- Email: [email protected]
Membrane channels activated by taurine in cultured mouse spinal cord neurons
Neuroscience Letters, 98 (1989) 229-233 Elsevier Scientific Publishers Ireland Ltd.
229
NSL 05954
Membrane channels activated by taurine in cultured mouse spinal cord neurons David Alexander Mathers, Anoop Grewal and Yihong Wang Department of Physiology, Faculty o[ Medicine, University of British Columbia, Vancouver, B.C. (Canada)
(Received 20 October 1988; Revised version received 25 November 1988; Accepted 25 November 19883 Key words'." Taurine; 7-Aminobutyric acid; Patch-clamp; Spinal cord; Neuron
Patch-clamp methods were used to compare biophysical properties of anion channels activated by taurine and 7-aminobutyric acid in the membrane of cultured mouse spinal neurons. Outside-out patches were voltage clamped at - 8 0 mV at a temperature of 21-23°C. Bath application of GABA (1.5-2/tM) or taurine (5-40/IM) induced chloride-dependent single-channel currents in 14/20 patches tested. Amplitude distributions of these currents showed peaks corresponding to conductance levels of 8, 16, 27 and 46 pS. Only a few percent of GABA-induced events reached the 46 pS level, while 30% of taurine-induced currents were of this size. The average lifetime of taurine-activated channels in the open state was 1.0+0.07 ms, significantly shorter than the corresponding value for GABA (1.6 +0.08 ms). Taurine-induced currents were abolished by 10 lzM strychnine, but persisted in the presence of 50 ,uM bicuculline.
The sulphur-containing amino acid taurine is f o u n d in millimolar a m o u n t s in m a m m a l i a n tissues. Within the central nervous system, taurine has been located in the cerebral cortex, cerebellum, retina and in the spinal cord [7, 13]. Taurine often inhibits the firing o f central neurons, as do other short chain o - a m i n o acids, including glycine, fl-alanine and 7-aminobutyric acid ( G A B A ) . Both taurine and G A B A suppress s p o n t a n e o u s discharge in the rat and guinea pig cerebellum [4, 14]. These suppressive effects are blocked by the G A B A antagonists picrotoxin and bicuculline, but are unaffected by the glycine antagonist strychnine. These observations indicate that taurine interacts with a G A B A - I i k e receptor in this preparation. In contrast, taurine induced depression o f activity in rat and h u m a n brainstem neurons is unaffected by G A B A antagonists, but is readily blocked by strychnine. In this preparation, taurine therefore appears to activate a glycine-like receptor [5]. Both taurine and fl-alanine hyperpolarize the ventral roots o f the frog spinal cord. This effect is selectively blocked by strychnine, suggesting the involvement o f a glycine-like receptor [12, 13]. However, taurine- and fl-alanine-induced depression o f the dorsal roots in the spinal cord can be antagonized by picrotoxin, bicuculline or Corre.sTondence: D.A. Mathers, Department of Physiology, Faculty of Medicine, University of British Columbia, 2146 Health Sciences Mall, Vancouver, B.C., Canada V6T 1W5.
