Properties of Human Brain Glycine Receptors Expressed in Xenopus oocytes Author(s): C. B. Gundersen, R. Miledi, I.

Parker Source: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 221, No. 1223 (Apr. 24, 1984), pp. 235-244 Published by: The Royal Society Stable URL: http://www.jstor.org/stable/35908 . Accessed: 14/07/2011 07:28
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Proc. R. Soc. Lond. B 221, 235-244 (1984) Printed in GreatBritain

Properties of human brain glycine receptors expressed in Xenopus oocytes

BY C. B.
GUNDERSEN,

R. MILEDI, F.R.S., AND I. PARKER

Departmentof Biophysics, UniversityCollegeLondon, GowerStreet, London WC1E6BT, U.K. (Received20 January 1984) Glycine and y-aminobutyric acid (GABA) receptors from the foetal human brain were 'transplanted' into the Xenopu8oocyte membraneby injecting the oocytes with poly(A)+-mRNA extracted from the cerebral cortex. Activation of both glycine and GABAreceptorsinducedmembrane currents carried largely by chloride ions. However, unlike the GABAactivated current, the glycine current was blocked by strychnine, and was not potentiated by barbiturate. At low doses, the glycine current increasedwith concentration following a 2.7th power relation, suggesting that binding of three moleculesof glycine may be requiredto open a single membranechannel. The currentinduced by steady application of glycine decreased with hyperpolarizationbeyond about -60 mV.
INTRODUCTION

Xenopus oocytes are very useful for the study of transmitter receptors, not only because they have native receptors (Kusano et al. I982) but, more importantly, because the injection of appropriateexogenous messengerRNA (mRNA) into the oocyte leads to the synthesis and incorporation into the membrane of foreign receptor-channel complexes. This 'transplantation' of receptors is particularly useful to study receptors pertaining to rather inaccessible cells, like those in the brain. For example, mRNA derived from the chick optic lobe induced the oocyte to acquire receptors to y-aminobutyricacid (GABA) (Milediet al. i982), rat brain mRNA induced receptors to serotonin, glutamate, kainate, acetylcholine and GABA (Gundersen et al.
I983

a,

i984

a), and a mRNA derived from foetal human

cerebralcortex induced receptorsto kainate and serotonin (Gundersenet al. i 984 b). We have now found that a different preparation of mRNA from foetal human brain induces, in addition, functional receptors for glycine and GABA.
METHODS

Procedures for the extraction of poly(A)+-mRNA and its injection into oocytes
of Xenopus laevis, were as described previously (Gundersen et al. I983 b; Miledi &

Sumikawa I982). The messenger RNA was extracted from the cerebral cortex of a 15-week-old foetus, obtained after termination of pregnancy with prostaglandin. Electrophysiological recording techniques were also as used before
[ 235 ]

236

C. B. Gundersen, R. Miledi and I. Parker

(Kusano et al. I982; Miledi I982). Briefly, oocytes were voltage clamped, and perfused continuously with Ringer solution (composition; NaCl, 120 mM; KCl, 2 mm; CaC12, 1.8 mm; HEPES, 5 mm, at pH 7.2) at room temperature (20-23 ?C). Drugs were applied in the perfusion solution, and the resulting membrane currents were recorded.
RESULTS

Membrane channels induced by mRNA from human cerebral cortex Injection of mRNA derived from the cerebral cortex of a 15-week-old foetus into Xenopus oocytes caused the synthesis and incorporation into the membrane of several types of drug- and voltage-activated channels. These included voltageactivated sodium channels, and channels activated by serotonin and kainate, which we have described previously (Gundersen et al. i984 b). In addition'ito these, the present messenger preparation induced the formation of channels activated by glycine and, in a few ooctyes, sensitivity to GABA was also observed (figure 1).
1.

TABLE

DRUG-

AND VOLTAGE-OPERATED WITH

MEMBRANE

CURRENTS

IN XENOPUS CORTEX

OOCYTES

INJECTED

mRNA

FROM FOETAL

HUMAN

CEREBRAL

(Figures give peak currents in nanoamperes (mean plus standard error of mean) and number of oocytes in brackets. Control (non-injected) and injected oocytes were all from the same donor. In the case of drug activated currents, measurements were made at a clamp potential of -100 mV, and the drug concentration was 10-3 M. The voltage activated sodium current was measured as the peak inward current elicited by depolarization to -10 from -100 mV. Similarly, the calcium activated transient outward (Tout)current, and the potassium current were measured for depolarizations to 0 and + 30 mV respectively (see Gundersen et al. 1983 b, I984b for further details). Sensitivity to serotcnin was not systematically tested, but a few injected oocytes examined all gave large responses.) control glycine kainate sodium potassium
Rout

human cortex mRNA 242 s.e. 105 (7) 10 s.e. 1.4 (4) 68 s.e. 26 (5) 0 (5) 9 s.e. 2.5 (5)

