 Hz (arrows); these values 100%. Error bars were usually smaller than the symbols and have correspond to mean channel durations of 5 and 14 ms, respectively. been omitted from the graph. Downloaded by guest on September 24, 2021 Neurobiology: Morales et A Proc. Nati. Acad. Sci. USA 91 (1994) 3099 L -40 -80 -120 1001

'N 10'- I A 1250nA 5 nA a- 10-32 250 s -2 C oC) 10-3 Membrane Potential (mV) E -120 -100 -80 -60 -40 -20 +20 .0.0 1 0-4 U) a) - _j CL 0 c a) C- o :H -2-0) o :L a) 0.1 1 10 100 A :W Frequency (Hz) 0 E i ) FIG. 5. Examples of power spectra (at -60 mV) derived from lU glycine-current noise in two oocytes injected with newborn L-GlyR (L) or H-GlyR (H) mRNA. Mean open time values derived from the FIG. 3. Voltage dependence of single-channel currents induced cornerfrequencies (arrows) were 290 ms for L-GlyRs and 140 ms and by glycine (100-200 1AM) from W-, H-, and L-GlyRs. (A) Samples of 7.7 ms for the slow and fast components of the H-GlyR spectrum, glycine (200 pM)-induced current fluctuations (lower trace of each pair of records) at the indicated holding potentials in an oocyte respectively. injected with H-GlyR mRNA. Note the peak current decrease and faster desensitization at -120 mV. (B) Elementary current-voltage channels gated by newborn H-GlyRs (i = 1.28 + 0.44 pA, y relationships for W-, H-, and L-GlyRs. Data come from more than = 32 pS, T = 6.94 ± 2.1 ms; n = 10, three donors) were not five donors in each group, and points are average values from 3-23 significantly different from those at -60 mV. oocytes. For newborn L-GlyRs, the power spectra of glycine- induced noise was well fitted by a single Lorentzian, as it was Unitary Conductance and Mean Open Time ofNewborn W-, for the adult. However, the corner frequencies (Jc) of the H-, and L-GlyRs. To see whether there were developmental spectra from newborn L-GlyRs were clearly lower than those changes in the properties of the GlyRs, we injected oocytes from the adult (cf. Figs. 1 and 5). Thus, the mean open time with W, H, or L mRNA from the newborn spinal cord. In all ofnewborn receptors (197 + 78 ms, n = 16, two donors) was cases, glycine activated Cl- channels that again rectified and approximately 11 times longer than that of adult L-GlyRs. had the same equilibrium potential as the channels expressed Moreover, the single-channel conductance of newborn by adult mRNAs. Furthermore, at -60 mV, the elementary L-GlyRs (i = 2.5 ± 1.3 pA, y = 62.5 pS, n = 16, same oocytes) current and conductance (i = 1.33 ± 0.48 pA,n= 17, five was twice as large as the corresponding value in adults (P < donors, 'y = 33 pS) and mean open time (7= 7.75 ± 2.01 ms, 0.01). Another important difference was that L-GlyR chan- n = 16, same oocytes) of the channel expressed by the nels from newborn rats were more voltage dependent than newborn H mRNA were again similar to the adult H-GlyRs. those induced by adult L mRNA. The mean open time of However, some spectra required a second Lorentzian curve newborn L receptors, determined from voltage jumps (Fig. to fit well at the low frequencies (Fig. 5), indicating that the 6), was markedly shorter at hyperpolarizing potentials. On channels may have complex kinetics with an additional slow average, T was 166 ± 56 ms at -60 mV (n = 7), whereas at 7 of about 130 ms. At -100 mV, the electrical properties of -100 mV it was 93 ± 28 (n = 6); P < 0.02. Glycine current fluctuations in oocytes injected with W O :H o :L A :W mRNA from newborn rats had a channel conductance of 41 25- + 8 pS (n = 6), which is similar to that found in adult rat W-GlyRs (38 ± 12 pS, n = 13). Moreover, as with adult 20- receptors, power spectra from receptors expressed by new- X born W mRNA could be well fitted by single Lorentzians, 15- T\ with corner frequencies quite different from those of receptors expressed by newborn L mRNA, which is by far the E *,10- predominant type in the newborn rat spinal cord. The mean open times for newborn W-GlyRs and L-GlyRs were close to n ,,§~~It 20 ms and 200 Ins, respectively. 