0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
230 strychnine. This suggests that a distinct taurine/fl-alanine receptor may exist in this preparation [12, 13]. In summary, these data suggest that taurine fairly generally inhibits neuronal firing, mimicking GABA, glycine or/%alanine dependent on the pathway being studied. To date, however, there have been no studies designed to investigate the action of taurine at the level of single channel currents in the neuronal membrane. In this paper, we have used the extracellular patch clamp method to reveal biophysical and pharmacological properties of membrane channels activated by taurine in spinal cord neurons. Experiments were performed on spinal cord neurons obtained from 13-day-old mouse (CD1) embryos and grown in primary dissociated culture [9, 10]. Cultures were kept for 2-3 weeks at 37°C in a 10% CO2 incubator prior to recording. Patchclamp recordings were carried out at 21-23°C using a List Electronic EPC-5 amplifier operating in the 'outside-out patch' mode [6, 1 I]. All patches clearly contained more than one agonist gated channel, as evidenced by the presence of occasional overlapping openings. After patch formation, the normal bathing solution was replaced by external Tris-saline containing (in mM): Tris-Cl 140, KC! 4, CaC12 1, MgCI2 1, 10 HEPES, pH 7.2. Patch electrodes were filled with a solution of composition (in mM): Tris-C1 140, NaC1 3, MgC12 1, I 1 EGTA, 10 HEPES, pH 7.2. Since the impermeant cation Tris + replaced the majority of Na + and K + ions across the isolated patch, membrane currents carried by these ions are negligible compared to agonist induced chloride fluxes [1, 10]. Patches were voltage-clamped to a potential of - 8 0 mV. GABA (1.5-2/tM, Sigma) and taurine (2 - 4 0 / t M , Sigma) were dissolved in external Tris-saline and applied to the membrane patches by bath perfusion. Patch currents were low-pass filtered at 1 kHz and recorded on Wideband FM tape. Cumulative frequency distributions of single-channel open times were constructed. Since we wished to compare the average kinetic parameters for GABA and taurine induced channels, separate distributions were not made for each channel substate in the present work. Open time distributions were fitted by the sum of two exponential terms using the Gauss-Newton-Marquadt algorithm [3]. Histograms of the amplitudes of single-channel currents were constructed. These distributions were fit by the sum of 1, 2, 3 or 4 normal curves using a Simplex method. At a holding potential of - 80 mV, application of 1.5/~M GABA induced inwardly directed single-channel currents in 14/20 patches tested. In all 14 of these cases, application of taurine at concentrations exceeding 5/~M also induced inwardly directed single channel currents (Fig. 1). The remaining 6 patches were insensitive to both amino acids. This insensitivity may simply reflect the limited membrane sampling achieved using the outside-out patch technique. Essentially all spinal cord neurons in these cultures show sensitivity to GABA when tested at the whole-cell level [1]. In the case of currents induced by both GABA and taurine, it was found that all events reversed polarity to outward charge flow on displacing the patch potential beyond 0 mV. This result is consistent with previous reports that both agonists gate chloride-selective channels in cultured spinal cord cells [1]. Amplitude distributions for GABA induced currents were well fit by the sum of
231
TAURINE flier
11
--
I t,
--.
J.3
-
[4
"
' v-rl--~'aa~t-rl
T'T
]~"
'
i r "~,'t
'
" ! "
"
'
' W"
'W p
r 'r
I
Jl
0
""I,,~' ~' r~ FrrflN,pr,qp
*'*
,, I
, ~,~v-r
"writ"
I,
12 13
_42p
I,,
Fig. 1. Single-channel currents activated by bath application o f 4 0 / a M taurine to an outside-out patch of m e m b r a n e excised from a mouse spinal cord neuron grown in culture for 3 weeks. The patch was voltage clamped to a potential of - 80 mV. Downward deflections from baseline (0) denote inward m e m b r a n e current. Representative events reaching current levels I,, 12, 13, and 14 are shown.
3 Gaussian terms (Fig. 2A). The modal values of these Gaussians occurred at current levels corresponding to conductance states of 7.7+0.29, 16.8_ 1.44 and 26.5+_1.32 pS (mean+_ S.E.M., 5 patches). All 5 patches tested with GABA displayed a small number (less than 2%) of openings which were considerably larger than expected (Fig. 2A). These events probably correspond to the rare, 46 pS conductance level reported to occur in GABA-sensitive channels from cultured spinal neurons [2].
eo.A o
GABA
Taurine
B
45 30
a
30 0
15
15 Z -5.0
-4.0
-a.o
-Z.o
-t.o
C u r r e n t A m p l i t u d e (pA)
o
-5.0
-4.0
-$.0
-2.0
- 1.0
0
C u r r e n t A m p l i t u d e (pA)
Fig. 2. Amplitude distributions for single-channel currents activated by 1.5/aM G A B A (A) or 40 p M taufine (B) in the same outside-out patch of spinal cord cell membrane, voltage clamped at - 80 mV. These distributions were fit by the s u m o f 3 (A) or 4 (B) Gaussian terms (smooth curves). In A, the modal values o f the 3-fit Gaussians occurred at current levels o f - 0 . 6 , - 1.4 and - 2 . 0 pA. In B, modal values o f the 4-fit Gaussians occurred at current levels o f - 0 . 8 , - 1.4, - 2 . 4 and - 3 . 4 pA.