1.5 s.e. 0.4 (5) 0 (4) 0 (5) 0 (5) 16 s.e. 4 (4)

Control (non-injected) oocytes showed either no response to high concentrations (1 mm) of glycine, or just detectable inward currents of 1-2 nA. In contrast, oocytes injected with human cortex mRNA gave large responses to 1 mm glycine, often with peak amplitudu of several hundred nA (table 1). The response to glycine was still present in oocytes which had been treated with collagenase to remove the follicular and other enveloping cells, which indicates that the glycine sensitivity resides in the oocyte membrane itself. Furthermore, sensitivity to glycine still ,Ug ml-') to inhibit developed in oocytes which were exposed to actinomycin D (50 transcription of mRNA from the oocyte's own genome. Thus, the membrane channels activated by glycine almost certainly arose as a result of translation of the human messenger RNA.

Human glycine receptors in Xenopus oocytes

Glycine and GABA responses from injected oocytes

237

Application of glycine to oocytes injected with the mRNA consistently elicited smooth inward membrane currents, and a few oocytes also showed similar currents in response to GABA (figure 1). The receptors underlying these responses were pharmacologically distinct. For example, the response to glycine was substantially reduced by 20 ,UM strychnine (figure 1 a-c), while the response to GABA was not appreciably changed by this concentration of strychnine. Also, the membrane current elicited by GABA was potentiated by barbiturate, similar to the potentiation of GABA responses induced by chick brain mRNA (Smart et al. I983) and

(a)
A

(bl
A

(c)
A

(d)
A

ge)
A

lycine

20nA (a)- (e) AA (f)- (g)

100 s

(f)
A

(g)
A

GABA

normal Ringer pentobarbital FIGURE 1. Membrane currents elicited by glycine and GABA in an oocyte injected with human cerebral cortex mRNA. Upward deflexions of the traces correspond to inward membrane currents. Drugs were applied by bath perfusion. Holding potential, -100 mV. Temperature, 23 ?C. (a)-(c) Response to glycine, and the blocking of the response by strychnine. In each frame, 10-3 Mglycine was applied as indicated by the arrows. Traces show: (a) response in normal Ringer, (b) response after adding 20 IgM strychnine, and (c) response 12 min after washing out the strychnine. (d)-(g) Effect of pentobarbital on the responses elicited by 3 x O-4 glycine (d), (e) and 10-4 GABA (f), (g). The records on the left (d) and (f) were obtained in normal Ringer, while those on the right (e) and (g) were obtained in the presence of 100 gm pentobarbital.

rat brain mRNA (C. B. Gundersen, R. Miledi and I. Parker, unpublished), while the glycine response changed little. Figure If, g illustrates an oocyte where the response to 10-4 M GABA was potentiated by a factor of about four times by 100 gM pentobarbital. The response to glycine, however, was not appreciably altered (figure 1 d, e). We have also recently recorded similar glycine and GABA responses from oocytes injected with mRNA derived from adult rat brain, in addition to the various other receptor types previously described (Gundersen et al. i 983 a, I 984 a).

238

C. B. Gundersen, R. Miledi and I. Parker

Effect of membrane potential on the glycine-activated current The current elicited by bath application of glycine became smaller as the membrane was depolarized, and inverted direction at about -22 mV in the oocyte illustrated in figures 2 and 3. A mean value for the reversal potential, obtained mV (+s.e.m.), which corresponds to the from four oocytes, was -22.7+0.9 chloride equilibrium potential in Xenopu-s oocytes (Kusano et al. I982). Furthermore, the currents elicited in the same oocytes by activation of muscarinic ACh receptors and receptors to serotonin and GABA, all of which are caused by increases in chloride conductance (Kusano et al. I982; Gundersen et al. I983a; Miledi et al. I982), reversed at about the same potential level as the glycine response. This indicates that chloride is the main ion permeating the membrane channels opened by the combination of glycine with the receptors.

