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MOLECULAR

and

Experimental

Therapeutics

39:691-696

PHARMACOLOGY,

ACCELERA

for Pharmacology
reserved.

TED

COMMUNICATION

Influence of Recombinant 7-Aminobutyric

ACid-A
Receptor
Subunit Composition on the Action of Allosteric Modulators
-y-Aminobutyric Acid-Gated CF Currents
GIULIA

PUIA,

STEFANO

VICINI,

PETER

H. SEEBURG,

Fidia-Georgetown
Institute for the Neurosciences,
Georgetown
Center for Molecular
Biology, University
of Heidelberg,
D6900

Received

November

28, 1990; Accepted

and ERMINIO
University
Heidelberg

of

COSTA

School of Medicine,
FRG (P.H.S.)

Washington

DC 20007

(G.P., S. V., E.C.),

and

March 14, 1991

SUMMARY
y-Aminobutyric
acid
rat cortical
neurons

(GABA)-activated
and in cultured

expression of specific molecular

3, and -y subunits,
were recorded
in the whole-cell

configuration.

C1 currents
in neonatal
cells engineered
for the

forms of the GABAA receptor

a,

with the patch-clamp

technique
The effects of various allosteric

modulators of GABAA receptors were determined. Diazepam and

clonazepam
showed greater efficacy as positive modulators
of
GABA-elicited
currents in a2$1 y2 or a391 y2 receptors
than in
al $1 y2 or a5f31 72 receptors
or in cortical neurons.
Alpidem
was more efficacious
at al /31 y2 or a2/31 y2 receptors
than at
al /91 y2 or a5f31 y2 receptors
or in cortical neurons.
Conversely,
zolpidem

was

equally

efficacious

for all these

receptors

except

for a5f31 y2. Both imidazopyridines

(alpidem and zolpidem) were

virtually ineffective
at modulating
the GABA response of a5f31 y2
receptors
and in almost all the receptors assembled
from al,

tor

The

GABAA

in

mammalian

receptor
brain;

is the most
this

receptor

abundant
has

inhibitory

recep-

a heteropolymeric

structure
that forms
a Cl channel
(1). To date,
12 distinct
receptor
subunits
have been identified
(six a, three 13, two y,
and one 5) (2-10).
The biophysical
properties
of receptors
assembled
from a number
of subunit
combinations
have been
defined
(11).
GABA binding
to its specific
recognition
site on the GABAA
receptor
opens
the ion channel
and allows
an inward
flow of
Cl into the cell. This Cl current
can be regulated
both by
drugs and by endogenous
ligands
that act as positive
or negative
allosteric
modulators.
GABA
action
can be modified
by isosteric
receptor
antagonists,
which
are thought
to bind to the extracellular
domain
of the receptor.
Allosteric
modulation
is elicited
by two classes
of compounds,
those that act on the extracellular
domain
and those
that
act on the channel
domain
of the

a3,
or a5 subunits together with f1 and yl subunits.
The 13carboline
derivatives
methyl-6,7-dimethoxy-4-ethyl-fl-carboline3-carboxylate
(DMCM) and methyl-fi-carboline-3-carboxylate
(13CCM)
elicited
a positive
allosteric
modulation
of al /3lyl
or
a2/3l yl receptors,
whereas they acted as negative allostenc
modulators
at nearly all other receptors
tested,
as they do in
cortical neurons. Although
the positive
allostenc
modulation
by
13-carbolines
never exceeded a doubling of the GABA response,
a2,

DMCM was more efficacious at a1fl1yl

receptors and fl-CCM
was more efficacious at a2131 yl receptors. DMCM was inactive
at a3f31 yl receptors,
whereas 13-CCM was virtually inactive at
a5131 yl receptors.
The benzodiazepine
4-chlorodiazepam,
which is a negative
modulator
resistent
to flumazenil
inhibition,
acted at all the various
GABAA receptors
that contained
a
subunit.

receptor

(1), with

allosteric

both

classes

modulators.

The

including

positive

positive

modulators

and

negative

acting

on

the

extracellular
whereas
the

domain
negative

include
BZs,
modulators

IZs, and triazolopyridazines,

mainly
comprise
BC deriva-

tives.

positive

and

allosteric

These

the extracellular
ited

by

flumazenil

modulator

(12).