5- DISCUSSION . I -120 -100 -80 -60 -40 -20 0 +20 Channel Conductances and Mean Open Times of Adult and Membrane Potential (mV) Newborn W-, H-, and L-GlyRs. The channel conductance of GlyRs described here is in good agreement with that de- FIG. 4. Channel open times of W (A), H (o), and L (o) GlyRs, scribed earlier for GlyRs from embryonic mouse spinal derived from power spectra obtained at various potentials. Data are neurons in culture (17). Since GlyR channels are character- from the same oocytes used for Fig. 3. ized by multiple conductance states in mouse and rat spinal Downloaded by guest on September 24, 2021 ,*,* 3100 Neurobiology: Morales et al. Proc. NatL Acad Sci. USA 91 (1994) A As values obtained for channel conductance and mean open time of H-GlyRs are in good agreement with those obtained for the al subunit (19), we can tentatively match the H-GlyRs with the al subunit. Voltage Dependence of GlyR Properties. We have shown that the channels expressed by W, H, and L mRNAs all B display a marked outward rectification. This is a fairly common characteristic of ligand-gated Cl- channels (i.e., y-aminobutyrate and glycine receptors) and could be due 102 either to a decrease in the probability of the channel being a) open, to a reduction in the mean open time, or to a smaller unitary conductance. We found that, like human fetal brain 0 GlyRs (13, 14), the newborn rat L-GlyRs open channels E -60 mV whose open time decreases markedly with hyperpolarization. E -80 mV Therefore, the rectification of L-GlyRs is probably caused -100 mV mainly by a shortening ofthe mean open time. In contrast, for o -120 mV newborn H-GlyRs and adult H- and L-GlyRs, the channel Zo0 101 o' conductance and mean open time did not change significantly -140 mV with voltage. Thus, it follows that, for these GlyRs, the rectification is mainly caused by a decrease in the number of channels that are open. 0 100 200 Developmental Regulation of GlyRs. Our results show that Time (ms) the single-channel characteristics of adult and newborn FIG. 6. Current relaxations following voltage steps during glycine H-GlyRs are very similar. Consequently, the fact that glycine application (200 AM) in an oocyte injected with newborn L-GlyR responses in oocytes injected with adult H-GlyR mRNA are mRNA. (A) Sample currents elicited by hyperpolarizing pulses much larger than those from oocytes injected with newborn applied from a holding potential of -60 mV. Records are the H-GlyR mRNA (2-4) is due to an increase in the number of difference between currents induced by voltage jumps to -80, -100, H-GlyRs expressed, resulting from the presence of a greater -120, and -140 mV, before and after glycine application. (B) Decay amount of mRNA molecules coding for H-GlyRs in the adult of currents shown in A plotted on semilogarithmic coordinates. spinal cord. This evidence for a developmental upregulation Potentials during the pulse are indicated at right. Decay time con- of H-GlyR mRNA correlates well with studies on the abun- stants at -60 to -140 mV (20-mV steps) were 195, 147, 112, 93, and dance of the al subunit mRNA in newborn or adult spinal 80 Ms. cord as shown by either Northern blot analysis (4, 22) or in neurons (12, 15, 18, 19), the channel conductance we ob- situ hybridization (23). tained reflects the average of the various subconductance In contrast to the H-GlyRs, the characteristics of adult states seen at the patch-clamp level. L-GlyRs differ markedly from those ofthe newborn L-GlyRs, The open times of GlyR-channels in spinal neurons of not only in channel conductance and open time but also in newborn and young rats (19) resemble our estimates for their channel sensitivity to voltage. Various hypotheses can be put forward to explain these differences, but perhaps the oocytes injected with W mRNA from newborn and adult simplest is to postulate the existence of different molecular spinal cord, respectively. Interestingly, despite the similarity forms of L-GlyRs in adult and newborn spinal cord. In this of X values obtained for GlyRs in newborn spinal cord case, would the adult L-GlyRs be made up ofany ofthe three neurons and newborn W-GlyRs expressed in oocytes, these a subunits cloned so far? Although short transcripts of al values do not correlate well with the mean open time ofGlyRs mRNA (=2 kb) can be detected in the adult spinal cord by expressed in oocytes injected with the