20 nA 100 s -10 -50 mV 0

-30

-20

2. FIGURE Reversalpotential of the glycine-activatedcurrent.Recordsshow currentselicited was by 3 x TO-4Mglycineappliedwhile the membrane clampedto the potentialsindicated (in millivolts). Figure 3 shows the currents elicited by a constant dose of glycine (3 x 10' m) applied with the membrane potential clamped to different levels. Between about 0 and -30 mV the current/voltage relation was nearly linear, but as the potential was made more negative the current reached a maximal value at about -70 mV, and then declined with further polarization. Concentration dependence of the glycine-activated current In the oocyte illustrated in figure 4, responses first became detectable at a glycine concentration of about 7 x 10-5 M, and then increased in size very steeply with increasing dose. At low concentrations, a doubling in dose gave more than a fourfold increase in peak current (figure 4a-d). However, 2 x 103 M glycine gave a nearly maximal response, and a further increase to 10-2 M gave little additional increase (figure 4f, g). The membrane current was well maintained throughout the duration of drug application at concentrations below about 2 x 10-4 M, but with higher doses the response declined progressively more rapidly. The relation between peak response size and glycine concentration, measured

Human glycine receptors in Xenopus

oocytes
_ 100

239

A A

~~~~A
f.
A

- 75

150 25 \lOA
A

1 i.

-120

-80

-40

membrane potential/mV

-50

FIGURE

3. Current/voltagerelation of the glycine response.Points show the peak membrane currentselicited by 3 x 10-4 M glycine at each potential. Same oocyte as figure2.

from one oocyte, is plotted on double logarithmic coordinates in figure 4h. The data points at concentrations below about 5 x 10-4 M fit well to a straight line, with a slope of 2.7. Thus, for low doses of glycine, the response size increases as the 2.7th power of the concentration. Similar measurements from five other oocytes injected with human cortex mRNA gave an overall mean slope value of 2.64 + 0.15 at low concentrations of glycine, with a range between 2.2 and 3.1 in different oocytes. Voltagejump relaxations To obtain information about the kinetics of the channels activated by glycine, we examined the relaxation of the clamp currents following step changes in membrane potential during steady application of glycine. This technique has been used to study acetylcholine activated channels in muscle (Neher & Sakmann 1975; Adams I 977), where the current was found to relax exponentially to a new steady value, with a time constant corresponding to the mean channel lifetime at the potential following the step. Figure 5 illustrates the membrane currents recorded when the potential was stepped to more negative values from a holding potential of -60 mV. In the absence of glycine, the current steps were rectangular, and varied linearly with potential (figure 5a). However, the same potential steps applied in the presence of glycine (5 x 10-4 m) gave an initially larger current, which was followed by a slow decline (figure 5b). As the potential was made more negative the initial current increased, but the decline became faster and at - 160 mV the current remaining at the end of the pulse was smaller than during the same pulse in the absence of glycine. The time course of current relaxations during glycine application are plotted on semi-logarithmic coordinates in figure 5 c. At potentials between -80 and

240

C. B. Gundersen, R. Miledi and I. Parker

(a)
A

(
A

(c)
A

(d)
A

i0-4 M

1.5X10M

2X 10 4M

3x 14 M

- 20nA(a)- (d) 100 nA (e) -(g) 100 s (e)

A

(f)
A

(g)
A

10-3M

2X 103M

10-2M

1000

(h)
A

A.
A

100

00

10A

l
10o4

!
io03 10-2

[glycine]/M

FIGuRE4. (a)-(g) Membrane currents elicited by different concentrations of glycine. All records were obtained at a potential of - 100 mV. Note the change in gain between the upper and lower sets of records. (h) Dose-response relation for the glycine activated current, plotted on double logarithmic coordinates. All measurements were made from the same oocyte as (a)-(g), which was clamped at -100 mV.

Human glycine receptors in Xenopus

(a) 10 nA (b) -160 mV

oocytes

241

100msO100

(c) 100

l~~~~~~~
50
-A

A AA

~-80
A

mV

200

AVA

v
A Ak V

_ 10 5~~~~~

200 40A A

A0
V

-160OmV A -140 mV-120 mV

5
[II

200

400