GABAA

receptor

allosteric

(1).
The

rates

and

lone

sulfate

13, 14).

The

presence
structure

modulators

within

to depend

of the

GABAA

only

BZ

is 4-chlorodiazepam,

resistant

bind

negative

domain

the

(4).

channel

steroid

hormone

and

picrotoxin)

on the
These

structural

compounds

but
or absence
can

known
its

In contrast,

and

derivatives)

to act

the

on

as negative

y2 subunit
action

the

effects

as positive

of the

on homopolymeric

in the

of positive

act

modulators

configuration

act

are inhib-

is flumazenil

or negative

allosteric
act

and

action

of the

influence

domain

also

modulators

receptor

of drugs

that

(barbitu(pregneno-

do not
receptor

appear
(3, 4,

receptors

ABBREVIATiONS:
GABAA receptor, type A -y-aminobutyric
acid receptor; DMCM, methyl-6,7-dimethoxy-4-ethyfi-carboIine-3-carboxylate;
GABA,
y-aminobutync
acid; fl-CCM, methyl-13-carboline-3-carboxylate;
zolpidem, N,N-6-tnmethyl-2-(4-methylphenyl)imidazo[1
,2-aJ-pyndine-3-acetamide
hemitartrate;
alpidem, N,N-dipropyl-6-chloro-2-(4-chlorophenyl)imidazo[1
,2-a]-pyndine-3-acetamidehemitartrate;
BZ, benzodiazepine; BC, fl-carboline;
IZ, imidazopyndine;
HEPES, 4-(2-hydroxyethyl)-1
-piperazineethanesulfonic
acid; EGTA, ethylene gycoI bis($-aminoethyl
ether)-N,N,N,Ntetraacetic
acid.
691

692

Pula ef al.

(3, 13, 14).

rodiazepam,

13, and

The activities
of BCs
require
a heteropolymeric

y subunits

and

BZs, including
4-chlostructure
containing
a,

(4, 12).

Recently,
the distinction
between
the
type
I and
type
II
classes
of BZs based
on binding
studies
(15) was shown
to be
related

to

different

However,

molecular

in situ

brain

areas

and

binding

the

assumption

BZ receptor

Our
three

present
BZs

studies

that

appears

of

studies

there

the

(5,

with

(4, 7, 13) provided

GABAA
receptor

subunit
assemblies
heterogeneity
of
and

forms

hybridization

various

only

(16).

of different

GABAA

evidence
subunits

are

subunit

17-19)

receptor

that the natural

is more
complex,

two

natural

types

of

to be reductionistic.

studies

address

(diazepam,

the

pharmacological

clonazepam,

and

profiles

of

4-chlorodiazepam),

two

BCs
(DMCM
and f3-CCM),
and two IZs (alpidem
and zolpidem).
We describe
the effects
of subunit
compositions
of heteropolymeric
recombinant
GABAA
receptors
on the efficacy
and potency
of these
allosteric
of the allosteric
modulation

modulators
they elicit.

and

on the

cation
GABA
pipette

of dimethylsulfoxide
(0.01% in bath medium)
failed to modify
responses.
To avoid uncontrolled
drug leakage,
we kept the
outside
the bath before the pressure
injection
and brought
it
into proximity
with the recorded
celljust
before
drug application.
Drugs
were applied
for 5 sec between
two GABA pulses
delivered
every 10
sec. The maximal
Cl current
measured
from each cell was larger (>1
nA) than the test response
of 150-200
pA we used, indicating
that the
percentages
of potentiation
we observed
were far below the maximal
efficacy
of the system.
Current
traces were recorded
by a patch-clamp
amplifier
(EPC-7;
List Electronics),
filtered
at 1500 Hz (eight-pole
lowpass Bessel;
Frequency
Devices),
and recorded
on a chart
recorder
(Gould 26005) for off-line
analysis.
Often the GABA response
was greater
in cells adjacent
to those from
which currents
were being recorded.
This observation
might relate to
electrical

coupling

easier
were

performed

Primary
rons

culture

were

of

and Methods

cortical

neurons.