232 Amplitude distributions for taurine induced currents were well fit by the sum of 4 Gaussian terms (Fig. 2B). The modal values of the 4 Gaussian terms tit to these distributions occurred at current levels corresponding to conductance states of 8.3 _+0.47, 16.5_ 0.99, 28.3 +_ 1.29 and 46.0 _4_-1.83 pS (5 patches). Statistical comparison of these conductances showed that the 3 smaller levels did not differ significantly from values derived for GABA activated channels ( P > 0.05, Wilcoxon test). Therefore, the major difference between the conductance of channels activated by the two amino acids lay in the marked ability of taurine to trigger large, 46 pS events. Currents of this size made up an average of 29.5 +8.3% of events triggered by taurine in the test patches. In the case of both GABA and taurine, the distribution of open channel lifetimes was well fit by an equation of the form y = Aft,.e t/TAUf+ N~.. e-t/TAU~ Here, Nr and N, denote the number of events in the fast and slow fit components respectively. The mean open time, TAUmean of membrane channels activated by GABA or taurine was calculated using the relation WAUmean Nf/(Nr+NO T A U t + Ns/(Nf+Ns)" WAUs [3]. The mean lifetime of taurine induced channels, 1.0+0.07 ms, was significantly briefer than the corresponding value calculated for GABA-activated channels (TAU mean= 1.6+0.08 ms, P < 0 . 0 1 , 5 patches). Taurine induced channels were completely and reversibly abolished in the presence of strychnine sulphate (10/tM), but were not blocked by bicuculline methiodide (50 /~M, 5 patches tested with each antagonist). Both GABA and taurine were found to activate channels with multiple conductance states. The 3 lowest conductance levels of these channels are apparently identical. The mean values obtained for these conductance levels are in very good agreement with previously published results [2]. Taurine-activated channels frequently entered a high conductance state of 46 pS conductance. This state may also be entered by GABA activated channels, but with much lower probability. It is possible that taurine activates channels associated with GABA receptors, and that taurine stabilises these channels in the 46 pS conductance state. This seems unlikely, in view of the insensitivity of taurine-induced events to bicuculline. Alternatively, taurine may activate glycine receptors, which are known to control a chlorideselective channel of 46 pS conductance in cultured spinal cord neurons [2]. The present data are fully consistent with this possibility, since taurine-induced events were highly sensitive to blockade by strychnine. Recent data strongly suggest that at least 3 types of taurine receptors may exist in spinal cord tissue. Two receptor subtypes apparently mediate the depolarising and hyperpolarising responses to taurine seen in the dorsal roots [8]. Pharmacological experiments suggest that these receptors are distinct from both GABA and glycine receptors in the spinal cord. A third type of taurine receptor, which resembles the glycine receptor pharmacologically, is apparently found in the ventral roots of the cord [12, 13]. The present data suggest that this ventral root class of taurine receptor may also be expressed on spinal cord neurons in culture. :
