gly paper 1

8/6/2019 Gly paper 1

1/15

Properties of Human Brain Glycine Receptors Expressed in Xenopus oocytesAuthor(s): C. B. Gundersen, R. Miledi, I. ParkerSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 221, No.1223 (Apr. 24, 1984), pp. 235-244Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/35908 .

Accessed: 14/07/2011 07:28

Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp. JSTOR's Terms and Conditions of Use provides, in part, that unlessyou have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you

may use content in the JSTOR archive only for your personal, non-commercial use.

Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at .http://www.jstor.org/action/showPublisher?publisherCode=rsl. .

Each copy of any part of a JSTOR transmission must contain the same copyright notice that appears on the screen or printed

page of such transmission.

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of

content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms

of scholarship. For more information about JSTOR, please contact [email protected].

The Royal Society is collaborating with JSTOR to digitize, preserve and extend access to Proceedings of the

Royal Society of London. Series B, Biological Sciences.

http://www.jstor.org
http://www.jstor.org/action/showPublisher?publisherCode=rslhttp://www.jstor.org/stable/35908?origin=JSTOR-pdfhttp://www.jstor.org/page/info/about/policies/terms.jsphttp://www.jstor.org/action/showPublisher?publisherCode=rslhttp://www.jstor.org/action/showPublisher?publisherCode=rslhttp://www.jstor.org/page/info/about/policies/terms.jsphttp://www.jstor.org/stable/35908?origin=JSTOR-pdfhttp://www.jstor.org/action/showPublisher?publisherCode=rsl


2/15

Proc. R. Soc. Lond. B 221, 235-244 (1984)Printed in GreatBritain

Properties of human brain glycine receptors expressedin Xenopus oocytesBY C. B. GUNDERSEN, R. MILEDI, F.R.S., AND I. PARKER

Departmentof Biophysics, UniversityCollegeLondon,GowerStreet,LondonWC1E6BT, U.K.(Received20 January 1984)

Glycine and y-aminobutyric acid (GABA) receptors from the foetalhuman brainwere 'transplanted' into the Xenopu8oocyte membranebyinjecting the oocytes with poly(A)+-mRNAextracted from the cerebralcortex.Activation ofbothglycine andGABAreceptors nducedmembranecurrents carried largely by chloride ions. However, unlike the GABA-activated current, the glycine current was blocked by strychnine, andwas not potentiated by barbiturate. At low doses, the glycine currentincreasedwith concentrationfollowinga 2.7th powerrelation,suggestingthat bindingof threemoleculesof glycinemay berequired o open a singlemembranechannel. The current nducedby steady applicationof glycinedecreased with hyperpolarizationbeyond about -60 mV.INTRODUCTION

Xenopus oocytes are very useful for the study of transmitter receptors,not onlybecause they have native receptors (Kusano et al. I982) but, more importantly,becausethe injectionof appropriateexogenous messengerRNA (mRNA) into theoocyte leads to the synthesis and incorporation into the membrane of foreignreceptor-channel complexes. This 'transplantation' of receptors is particularlyuseful to study receptorspertaining to rather inaccessible cells, like those in thebrain. For example, mRNA derived from the chick optic lobe induced the oocyteto acquirereceptorsto y-aminobutyricacid (GABA)(Milediet al. i982), rat brainmRNA induced receptors to serotonin, glutamate, kainate, acetylcholine andGABA (Gundersen et al. I983 a, i984 a), and a mRNA derived from foetal humancerebralcortex inducedreceptorsto kainate andserotonin(Gundersen t al. i 984 b).We have now found that a different preparationof mRNA from foetal humanbrain induces, in addition, functional receptorsfor glycine and GABA.