trypsin

(0.25

mg/ml;

35-mm

Nunc

dishes

rat cortical

neu-

(20). Briefly,
cells were dispersed
with
Sigma)
and plated at a density
of 0.8-1 x 106 on
that were coated
with poly-L-lysine
(10 tg/ml;
were maintained
in basal Eagles
medium,
10%
(GIBCO),
25 mM KCI, 2 mM glutamine
(Sigma),
(GIBCO),
for 1 to 3 weeks.
After
24 hr, the

Sigma).
The cultures
fetal bovine
serum
100 tg/ml
gentamicin
incubation
medium
was replaced
was added
to inhibit
replication
Culture

Neonatal

of kidney

and

1 gM cytosine

of nonneuronal

embryonic

cell

line

arabinofuranoside

eDNA

transfeetion.

Transformed
human
embryonic
kidney 293 cells (American
Type Culture Collection
no. CRL 1573) were grown in minimal
essential
medium
(GIBCO),
supplemented
with 10% fetal bovine
serum,
100 units/ml
penicillin
(GIBCO),
and 100 units/ml
streptomycin
(GIBCO),
in a 6%
CO2 incubator.
Exponentially
growing
cells were dispersed
with trypsin
and seeded
at 2 x iO cells/35-mm
dish, in 2 ml of culture
medium.
Transfection

was

performed

with

the

calcium

phosphate

precipitation

technique
(21). Human
(al, a2, a3, flu, -yi, and y2) and rat (aS) GABAA
receptor
subunit
cDNAs
singly inserted
into the eukaryotic
expression
vector pCIS2
(22) were used to perform
the transfections.
Cells were
incubated
in the presence
(3 gg/35-mm
dish) ofone or more supercoiled
plasmids
for 12-16
hr at 37, under
3% CO2. The medium
was removed,
and the cells were rinsed twice with culture
medium
and then incubated
in the same
medium
for 24 hr at 37, under
6% CO2, before electrophysiological

studies.

subunit

combinations:

a2fllyl,

a3fllyl,

Cells

and
were

the

whole-cell

microscope

(Zeiss

in

inverted

pipette
contained
mM HEPES-CsOH
KC1, 2 mM CaCl2,
adjusted

to 325

pH 4 with
positive

range,
in such

outward
a way

145

expressed

a3flly2,

the

following

a5fily2,

cultures

studied

with

of cortical

the

single-electrode

configuration
IM-35)

mM

CsC1,

neurons

at room
1 mM

on

the

temperature.
MgCl2,

stage
The

11 nM

M with

sucrose.

was applied
With

of
and

generated

a peak

in neurons

amplitude

of

in the
pA.

was
to

pulses

of

25-50

or transfected
150-200

10

5 mrsi

adjusted

30-msec

currents

an

recording

EGTA,

M in H2O,

with

iontophoretic

were

as to obtain

(0.5

by iontophoresis

GABA

currents

GABA

BZs

nA

cells
were

a gift from Hoffman

La Roche, DMCM
and 13-CCM were from Ferrosan, and the IZs were from Syntelabo.
All drugs were dissolved
in bath
solution

containing

of 0.01%.
of the

Drugs
cell

body,

dimethylsulfoxide
were
with

applied

at a maximal

by pressure

micropipettes

final

(2 to 4 psi),

of 5-10

gm

concentration
in the

diameter.

3 days

proximity
The

This

appli-

phenomenon

receptor

made

subunits.

it

Recordings

of transfection.

neurons

and

shown

in Fig.

diazepam
and

and

diazepam

of the positive
(both
at 10 zM)
on

of the
(i0

little

GABA

of

by

GABAA

receptors

are

records,

alpidem

and

toward

a5131y2

diazepam

mediated

by

diazepam

(Fig.

had

similarly

Diazepam

was

receptors,

much

greater

Alpidem
apas at native

different

concentrations

2) in cortical
low

and

in axflFy2

efficacy

at

a1f31y2,

a5/31y2,
and native
GABAA
receptors,
and its lowest
was at a5fl1y2
receptors.
Diazepam
efficacy
was nearly
greater
for the a3131-y2
assemblage
than
for a1131y2,
and

native

receptors,

an intermediate
mately
the same
and

a2f31-y2

a5f31y2

and

at a2/31y2

efficacy.
for the

receptors

receptor

The
native

(-.-50

(-4000

Differences
tuted
GABAA

receptors

expressed
efficacious
cacy
and

but

was

much

Zolpidem

chose
such

alpidem

between

ceptors

(Fig.

a5131y2

receptors.