233 I Barker, J.L. and Mathers, D.A., GABA analogues activate channels of different duration on cultured mouse spinal neurons, Science, 212 (I 98 l) 358-361. 2 Bormann, J., Hamill, O.P. and Sakmann, B., Mechanism of anion permeation through channels gated by glycine and y-aminobutyric acid in mouse cultured spinal neurones, J. Physiol. (Lond.), 385 (1987) 243-286. 3 Chow, P. and Mathers, D.A., Convulsant doses of penicillin shorten the lifetime of GABA induced channels in cultured central neurones, Br. J. Pharmacol., 88 (1986) 541-548. 4 Frederickson, R.C.A., Neuss, M., Morzorati, S.L. and McBride, W.J., A comparison of the inhibitory effects of taurine and GABA on identified Purkinje ceils and other neurons in the cerebellar cortex of the rat, Brain Res., 145 (1978) 117-126. 5 Haas, H.L. and Hosli, L., The depression of brain stem neurones by taurine and its interaction with strychnine and bicuculline, Brain Res., 52 (1973) 399-402. 6 Hamill, O.P., Marry, 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, Pfluegers Arch., 391 (1981) 85-100. 7 Huxtable, R.J., Franconi, F. and Giotti, A., The Biology of Taurine. Advances in Experimental Medicine, Vol. 217, Plenum, New York, 1987. 8 Kuba, Y., Akiyoshi, E. and Akagi, H., Identification of two taurine receptor subtypes on the primary afferent terminal of frog spinal cord, Br. J. Pharmacol., 94 (1988) 1051-1056. 9 Mathers, D.A. and Yoshida, H., The benzodiazepine triazolam: direct and GABA depressant effects at the membrane of cultured mouse spinal cord neurons, Eur. J. Pharmacol., 139 (1987) 53-60. 10 Mathers, D.A., Spontaneous and GABA-induced single channel currents in cultured murine spinal cord neurons, Can. J. Physiol. Pharmacol., 63 (1985) 1228-1233. 11 Mathers, D.A., The GABAA receptor: new insights from single-channel recording, Synapse, 1 (1987) 9(~101. 12 Nicoll, R.A., Padjen, A. and Barker, J.L., Analysis of amino acid responses on frog motoneurones, Neuropharmacol., 15 (1976) 45-53. 13 Oja, S.S. and Kontro, P., Taurine. In A. Lajtha (Ed.), Handbook of Neurochemistry, 2nd Edn., Plenum, New York, 1983, pp. 501-521. 14 Okamoto, K. and Sakei, Y., Augmentation by chlordiazepoxide of the inhibitory effects of taurine, fl-alanine and 7-aminobutyric acid on spike discharges in guinea pig cerebellar slices, Br. J. Pharmacol., 65 (1980) 277 285.
229
NSL 05954
Membrane channels activated by taurine in cultured mouse spinal cord neurons David Alexander Mathers, Anoop Grewal and Yihong Wang Department of Physiology, Faculty o[ Medicine, University of British Columbia, Vancouver, B.C. (Canada)
(Received 20 October 1988; Revised version received 25 November 1988; Accepted 25 November 19883 Key words'." Taurine; 7-Aminobutyric acid; Patch-clamp; Spinal cord; Neuron
Patch-clamp methods were used to compare biophysical properties of anion channels activated by taurine and 7-aminobutyric acid in the membrane of cultured mouse spinal neurons. Outside-out patches were voltage clamped at - 8 0 mV at a temperature of 21-23°C. Bath application of GABA (1.5-2/tM) or taurine (5-40/IM) induced chloride-dependent single-channel currents in 14/20 patches tested. Amplitude distributions of these currents showed peaks corresponding to conductance levels of 8, 16, 27 and 46 pS. Only a few percent of GABA-induced events reached the 46 pS level, while 30% of taurine-induced currents were of this size. The average lifetime of taurine-activated channels in the open state was 1.0+0.07 ms, significantly shorter than the corresponding value for GABA (1.6 +0.08 ms). Taurine-induced currents were abolished by 10 lzM strychnine, but persisted in the presence of 50 ,uM bicuculline.