time/ms FIGURE Current relaxations following voltage steps imposed during glycine application. 5. (a), (b) Traces show membrane currents elicited by hyperpolarizing pulses applied from a holding potential of -60 mV. Records in (a) were obtained in normal Ringer, at potentials of (from bottom to top) -80, - 100, - 120, -140 and - 160 mV. Records in (b) were from the same oocyte, but obtained at the peak of the response elicited by 5 x 10-4 M glycine. At -60 mV the maximal glycine induced current was 87 nA. Pulse potentials are indicated in millivolts next to the traces. Temperature, 23 'C. (c) Decay of the membrane current during hyperpolarizing pulses in the presence of 5 X 10-4 M glycine, plotted on semi-logarithmic coordinates. Data from the same oocyte as (a), (b). Potentials during the pulse are indicated next to the data. Holding potential was -60 mV. Decay time constants are: -80 mV, 164 ms; -100 mV, 147 ms; -120 mV, 129 ms; -140 mV, 106 ms; -160 mV, 91 ms.
- 160 mV the points lie on straight lines, indicating that the current relaxations largely follow single exponential time courses. The time constants derived from the lines became shorter at more negative potentials, and for the oocyte illustrated the time constant was 164 ms at -80 mV, and 91 ms at - 160 mV (temperature 23 ?C).

242

C. B. Gundersen, R. Miledi and I. Parker

The current at the beginning of the voltage steps in figure 5 increased linearly with polarization (figure 6), while the steady state current measured at the end of the pulse declined as the potential was made more negative than about -80 mV (figure 6 and see also figure 3). A likely explanation for this is that the current flowing through single glycine-activated channels shows an ohmic behaviour (that is, the conductance is independent of voltage), but the relative number of open channels decreases with time when the potential is made more negative.

300
A

XA

1200

100

-160

-120 -80 -40 membrane potential/mV

FIGURE

6. Current/voltagerelations of the instantaneous(A) and steady state (A) current activated by glycine. Data from the same oocyte as figure 6, using a glycine concentration of 5 x 10-4 M. Steady state current was measured at the end of 3 s duration pulses. Instantaneous current was measured at the onset of the pulses. In both cases, correction was made for the passive leakage current of the oocyte. The reversal potential (star) was directly measured, as in figure 2.
DISCUSSION

The properties of 'human brain' glycine- and GABA-activated channels transplanted into the Xenopus oocyte membrane show many similarities with those of (native) glycine channels in the neurons of the rat and cat central nervous system. For example, in both neurons and oocytes glycine- and GABA-activated currents are carried largely by chloride ions (see, for example, Barker & Ransom I978 a) although, of course, the responses in the neurons are inhibitory, rather than depolarizing as in the oocyte, because of the more negative equilibrium potential for chloride. Also, the neuronal glycine channels are blocked selectively by

Human glycine receptors in Xenopus

oocytes

243

strychnine (Barker et al. I 983), while the GABA channels are selectively potentiated by barbiturates (Barker & Ransom I978 b). However, a marked difference is apparent in estimates of the lifetime of the glycine-activated channels. Using noise analysis, Barker et al. (I982) estimated the mean lifetime of glycine channels in mouse spinal neurons as about 8 ms, and found little voltage dependence of the lifetime. In contrast, the channel lifetime in the oocyte, derived from voltage jump experiments, was much longer (160 ms at -80 mV) and became shorter with hyperpolarization. Although the relaxation in current following voltage steps probably reflects the channel lifetime, other factors, such as channel blocking or a slow decrease in single channel conductance may also contribute significantly. We hope to investigate the properties of the single glycine channel in more detail using noise analysis and patch clamp recording (Miledi et al. I983). However, the presence of a variety of spontaneously active membrane channels in the oocyte complicates single channel recording. An interesting feature of the human glycine receptor-channel complex induced in the oocyte is its steep concentration dependence. At low doses, the current activated by glycine increases roughly as the 2.7th power of the concentration, which suggests that at least three molecules of glycine must be simultaneously bound to the receptor sites to open their associated ion channel. This concentration dependence is much steeper than for the activation of chick brain GABA receptors (Miledi et al. I982) and human and rat kainate receptors (Gundersen et al. I984 a, b) induced in the oocyte. In these cases, channel activation increases as the square, or less, of agonist concentration, suggesting that binding of two molecules of agonist