3A)

but

failed

sensitivity
y 1 subunit.

lation

GABA

of the

expressing

did

alfll-y2,

Decreased
taming
the
in cells

for

the

reconstielicited
by

the
alf3Fy2
combination
as
a receptor
seems
to be frequently
clonazepam
receptors,

were equally
but their
effi-

subunit
was present
However,
a signifi-

potentiation
at the a3f3Fy2
receptors,
compared
receptors
(Duncan
test),
could
be observed.
and

in efficacy

greater

of 10 M, in cortical
a2f3Fy2,
a3f31y2,
or

considerably
when
the a2
greater
in a3fl1y2
receptors.

cantly
greater
with a1/31y2

showed

was approxifor the a1f31y2

BZ efficacy
at various
Fig. 3A shows the effects

in brain
(18). Diazepam
and
in cortical
and in a1131-y2

increased
was even

diazepam

EC50 of diazepam
receptors
and

nM)

potency
3 times
a5f31y2,

nM).

in IZ and
receptors.

receptors.
We
point
because

and

of cortical

efficacy
and potency
of diazepam
We measured
the allosteric
modulation

response
M)

receptors

these

activity

receptors.

of diazepam

than
for other
receptors.
as efficacious
at a1f31y2
at a3f31y2
receptors.

in the
receptors.

receptors.

from

of facilitation

to 10

GABAA

reconstituted

evident

for a3flFy2
receptors
peared
to be nearly
receptors
and lower

on ax13ly2
effects

on native

very

extent

alpidem

modulatory

various

1. As

showed

the

a5131y2
reference

or cultures

voltage-clamp

(23),

(13).

two BZs and two IZs, all at a concentration

cells
and
in cells
expressing
a1fl1y2,

a131y1,

(pH 7.2). Cells were bathed

in 145 mM NaCl,
and 5 mM HEPES-NaOH
(pH 7.4); osmolarity

mOs

HC1)

current.

analyzed

a2f31-y2,

Primary

cells

technique

were

cs5f.Thyl.

Eleetrophysiology.
of transfected

that

cr131-y2,

of

Differences
at ax131y2

cells.
and

within

cells

the GABAA

Results
Examples
alpidem

as described

prepared

293

direction
Action

Materials

between

to find cells expressing

show

yl

significant

to potentiate

by the
subunit

differences

a3f31y2,

to BZs and
We further

response
the

not

a2/3Fy2,

and

GABA

cortical

re-

responses

at

IZs at receptors
investigated
the
two

together

BZs

and
with

the
alfll,

conmodutwo

IZs

a2131,

Heterogenefty

Pharmacological

of GABAA Receptors

693

IJIIIIIIIIIIIIIIIIJIIIIIIIIHILIIILILIHLLLL
Cortex

alJ3ly2

Fig. 1. Differential sensitivity to diazepam and

alpidem of neurons expressing native GABAA
receptors
(cortex) and of cells engineered to
express
al/ly2,
a3ly2,
or a591-y2 receptors. The upper trace marks the repetitive iontophoretic
application
of GABA (50 nA for 30
msec). GABA elicited an inward Cl current.
Diazepam and alpidem, applied by pressure at
a concentration
of 1 0 M for 2 sec (arrowheads), potentiated the inward current.

a3f3 1y2

V Diazeparn

-Tin_mT
r-1-nl-mTmTrmTnTrffrnTn
-

yAlpidem

a513 ly2JlOOpA
10

Negative
receptors.

c(nM)