METHODSProcedures for the extraction of poly(A)+-mRNA and its injection into oocytesof 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 prosta-glandin. Electrophysiological recording techniques were also as used before[ 235 ]


3/15


4/15

Human glycine receptors in Xenopus oocytes 237Glycine and GABA responses from injected oocytes

Application of glycine to oocytes injected with the mRNA consistently elicitedsmooth inward membrane currents, and a few oocytes also showed similar currentsin response to GABA (figure 1). The receptors underlying these responses werepharmacologically distinct. For example, the response to glycine was substantiallyreduced by 20 ,UMstrychnine (figure 1a-c), while the response to GABA was notappreciably changed by this concentration of strychnine. Also, the membranecurrent elicited by GABA was potentiated by barbiturate, similar to the poten-tiation of GABA responses induced by chick brain mRNA (Smart et al. I983) and

(a) (bl v (c)A A A

v v

(d) ge) lycine 20nA (a)- (e)A A AA(f)- (g)(f) (g) GABA 100 s

A A

normal Ringer pentobarbitalFIGURE 1. Membrane currents elicited by glycine and GABA in an oocyte injected with humancerebral cortex mRNA. Upward deflexions of the traces correspond to inward membranecurrents. Drugs wereapplied by bath perfusion. Holding potential, -100 mV. Temperature,23 ?C.(a)-(c) Response to glycine, and the blocking of the response by strychnine. In eachframe,10-3 Mglycine was applied as indicated by the arrows. Traces show: (a) response in normal

Ringer, (b) response after adding 20 IgMtrychnine, and (c) response 12 min after washingout the strychnine.(d)-(g) Effect of pentobarbital on the responses elicited by 3 x O-4 glycine (d), (e) and10-4 GABA (f), (g).The records on the left (d)and (f) were obtained in normal Ringer, whilethose 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), whilethe glycine response changed little. Figure If, g illustrates an oocyte where theresponse to 10-4 M GABA was potentiated by a factor of about four times by 100 gMpentobarbital. The response to glycine, however, was not appreciably altered(figure 1d, e). We have also recently recorded similar glycine and GABA responsesfrom oocytes injected with mRNA derived from adult rat brain, in addition to thevarious other receptor types previously described (Gundersen et al. i 983 a, I984 a).


5/15

238 C. B. Gundersen, R. Miledi and I. ParkerEffect of membrane potential on the glycine-activated current

The current elicited by bath application of glycine became smaller as themembrane was depolarized, and inverted direction at about -22 mV in the oocyteillustrated in figures 2 and 3. A mean value for the reversal potential, obtainedfrom four oocytes, was -22.7+0.9 mV (+s.e.m.), which corresponds to thechloride equilibrium potential in Xenopu-soocytes (Kusano et al. I982). Further-more, the currents elicited in the same oocytes by activation of muscarinic AChreceptors and receptors to serotonin and GABA, all of which are caused byincreases in chloride conductance (Kusano et al. I982; Gundersen et al. I983a;Miledi et al. I982), reversed at about the same potential level as the glycineresponse. This indicates that chloride is the main ion permeating the membranechannels opened by the combination of glycine with the receptors.

20 nA100 s

-10 0-50 mV -30 -20

FIGURE. Reversalpotentialof the glycine-activated urrent.Recordsshow currentselicitedby 3 x TO-4Mglycineappliedwhile the membranewasclamped o the potentials ndicated(inmillivolts).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 about0 and -30 mV the current/voltage relation was nearly linear, but as the potentialwas 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 currentIn 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 withincreasing dose. At low concentrations, a doubling in dose gave more than afourfold increase in peak current (figure 4a-d). However, 2 x 103 M glycine gavea nearly maximal response, and a further increase to 10-2 M gave little additionalincrease (figure 4f, g). The membrane current was well maintained throughout theduration of drug application at concentrations below about 2 x 10-4 M, but withhigher doses the response declined progressively more rapidly.The relation between peak response size and glycine concentration, measured


6/15

Human glycine receptors in Xenopus oocytes 239_ 100

A - 75A ~~~~A, f. 150

A 25

I 1 , 1 . I . I \l _OA-120 -80 -40 A 0membranepotential/mV | t

-50

FIGURE 3. Current/voltage elation of the glycineresponse.Points show the peak membranecurrentselicitedby 3 x 10-4 M glycineat each potential.Sameoocyte as figure2.from one oocyte, is plotted on double logarithmic coordinates in figure 4h. Thedata points at concentrations below about 5 x 10-4 M fit well to a straight line, witha slope of 2.7. Thus, for low doses of glycine, the response size increases as the 2.7thpower of the concentration. Similar measurements from five other oocytes injectedwith human cortex mRNA gave an overall mean slope value of 2.64 + 0.15 at lowconcentrations of glycine, with a range between 2.2 and 3.1 in different oocytes.