clone a2, the combi- Northern blot hybridization (4, 22), it is unlikely that the adult nation al plus a2, or newborn H or L mRNA fractions (refs. L-GlyR mRNA corresponds to these, since the electrophys- 17 and 19 and this study). This unexpected discrepancy iological properties of al (19) and adult H-GlyRs (this study) indicates that, despite the availability of several clones, clearly differ from those of the receptors expressed by adult injection into oocytes oftissue-derived mRNA is still worthy L-GlyR mRNA. A correspondance to the a2 subunit has to of use and suggests that the differences between receptors be ruled out too, since our study shows that the adult expressed from W mRNA and fractions or clones may be due L-GlyRs have an open time which is 1/10th that of a2 to the coassembly of heteromeric subunits. In view of our receptors (19). It could be that the adult L-GlyRs correspond results, it would not be surprising if coexpression with the (3 to the rat brain a3 subunit (24), since their mRNAs are similar subunits or other clones were to give values similar to the in size, and the adult L-GlyRs are moderately sensitive to ones we observed. This is supported by the fact that the (-alanine and are very weakly activated by taurine (data not presence of ,3 subunits in heteromeric a/P receptors deter- shown), like a3 subunits expressed in oocytes (24). However, mines their channel conductance and sensitivity to picrotoxin a3 mRNA is present at only a very low level in the spinal cord (1, 20). (23), which does not agree well with our data, since adult Determination of channel characteristics of newborn L-GlyRs expressed in oocytes generate currents whose av- L-GlyRs, either by voltage-jump relaxation or by noise erage amplitude, although smaller than those of H-GlyRs, is analysis, indicates a relatively high value for the mean open substantial (-200 nA). Therefore, adult L-GlyRs may differ time. Our estimate of a long value for newborn L-GlyRs is from the a3 subunit and thus represent a hitherto uncloned in good agreement with that obtained for the a2 subunit (19) subunit, additional to the one previously postulated by Akagi cloned from the L-GlyR mRNA fraction of young rats (21); et al. (4). It is also possible that native mRNAs lead to the this suggests strongly that the properties ofnewborn L-GlyRs expression ofheteromeric GlyRs whose subunit composition are due to the a2 subunit. A similar variety ofGlyRs probably is developmentally regulated. occurs in the human nervous system since, as shown earlier The mechanisms underlying these complex regulations of (13), human fetal brain GlyRs expressed in oocytes are also quantities and types ofGlyRs expressed during development characterized by a long channel open time, comparable to still remain to be elucidated. Nonetheless, it is interesting to that found here for newborn rat L-GlyRs. recall that the longer open time of newborn versus adult Downloaded by guest on September 24, 2021 Neurobiology: Morales et al. Proc. Natl. Acad. Sci. USA 91 (1994) 3101 GlyRs resembles the situation found previously for acetyl- 13. Gundersen, C. B., Miledi, R. & Parker, I. (1984) Proc. R. Soc. choline receptors in developing or denervated muscles (5, 25, London Ser. B 221, 235-244. 26). Thus, it is possible that during development, changes in 14. Gundersen, C. B., Miledi, R. & Parker, I. (1986) J. Physiol. the kinetics of receptor actions play a general role in deter- (London) 377, 40P. mining the properties of the cells innervated. 15. Bormann, J., Hamill, 0. P. & Sakmann, B. (1987) J. Physiol. (London) 385, 243-286. We thank Drs. Ian Parker and I. Ivorra for helpful comments on 16. Akaike, N. & Kaneda, M. (1989) J. Neurophysiol. 62, 1400- this manuscript, Rico Miledi for computer programmin; and the 1409. National Institutes ofHealth (Public Health Service Grant NS23284) 17. Barker, J. L., McBurney, R. N. & MacDonald, J. F. (1982) J. for support. A.M. was a recipient of an International Fogarty Physiol. (London) 322, 365-387. Fellowship. 18. Takahashi, T. & Momiyama, A. (1991) Neuron 7, 965-969. 19. Takahashi, T., Momiyama, A., Hirai, K., Hishinuma, F. & 1. Betz, H. 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