Cortex

so

a1l1Pr2

53

u2111fz

60

u3111Y2
a5l1lp2

140
4000

a3ly2

a2ly2
I-

zLU

-1

-7
Log

-6

-5

cs

behaved

as

receptors

containing

a3f31, or a5131 subunit

combinations
(Fig. 3B). Each drug was
much
more efficacious
at receptors
containing
the y2 rather
than the )1 subunit.
The efficacies
ofdiazepam
and clonazepam
at GABAA
receptors
containing
the y1 subunit
were
reduced
by about 50%, compared
with al$iy2
receptors.
Alpidem
and
zolpidem
efficacies
were markedly
decreased
(approximately
85% for alpidem
and 75% for zolpidem)
at a1131yl
receptors,
compared
with a1/31y2
receptors,
and there
was virtually
no
of the
or a3131yl

GABA
receptors.

response

in cells

expressing

a1131y1,

receptors
containing
a2131, a3$1,
or a5131

at

negative

GABAA
as 4-chlo-

the yl or y2
subunit
com-

decreased
GABA-actiand in all the recombiDMCM
and 13-CCM

modulators
in cortical
neurons
and
at
y2 subunit
but acted
as positive
modand
a2131-yl
receptors.
DMCM
was
more

the

alfllyl

efficacious
at ali3lyl
than
at a2f3Fyl
CCM
showed
a higher
efficacy
at

receptors;
a2fl1y1

than

13-

conversely,
at alfllyl

receptors.
At a3$1-yl
receptors,
fl-CCM
was a negative
modulator, whereas
DMCM
was almost
inactive.
DMCM
elicited
a
weak positive
modulation
at a5131yl receptors,
whereas
13-CCM
was inactive.
Thus, BC derivatives
might behave
as positive
or
negative
modulators
according
to the receptor
structure.
Fig. 5
shows

Fig. 2. Dose-dependent
potentiation by diazepam of GABA-evoked
C1
current in cortical cells and cells expressing cr1 131y2, a2$1 y2, cr3131 y2,
and a5131 y2 transfected
receptors. Each concentration
point is mean
standard error of six different cells. The ECse of each curve is shown in
the inset. The maximal potentiation values for the different receptors are
as follows: a5$1 y2, 1 1 0%; al$1 y2 and cortex, 120%; a2131 y2, 270%;
and a3131y2, 400%.

at recombinant
/3-CCM,
as well

(Fig.
4). 4-Chlorodiazepam
C
currents
in cortical
neurons
receptors
by 30-70%.
In contrast,

binations

ulators
ally2
Cortex
a5pj

a2$lyl,

modulatory
activity
The BCs DMCM
and

rodiazepam,
were tested
on
subunit
together
with
a1131,

vated
nant

potentiation

the

DMCM
and the
same

dose-dependent

positive

of GABA-activated
dose-dependent

drug

Cl
negative

of GABA-activated

Cl

allosteric
currents

allosteric
currents

modulation

in a1$1y1

by

receptors

modulation

by the

in al$1-y2

receptors.

Discussion
Recent
cloning
various
subunits
a search
for the
erogeneity.
Our
causes

a diversity

and IZ
currents.
structure
spite

acting
This
of the

of the

fact

of the cDNAs
encoding
for the mRNA
for
of the GABAA receptor
(2-10)
has prompted
functional
significance
of this structural
hetexperiments
show
that
this
heterogeneity
in the

pharmacological

profiles

of BZ,

as allosteric
modulators
of GABA-gated
diversity
may arise
from
differences
modulatory
that

differences

translational
modifications
underlie
some of the diversity

center

where
in the

of reconstituted
we observed,

these

drugs

stochiometry

receptors
we believe

BC,

in

Cl
the

act.

In

or post-

might
that our

694

Puia et a!.
500

400

a113172
Cortex

#{149}

a2131y2
C

300

a3131-y2

Cu
C
0)
0

a5131-y2

200

100

Fig. 3. A, BZ and lZ modulation of the

response of native receptors and
of reconstituted
receptors
containing
a2131
and a5fl1
subunit
combinations
together with the y2 subunit. B, BZ and IZ modulation
of the
GABA

Diazepam

Clonazepam

Alpidem

Zolpidem

GABA

responses

of al/3ly1

a2f31y1,

a3131 yl
or a5$1 yl receptors.
Each
value is the mean standard error of 812 cells.
Subunit combinations showing
a significant (p < 0.05, Duncan test) difference
in potentiation of the GABA response, compared with that measured for
al$1 y2 receptors. Drug concentrations
in all experiments
were 1 0 PA.
,

400

alf3ly2

a1j3171

300

a21y1
C

a3171

200

.2
Cu

af3171

C
0)
0

a.