Voltagejump relaxationsTo 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 beenused to study acetylcholine activated channels in muscle (Neher & Sakmann 1975;Adams I 977), where the current was found to relax exponentially to a new steadyvalue, with a time constant corresponding to the mean channel lifetime at thepotential following the step.Figure 5 illustrates the membrane currents recorded when the potential wasstepped to more negative values from a holding potential of -60 mV. In theabsence of glycine, the current steps were rectangular, and varied linearly withpotential (figure 5a). However, the same potential steps applied in the presenceof glycine (5 x 10-4 m) gave an initially larger current, which was followed by a slowdecline (figure 5b). As the potential was made more negative the initial currentincreased, but the decline became faster and at - 160 mV the current remainingat the end of the pulse was smaller than during the same pulse in the absence ofglycine.

The time course of current relaxations during glycine application are plotted onsemi-logarithmic coordinates in figure 5c. At potentials between -80 and


7/15

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

(a) ( (c) (d)A A A A

i0-4 M 1.5X10M 2X 10 4M 3x 14 M

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

(e) (f) (g)A A A10-3M 2X 103M 10-2M

1000 (h) A.AA

100

o A

00

10A

l !10o4 io03 10-2

[glycine]/MFIGuRE4. (a)-(g) Membrane currents elicited by different concentrations of glycine. All recordswere obtained at a potential of - 100 mV. Note the change in gain between the upper andlower sets of records. (h) Dose-response relation for the glycine activated current, plottedon double logarithmic coordinates. All measurements were made from the same oocyte as

(a)-(g), which was clamped at -100 mV.


8/15


9/15

242 C. B. Gundersen, R. Miledi and I. ParkerThe 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 endof 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 currentflowing through single glycine-activated channels shows an ohmic behaviour (thatis, the conductance is independent of voltage), but the relative number of openchannels decreases with time when the potential is made more negative.

A

300A

XA 1200

A

, 100

I I I I I I I *-160 -120 -80 -40 0membrane potential/mV

FIGURE 6. Current/voltagerelationsof the instantaneous(A) and steady state (A) currentactivated by glycine. Data from the same oocyte as figure 6, using a glycine concentrationof 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, correctionwas made for the passive leakage current of the oocyte. The reversal potential (star) wasdirectly measured, as in figure 2.

DISCUSSIONThe properties of 'human brain' glycine- and GABA-activated channels trans-

planted 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 currentsare carried largely by chloride ions (see, for example, Barker & Ransom I978 a) -although, of course, the responses in the neurons are inhibitory, rather thandepolarizing as in the oocyte, because of the more negative equilibrium potentialfor chloride. Also, the neuronal glycine channels are blocked selectively by


10/15

Human glycine receptors in Xenopus oocytes 243strychnine (Barker etal. I 983), while the GABA channels are selectively potentiatedby barbiturates (Barker & Ransom I978 b). However, a marked difference isapparent in estimates of the lifetime of the glycine-activated channels. Using noiseanalysis, Barker et al. (I982) estimated the mean lifetime of glycine channels inmouse spinal neurons as about 8 ms, and found little voltage dependence of thelifetime. In contrast, the channel lifetime in the oocyte, derived from voltage jumpexperiments, was much longer (160 ms at -80 mV) and became shorter withhyperpolarization.