The sulphur-containing amino acid taurine is f o u n d in millimolar a m o u n t s in m a m m a l i a n tissues. Within the central nervous system, taurine has been located in the cerebral cortex, cerebellum, retina and in the spinal cord [7, 13]. Taurine often inhibits the firing o f central neurons, as do other short chain o - a m i n o acids, including glycine, fl-alanine and 7-aminobutyric acid ( G A B A ) . Both taurine and G A B A suppress s p o n t a n e o u s discharge in the rat and guinea pig cerebellum [4, 14]. These suppressive effects are blocked by the G A B A antagonists picrotoxin and bicuculline, but are unaffected by the glycine antagonist strychnine. These observations indicate that taurine interacts with a G A B A - I i k e receptor in this preparation. In contrast, taurine induced depression o f activity in rat and h u m a n brainstem neurons is unaffected by G A B A antagonists, but is readily blocked by strychnine. In this preparation, taurine therefore appears to activate a glycine-like receptor [5]. Both taurine and fl-alanine hyperpolarize the ventral roots o f the frog spinal cord. This effect is selectively blocked by strychnine, suggesting the involvement o f a glycine-like receptor [12, 13]. However, taurine- and fl-alanine-induced depression o f the dorsal roots in the spinal cord can be antagonized by picrotoxin, bicuculline or Corre.sTondence: D.A. Mathers, Department of Physiology, Faculty of Medicine, University of British Columbia, 2146 Health Sciences Mall, Vancouver, B.C., Canada V6T 1W5.
0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
230 strychnine. This suggests that a distinct taurine/fl-alanine receptor may exist in this preparation [12, 13]. In summary, these data suggest that taurine fairly generally inhibits neuronal firing, mimicking GABA, glycine or/%alanine dependent on the pathway being studied. To date, however, there have been no studies designed to investigate the action of taurine at the level of single channel currents in the neuronal membrane. In this paper, we have used the extracellular patch clamp method to reveal biophysical and pharmacological properties of membrane channels activated by taurine in spinal cord neurons. Experiments were performed on spinal cord neurons obtained from 13-day-old mouse (CD1) embryos and grown in primary dissociated culture [9, 10]. Cultures were kept for 2-3 weeks at 37°C in a 10% CO2 incubator prior to recording. Patchclamp recordings were carried out at 21-23°C using a List Electronic EPC-5 amplifier operating in the 'outside-out patch' mode [6, 1 I]. All patches clearly contained more than one agonist gated channel, as evidenced by the presence of occasional overlapping openings. After patch formation, the normal bathing solution was replaced by external Tris-saline containing (in mM): Tris-Cl 140, KC! 4, CaC12 1, MgCI2 1, 10 HEPES, pH 7.2. Patch electrodes were filled with a solution of composition (in mM): Tris-C1 140, NaC1 3, MgC12 1, I 1 EGTA, 10 HEPES, pH 7.2. Since the impermeant cation Tris + replaced the majority of Na + and K + ions across the isolated patch, membrane currents carried by these ions are negligible compared to agonist induced chloride fluxes [1, 10]. Patches were voltage-clamped to a potential of - 8 0 mV. GABA (1.5-2/tM, Sigma) and taurine (2 - 4 0 / t M , Sigma) were dissolved in external Tris-saline and applied to the membrane patches by bath perfusion. Patch currents were low-pass filtered at 1 kHz and recorded on Wideband FM tape. Cumulative frequency distributions of single-channel open times were constructed. Since we wished to compare the average kinetic parameters for GABA and taurine induced channels, separate distributions were not made for each channel substate in the present work. Open time distributions were fitted by the sum of two exponential terms using the Gauss-Newton-Marquadt algorithm [3]. Histograms of the amplitudes of single-channel currents were constructed. These distributions were fit by the sum of 1, 2, 3 or 4 normal curves using a Simplex method. At a holding potential of - 80 mV, application of 1.5/~M GABA induced inwardly directed single-channel currents in 14/20 patches tested. In all 14 of these cases, application of taurine at concentrations exceeding 5/~M also induced inwardly directed single channel currents (Fig. 1). The remaining 6 patches were insensitive to both amino acids. This insensitivity may simply reflect the limited membrane sampling achieved using the outside-out patch technique. Essentially all spinal cord neurons in these cultures show sensitivity to GABA when tested at the whole-cell level [1]. In the case of currents induced by both GABA and taurine, it was found that all events reversed polarity to outward charge flow on displacing the patch potential beyond 0 mV. This result is consistent with previous reports that both agonists gate chloride-selective channels in cultured spinal cord cells [1]. Amplitude distributions for GABA induced currents were well fit by the sum of
231
TAURINE flier
11
--
I t,
--.