-100

Diazepam
results
in

are

a good

first

pharmacological

efficacy
the
the

not

Clonazepam

approximation
profiles

only

a subunit
y subunit.

of

changes

of structural
GABAA

in relation

but also depends

on
Indeed,
the direction

BC derivatives

seems

Alpidem

Zolpidem
differences

receptors.

to the

The
form

of

insensitive

of
of

Ligand

dependent

on the type

of

V subunit.

Drug

potency

to be related
in

contrast

ligand

for the

was

studied

only

for

diazepam

to changes
in the molecular
form
the efficacy
of 4-chlorodiazepam,

and
of the

to

peripheral

BZ receptor

similar;

that

also

appeared
a subunit,
a selective

decreases

GABA-

4 -chlorodiazepam

transmembrane

the molecular
structure
of the modulatory
effect

to be markedly

molecular

be

drug

distinct
exist

domain
to flumazenil
binding

in the

brain.

or a2

or a3

more,

in those

the

(type

presence

rather

than

al

first

ent

BZ sensitivities

This

are

not

are

operative

suggests
in the

two

that
types

the

molecular

of allosteric

mechanisms
modulation

that
may

not

tional

II)

differences

GABAA

differences

that
but
Our
and

131, y2, and

diazepam
to

via

the
to be

binding

also
expand

receptor

suggested
verify
on

type

have

been

either

al

the

This

relation

a3

not

repro-

(type

I)

Furtherby

subunit,

only

have

BZ type

existence

two

II (15),

facilitated

if the
subtypes

that
the

(16).
was

extent

was present.

distinct

at least

I and

subunits

a greater

results

that

type

receptor

or a2 subunits,

indication
uniform.

binding

receptors,

the

act

is known

indicated

termed

containing

of GABA

gated
Cr currents
in a manner
that is insensitive
to flumazenil
(12). Moreover,
the direction
of its modulatory
action
appeared
to be independent
of the molecular
form
of a and y subunits.
difference

have

receptors,

These

receptors

which

inhibiton.

of BZ

with

may

receptor,

experiments

classes

duced

modulation

of the

was

differ-

II receptors
of such

between

funcstruc-

Pharmacological

120

Heterogeneity

of GABAA

695

Receptors

alali2
Cortex

80

E:J

a2172

40

a3

172

a5

172

Fig. 4. DMCM and f3-CCM modulation of

GABA responses
in cells expressing dif-

ferent

0)

subunit assemblies.
Drugs
at a concentration
of iO
Significant (p < 0.05, Duncan test)
ence relative to the effect on al
receptors.
Each value is an average
cells.

a1171

a2fl171

50
0

were

applied

a3jIlyl

PA.

differflu y2
of six

-40

a5j3171

-80

4-chlorodiazepam

f3-CCM

DMM

100

al1yl
C
0

expressing

a3131y2

potentiation

with

4.,

for a1f31y2
and a2fl1y2
likely,
GABAA
receptors

consist

of a heterogeneous

C-

receptors

actions

with

Our

CO

data

results
of zolpidemofprevious
for
a3

Fig. 5. Dose-response

to DMCM of GABA-evoked
Cl current in cells
and al flu yl transfected
receptors.
Each concentra-

expressing cr1131y2
tion point is mean standard

error of four different

tural
heterogeneity
and functional
diversity
in the allosteric
modulation
of the GABAA receptor
by BZs, BCs, and IZs.
The extent
of GABA
activity
potentiation
by maximally
efficacious
doses
of diazepam
and clonazepam
(10 M)
was
similar
in rat cortical
neurons
expressing
native
receptors
and
in cells engineered
for the expression
of al13Fy2
and a5f31y2
receptors.
However,
both drugs were more efficacious
when
a2
substituted

for

al,

al.