Although the relaxation in current following voltage steps probably reflects thechannel lifetime, other factors, such as channel blocking or a slow decrease in singlechannel conductance may also contribute significantly. We hope to investigate theproperties of the single glycine channel in more detail using noise analysis and patchclamp recording (Miledi et al. I983). However, the presence of a variety ofspontaneously active membrane channels in the oocyte complicates single channelrecording.An interesting feature of the human glycine receptor-channel complex inducedin the oocyte is its steep concentration dependence. At low doses, the currentactivated by glycine increases roughly as the 2.7th power of the concentration,which suggests that at least three molecules of glycine must be simultaneouslybound to the receptor sites to open their associated ion channel. This concentrationdependence is much steeper than for the activation of chick brain GABA receptors(Miledi etal. I982) and human and rat kainate receptors (Gundersen etal. 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 ofagonist may be required to open the channel. An important consequence of thishigh degree of cooperativity is that the inhibitory action of glycine on brainneurons is expected to be steeply graded over a narrow concentration range. Forexample, 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 demonstrationthat functional glycine- and GABA-activated membrane channels can be trans-planted from the human brain into the Xenopus oocyte. The use of this simplifiedsystem 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 workwas supported by the M.R.C. and the Royal Society.

REFERENCESAdams, P. R. I977 Relaxation experiments using bath applied suberyldicholine. J. Phy8iol.,Lond. 268, 271-289.Barker, J. L., McBurney, R. N. & Macdonald, J. F. I982 Fluctuation analysis of neutral aminoacid 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 aminoacid responses in cultured mouse spinal neurones studied under voltage clamp. Br. J.Pharmac. 80, 619-629.


11/15

244 C. B. Gundersen, R. Miledi and I. ParkerBarker, J. L. & Ransom, B. R. 1978a Amino acid pharmacology of mammalian centralneurones grown in tissue culture. J. Physiol., Lond. 280, 331-354.Barker, J. L. & Ransom, B. R. 1978b Pentobarbitone pharmacology of mammalian centralneurones 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 foreignmessenger 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 byrat 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 inducesdrug- and voltage-operated channels in Xenopus oocytes. Nature, Lond. (In the press.)Hamill, 0. P., Bormann, J. & Sakmann, A. I983 Activation of multiple-conductance statechloride channels in spinal neurones by glycine and GABA. Nature, Lond. 305, 805-808.Kusano, K., Miledi, R. & Stinnakre, J. I982 Cholinergic and catecholaminergic receptors inthe Xenopus oocyte membrane. J. Physiol., Lond. 328, 143-170.Miledi, R. 1982 A calcium-dependent transient outward currentin 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 frogoocytes. Proc. R. Soc. Lond. B 266, 509-515.Miledi, R., Parker, I. & Sumikawa, K. I983 Recording of single gamma-amino-butyrate andacetylcholine activated channels translated by exogenous messenger RNA in Xenopusooctyes. Proc. R. Soc. Lond. B 218, 481-484.Miledi, R. & Sumikawa, K. 1982 Synthesis of cat muscle acetylcholine receptors by Xenopusoocytes. Biomed. Res. 3, 390-399.Neher, E. & Sakmann, B. 1975 Voltage dependence of drug-induced conductance in frogneuromuscular 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 offunctional chick brain GABA-benzodiazapine-barbiturate/receptor complexes in mRNAinjected Xenopus oocytes. Neurosci. Lett. 40, 55-59.