J.3
-
[4
"
' v-rl--~'aa~t-rl
T'T
]~"
'
i r "~,'t
'
" ! "
"
'
' W"
'W p
r 'r
I
Jl
0
""I,,~' ~' r~ FrrflN,pr,qp
*'*
,, I
, ~,~v-r
"writ"
I,
12 13
_42p
I,,
Fig. 1. Single-channel currents activated by bath application o f 4 0 / a M taurine to an outside-out patch of m e m b r a n e excised from a mouse spinal cord neuron grown in culture for 3 weeks. The patch was voltage clamped to a potential of - 80 mV. Downward deflections from baseline (0) denote inward m e m b r a n e current. Representative events reaching current levels I,, 12, 13, and 14 are shown.
3 Gaussian terms (Fig. 2A). The modal values of these Gaussians occurred at current levels corresponding to conductance states of 7.7+0.29, 16.8_ 1.44 and 26.5+_1.32 pS (mean+_ S.E.M., 5 patches). All 5 patches tested with GABA displayed a small number (less than 2%) of openings which were considerably larger than expected (Fig. 2A). These events probably correspond to the rare, 46 pS conductance level reported to occur in GABA-sensitive channels from cultured spinal neurons [2].
eo.A o
GABA
Taurine
B
45 30
a
30 0
15
15 Z -5.0
-4.0
-a.o
-Z.o
-t.o
C u r r e n t A m p l i t u d e (pA)
o
-5.0
-4.0
-$.0
-2.0
- 1.0
0
C u r r e n t A m p l i t u d e (pA)
Fig. 2. Amplitude distributions for single-channel currents activated by 1.5/aM G A B A (A) or 40 p M taufine (B) in the same outside-out patch of spinal cord cell membrane, voltage clamped at - 80 mV. These distributions were fit by the s u m o f 3 (A) or 4 (B) Gaussian terms (smooth curves). In A, the modal values o f the 3-fit Gaussians occurred at current levels o f - 0 . 6 , - 1.4 and - 2 . 0 pA. In B, modal values o f the 4-fit Gaussians occurred at current levels o f - 0 . 8 , - 1.4, - 2 . 4 and - 3 . 4 pA.
232 Amplitude distributions for taurine induced currents were well fit by the sum of 4 Gaussian terms (Fig. 2B). The modal values of the 4 Gaussian terms tit to these distributions occurred at current levels corresponding to conductance states of 8.3 _+0.47, 16.5_ 0.99, 28.3 +_ 1.29 and 46.0 _4_-1.83 pS (5 patches). Statistical comparison of these conductances showed that the 3 smaller levels did not differ significantly from values derived for GABA activated channels ( P > 0.05, Wilcoxon test). Therefore, the major difference between the conductance of channels activated by the two amino acids lay in the marked ability of taurine to trigger large, 46 pS events. Currents of this size made up an average of 29.5 +8.3% of events triggered by taurine in the test patches. In the case of both GABA and taurine, the distribution of open channel lifetimes was well fit by an equation of the form y = Aft,.e t/TAUf+ N~.. e-t/TAU~ Here, Nr and N, denote the number of events in the fast and slow fit components respectively. The mean open time, TAUmean of membrane channels activated by GABA or taurine was calculated using the relation WAUmean Nf/(Nr+NO T A U t + Ns/(Nf+Ns)" WAUs [3]. The mean lifetime of taurine induced channels, 1.0+0.07 ms, was significantly briefer than the corresponding value calculated for GABA-activated channels (TAU mean= 1.6+0.08 ms, P < 0 . 0 1 , 5 patches). Taurine induced channels were completely and reversibly abolished in the presence of strychnine sulphate (10/tM), but were not blocked by bicuculline methiodide (50 /~M, 5 patches tested with each antagonist). Both GABA and taurine were found to activate channels with multiple conductance states. The 3 lowest conductance levels of these channels are apparently identical. The mean values obtained for these conductance levels are in very good agreement with previously published results [2]. Taurine-activated channels frequently entered a