the

diazepam

In fact,

of GABA
al/31y2

activity
or a5f31y2

and

even

more

so when

dose-response

in cortical
receptors

a3

curves

neurons

and

demonstrate

substituted

in cells
low

(10)

tors.

but
action

modulation

on

efficacies

certain

have

neity

GABAA

and

the

drugs

therapeutic

targeting

produced

ture,

potentiation

of

GABA

activity

in

cells

molecular

structure

was

investigated

in Xenopu.s

oocytes

(25).

sensitivity

BZ

subtypes

a preference

seem

to be located
the

relation
heteroge-

anxiolytic

to specific

and

between

for

GABAA

for certain

to BZs,

in

the -yl
cells.

implications

Differences

of

a positive

was described

of various

in

in astrog-

structural

to

-yl

was

modulation

(17-19),

responses

was

the

However,

by DMCM

drugs

the

conferring

be expressed

important

perhaps

of

IZs

negative
modulaor a3f31y1
recep-

and

IZ

the

there

might

potency

Recently,
pressed

with

and

carried

Furthermore,

receptor

subtypes,

diversity

the

shows

BZs
that

structures

have

of these

more

subunit.

of

receptor

and

may

are
support

(24), which
suggests
that
GABAA
receptors
of glial

between

efficacy

anxiogenic

both

is lacking.

brain

demonstrated

al

to elicit
a2131yl

response

in certain

of the drug, whereas

diazepam
efficacy
was much greater
(4fold potentiation
versus
1.5-fold)
in a3f31-y2
receptors.
One
might
surmise
that this difference
could be due to a greater
number
of cellularly
expressed
a3131y2 receptors
than alflFy2
or a5/31y2
receptors.
However,
this possibility
can be excluded
because,
if this were the case, all the drugs tested
should
have
a maximal

cells

cultures
of astrocytes
may be present
in the

Because
we

3B).

study

these

and

zolpidem
the

by

combination

of the GABA

primary
subunit
preferentially

expressing

that

receptors

(Fig.

a complete

inter-

(7) aSshowing
is lower thatthan the foraffinity
a2 or

currents

of y2

a subunit

cells,

GABA

instead

these

drug

assemblages

and

GABAA

to

at

of the

by BZs

containing

of C
in

regard

subtypes.

for zolpidem
and alpidem
applied
to cells expressing

Such

hal

receptors

reduced

average

than

studies
containing

neurons

with
measured

subunit
IZs

receptors
for

modulation

tendency
tion when

for

for potentiation

similar

The
subunit

cells.

binding
receptors

affinity

markedly

an

receptor
by

in cortical

responses

certain

subunit-containing

highest

-100

that

to modulation

show
maximal
of this IZ was

population

reflect

GABAA

not
efficacy

receptors.
expressed

the

probably

suggest

susceptible
a in! y2

and

diverse

did
the

receptor

compositions,

native

Co
--4

Alpidem
receptors;

greatest
Very
subunit

.,-4

receptors.
a3flFy2

brain
BZs

GABAA

structures.

related

to

receptors

in molecular
our

the

receptor

reconstitution

the
ex-

struc-

696

Pula et a!.

model

and the

difference

Xenopus

between

cies differences
human).

might

oocytes

the

two

expression

in recombinant

be related

to the

systems

and/or

subunit

structures

Differences

intrinsic
to spe-

(rat

versus

Acknowledgments

We would like to thank

helpful

Dr.

G. David

Lange

and

Dr.

Keith

Blockehurst

for

comments.

in the

negative

allosteric

modulation

of -y-aminobutyric

acid

receptors
elicited
by 4-chlorodiazepam
and by a fl-carboline-3-carboxylate
ester:
a study with natural
and reconstituted
receptors.
Proc. NatL Aced.

Sd.
USA 86:7275-7279
(1989).
13. Pritchett,
D. B., H. Sontheimer,
C. M. Gorman,
H. Kettenmann,
P. H.
Seeburg,
and P. R. Schofield.
Transient
expression
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allosteric
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(1988).
14. Puia, G., M. R. Santi, S. Vicini, D. B. Pritchett,
R. H. Purdy,
S. M. Paul, P.
H. Seeburg, and E. Costa. Neurosteroids
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