12/15


13/15

PROCEEDINGS AND PHILOSOPHICAL TRANSACTIONSOF THE ROYAL SOCIETY

Notice to contributorsThe Royal Society welcomes suitable communications for publication in itsscientificjournals: papers estimatedto occupy up to 24 printed pages are con-sidered for the Proceedingsnd longer papers and those with numerous or largeillustrations for the Philosophicalransactions.Detailed advice on the preparation of papers to be submitted to the Societyis given in a leaflet available from the ExecutiveSecretary,The Royal Society,6 Carlton House Terrace, London SWIY 5AG. The 'Instructions to authors' arealso printed in every fifth volume of the Proceedings and B (volume numbersending in o or 5). The basic requirementsare: a paper should be as concise asits scientific content allowsand grammatically correct;standardnomenclature,units and symbolsshould be used; the text (including the abstract, the list ofreferences and figure descriptions) should be in double spaced typing on oneside of the paper. A leaflet giving detailed advice on the preparationof illus-trations is availablefromthe Executive Secretary; diagramsshould be expertlydrawn at about twice the proposedfinal size, preferablywith lettering in thecorrect style but if this is not possible the lettering should be inserted not onthe original drawings but on a set of copies; where photographs are essentialthe layout should be designed to give the most effective presentation.The initial submission of a paper must be through a Fellow or ForeignMember of the Society, but subsequent correspondence will be conducteddirect with the author. The latest lists of Fellows and Foreign Members are tobe found in the current edition of the YearBookof theRoyal Society.A copy of'Notes for the guidance of Fellowscommunicatingpapers' is available from theExecutive Secretary. In the event of any difficulty, an author is invited to seekthe assistance of the Executive Secretary.No page charge is levied, and the first50 offprintsof a paper are supplied tothe author gratis.The Editors particularly welcome short communications to Proceedings;sfar as possible they will be given expeditious treatment both in considerationand in printing, and this will be facilitated if a paper is submitted with a firmrecommendation by a Fellow.

Associate Editors: series B, Biological Sciences(For Standing Orders see current Year Book.)

ProfessorP. Allen Sir Cyril Clarke ProfessorJ. M. MitchisonDr W. F. Bodmer ProfessorJ. P. Cooper Dr D. NobleProfessorB. B. Boycott Dr K. Dalziel ProfessorJ. G. PhillipsProfessorF. W. Campbell Dr L. L. Iversen ProfessorD. J. WeatherallCopyright

? I984 The Royal Society and the authors of individual papers.It is the policy of the Royal Society not to charge any royaltyfor the productionof a single copy of any one article made for privatestudy or research.Requestsfor the copying or reprintingof any articlefor any other purpose should be sentto the Royal Society.


14/15

HIGHLIGHTS OF BRITISH SCIENCE'Britain has a great tradition in natural science. From the time of the so-calledscientific revolution of the mid-seventeenth century with the foundation of theRoyal Society at the heart of it, British scientists have been in the forefront ofadvances not merely in fundamental science but also in its application to thepractical problems of everyday life. Science, of course, knows no national boun-daries and it owes much of its progress and vitality to the free exchange of in-formation and ideas between scientists in all countries. It is nevertheless true that,for a varietyof reasons, some countries, of which Britain is one, have made contri-butions out of all proportion to their physical size and population. Today we are inthe midst of a worldwide economic recession which has indeed affected Britainmore severely than some other highly industrialized nations. Since economicprogress and with it our living standards depend nowadays almost entirely onadvances in science and in technology based upon it, it is perhaps not surprisingthat some members of the public should wonder whether we have fallen behindother nations and whether our expenditure on science and scientific research isbeing misdirected. Nothing could be furtherfrom the truth. Our record during thepast twenty-five years is an enviable one and our scientific research is vigorous asever, flushed with success and full of promise. Britain has made and continues tomake outstanding contributions in many and diverse fields of science. Some of theseare set out in this book and it is my hope that its contents will not only stimulateappreciation of some highlights in Britishscience but will indicate also its promisefor the future.' Extract rom LordTodd's Preface

CONTENTSDiscoveries about the universeRecent advances in weather forecastingResearch in seas and oceansContributionsof scientific discoveries to increases in agriculturalproductivityScience and the development of nuclear energyThe jubilantelectronDevelopments in electron microscopy and microanalysisChemistry in microtimeThe intracellularelectrode: 25 years of research in cellular electro-physiologyMolecules of lifeHigh blood pressure: the evolution of drug treatment: Britishcontribution

240 pages 44 platesISBN 085403 1049

Price including packing and postage?8.00 (U.K. addresses) ?8.25 (overseas addresses)

The Royal Society6 Carlton House Terrace, London SW1Y 5AG


15/15

gly paper 1

Documents