may be required to open the channel. An important consequence of this high degree of cooperativity is that the inhibitory action of glycine on brain neurons is expected to be steeply graded over a narrow concentration range. For example, a concentration of 10-4 M in the synaptic cleft would give little effect, while 10-3 M would give almost maximal inhibition. Perhaps the most important feature of the present results is the demonstration that functional glycine- and GABA-activated membrane channels can be transplanted from the human brain into the Xenopus oocyte. The use of this simplified system should greatly facilitate the study of these receptor-channel complexes, and their modulation by convulsant and depressant drugs. We are grateful to Sister J. McPherson for help in obtaining the foetal material. and to Louise Morgan and Patricia Harkness for technical assistance. This work was supported by the M.R.C. and the Royal Society.
REFERENCES

Adams, P. R. I977 Relaxation experiments using bath applied suberyldicholine. J. Phy8iol., Lond. 268, 271-289. Barker, J. L., McBurney, R. N. & Macdonald, J. F. I 982 Fluctuation analysis of neutral amino acid responses in cultured mouse spinal neurones. J. Phy8iol., Lond. 322, 365-387. Barker, J. L., McBurney, R. N. & Mathers, D. A. I 983 Convulsant induced depression of amino acid responses in cultured mouse spinal neurones studied under voltage clamp. Br. J. Pharmac. 80, 619-629.

244

C. B. Gundersen, R. Miledi and I. Parker

Barker, J. L. & Ransom, B. R. 1978a Amino acid pharmacology of mammalian central neurones grown in tissue culture. J. Physiol., Lond. 280, 331-354. Barker, J. L. & Ransom, B. R. 1978b Pentobarbitone pharmacology of mammalian central neurones grown in tissue culture. J. Physiol., Lond. 280, 355-372. Gundersen, C. B., Miledi, R. & Parker, I. I983 a Serotonin receptors induced by exogenous messenger RNA in Xenopus oocytes. Proc. R. Soc. Lond. B 219, 103-109. Gundersen, C. B., Miledi, R. & Parker, I. I 983 b Voltage-operated channels induced by foreign messenger RNA in Xenopus oocytes. Proc. R. Soc. Lond. B 220, 131-140. Gundersen, C. B., Miledi, R. & Parker, I. I984a Glutamate and kainate receptors induced by rat brain messenger RNA in Xenopus oocytes. Proc. R. Soc. Lond. B (In the press.) Gundersen, C. B., Miledi, R. & Parker, I. i984b Messenger RNA from human brain induces drug- and voltage-operated channels in Xenopus oocytes. Nature, Lond. (In the press.) Hamill, 0. P., Bormann, J. & Sakmann, A. I983 Activation of multiple-conductance state chloride channels in spinal neurones by glycine and GABA. Nature, Lond. 305, 805-808. Kusano, K., Miledi, R. & Stinnakre, J. I982 Cholinergic and catecholaminergic receptors in the Xenopus oocyte membrane. J. Physiol., Lond. 328, 143-170. Miledi, R. 1982 A calcium-dependent transient outward current in Xenopus laevis oocytes. Proc. R. Soc. Lond. B 215, 491-497. Miledi, R., Parker, I. & Sumakawa, K. I982 Synthesis of chick brain GABA receptors by frog oocytes. Proc. R. Soc. Lond. B 266, 509-515. Miledi, R., Parker, I. & Sumikawa, K. I983 Recording of single gamma-amino-butyrate and acetylcholine activated channels translated by exogenous messenger RNA in Xenopus ooctyes. Proc. R. Soc. Lond. B 218, 481-484. Miledi, R. & Sumikawa, K. 1982 Synthesis of cat muscle acetylcholine receptors by Xenopus oocytes. Biomed. Res. 3, 390-399. Neher, E. & Sakmann, B. 1975 Voltage dependence of drug-induced conductance in frog neuromuscular junction. Proc. natn. Acad. Sci. U.S.A. 72, 2140-2144. Smart, T. G., Constanti, A., Bilbe, G., Brown, D. A. & Barnard, E. A. I983 Synthesis of functional chick brain GABA-benzodiazapine-barbiturate/receptor complexes in mRNA injected Xenopus oocytes. Neurosci. Lett. 40, 55-59.

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Number 1223

CONTENTS C. B., MILEDI, R. & PARKER, I. Glutamate and kainate receptors induced by rat brain messenger RNA in Xenopus oocytes CHUNG, S. H. & JOHNSON, MELANIE S. Studies on sound-induced epilepsy in mice ROUND, F. E. & CRAWFORD, R. M. The lines of evolution of the Bacillariophyta. II. The centric series. [Plates 1-7]
GUNDERSEN, HILDRETH, ELLEN

pages 127-143 145-168 169-188

C.

189-220 221-233

The computation of the velocity field. [Plate 1] BEDFORD, J. M., RODGER, J. C. & BREED, W. G. Why so many mammalian spermatozoa - a clue from marsupials? [Plate 1] GUNDERSEN, C. B., MILEDI, R. & PARKER, I. Properties of human brain glycine receptors expressed in Xenopus oocytes

235-244

Published by the Royal Society, 6 Carlton House Terrace, London SW1 Y 5AG Printed in GreatBritain for the Royal Society at the University Press, Cambridge