high conductance state of 46 pS conductance. This state may also be entered by GABA activated channels, but with much lower probability. It is possible that taurine activates channels associated with GABA receptors, and that taurine stabilises these channels in the 46 pS conductance state. This seems unlikely, in view of the insensitivity of taurine-induced events to bicuculline. Alternatively, taurine may activate glycine receptors, which are known to control a chlorideselective channel of 46 pS conductance in cultured spinal cord neurons [2]. The present data are fully consistent with this possibility, since taurine-induced events were highly sensitive to blockade by strychnine. Recent data strongly suggest that at least 3 types of taurine receptors may exist in spinal cord tissue. Two receptor subtypes apparently mediate the depolarising and hyperpolarising responses to taurine seen in the dorsal roots [8]. Pharmacological experiments suggest that these receptors are distinct from both GABA and glycine receptors in the spinal cord. A third type of taurine receptor, which resembles the glycine receptor pharmacologically, is apparently found in the ventral roots of the cord [12, 13]. The present data suggest that this ventral root class of taurine receptor may also be expressed on spinal cord neurons in culture. :
233 I Barker, J.L. and Mathers, D.A., GABA analogues activate channels of different duration on cultured mouse spinal neurons, Science, 212 (I 98 l) 358-361. 2 Bormann, J., Hamill, O.P. and Sakmann, B., Mechanism of anion permeation through channels gated by glycine and y-aminobutyric acid in mouse cultured spinal neurones, J. Physiol. (Lond.), 385 (1987) 243-286. 3 Chow, P. and Mathers, D.A., Convulsant doses of penicillin shorten the lifetime of GABA induced channels in cultured central neurones, Br. J. Pharmacol., 88 (1986) 541-548. 4 Frederickson, R.C.A., Neuss, M., Morzorati, S.L. and McBride, W.J., A comparison of the inhibitory effects of taurine and GABA on identified Purkinje ceils and other neurons in the cerebellar cortex of the rat, Brain Res., 145 (1978) 117-126. 5 Haas, H.L. and Hosli, L., The depression of brain stem neurones by taurine and its interaction with strychnine and bicuculline, Brain Res., 52 (1973) 399-402. 6 Hamill, O.P., Marry, 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, Pfluegers Arch., 391 (1981) 85-100. 7 Huxtable, R.J., Franconi, F. and Giotti, A., The Biology of Taurine. Advances in Experimental Medicine, Vol. 217, Plenum, New York, 1987. 8 Kuba, Y., Akiyoshi, E. and Akagi, H., Identification of two taurine receptor subtypes on the primary afferent terminal of frog spinal cord, Br. J. Pharmacol., 94 (1988) 1051-1056. 9 Mathers, D.A. and Yoshida, H., The benzodiazepine triazolam: direct and GABA depressant effects at the membrane of cultured mouse spinal cord neurons, Eur. J. Pharmacol., 139 (1987) 53-60. 10 Mathers, D.A., Spontaneous and GABA-induced single channel currents in cultured murine spinal cord neurons, Can. J. Physiol. Pharmacol., 63 (1985) 1228-1233. 11 Mathers, D.A., The GABAA receptor: new insights from single-channel recording, Synapse, 1 (1987) 9(~101. 12 Nicoll, R.A., Padjen, A. and Barker, J.L., Analysis of amino acid responses on frog motoneurones, Neuropharmacol., 15 (1976) 45-53. 13 Oja, S.S. and Kontro, P., Taurine. In A. Lajtha (Ed.), Handbook of Neurochemistry, 2nd Edn., Plenum, New York, 1983, pp. 501-521. 14 Okamoto, K. and Sakei, Y., Augmentation by chlordiazepoxide of the inhibitory effects of taurine, fl-alanine and 7-aminobutyric acid on spike discharges in guinea pig cerebellar slices, Br. J. Pharmacol., 65 (1980) 277 285.