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Structural Mechanisms Underlying Benzodiazepine Modulation of the GABA Cellular/Molecular
Structural Mechanisms Underlying Benzodiazepine
Modulation of the GABA
A
Receptor
Susan M. Hanson and Cynthia Czajkowski
Department of Physiology, University of WisconsinMadison, Madison, Wisconsin 53711
Many clinically important drugs target ligand-gated ion channels; however, the mechanisms by which these drugs modulate channel
function remain elusive. Benzodiazepines (BZDs), anesthetics, and barbiturates exert their CNS actions by binding to GABA
A
receptors
and modulating their function. The structural mechanisms by which BZD binding is transduced to potentiation or inhibition of GABA-
induced current (
I
GABA
) are essentially unknown. Here, we explored the role of the
2
Q182-R197 region (Loop F/9) in the modulation of
I
GABA
by positive (flurazepam, zolpidem) and negative [3-carbomethoxy-4-ethyl-6,7-dimethoxy- -carboline (DMCM)] BZD ligands.
Each residue was individually mutated to cysteine, coexpressed with wild-type
1
and
2
subunits in
Xenopus oocytes, and analyzed using
two-electrode voltage clamp. Individual mutations differentially affected BZD modulation of
I
GABA
. Mutations affecting positive modu-
lation span the length of this region, whereas
2
W183C at the beginning of Loop F was the only mutation that adversely affected DMCM
inhibition. Radioligand binding experiments demonstrate that mutations in this region do not alter BZD binding, indicating that the
observed changes in modulation result from changes in BZD efficacy. Flurazepam and zolpidem significantly slowed covalent modifica-
tion of
2
R197C, whereas DMCM, GABA, and the allosteric modulator pentobarbital had no effects, demonstrating that
2
Loop F is a
specific transducer of positive BZD modulator binding. Therefore,
2
Loop F plays a key role in defining BZD efficacy and is part of the
allosteric pathway allowing positive BZD modulator-induced structural changes at the BZD binding site to propagate through the protein
to the channel domain.
Key words: GABA; GABA
A
receptor; benzodiazepine; efficacy; allosteric modulation; Loop F; zolpidem
Introduction
Members of the Cys-loop family of ligand-gated ion channels
(LGICs) control ion permeability of cell membranes by coupling
agonist binding to channel opening. Gating of these channels can
be allosterically modulated by a number of clinically important
drugs. Within the LGIC family, the GABA
A
receptor (GABA
A
R)
is an excellent model for studying mechanisms underlying allo-
steric modulation because of the large number of drugs that tar-
get this receptor, including benzodiazepines (BZDs), barbitu-
rates, anesthetics, and ethanol. BZDs are among the most widely
prescribed drugs in the United States, with
80 million prescrip-
tions written each year, and are used for sedation, sleep induc-
tion, anxiety relief, muscle spasm relief, epileptic seizure control,
and treating some forms of depression (for review, see Mohler et
al., 2002).
BZDs exert their effects on the CNS by binding to the
GABA
A
R and allosterically modulating GABA-induced current
(I
GABA
) responses. The BZD binding site is located on the extra-
cellular surface of the GABA
A
R and is formed by residues located
in at least six noncontiguous regions at the / interface histori-
cally designated Loops A-F (Fig. 1 A) (for review, see Sigel, 2002).
This site binds a large selection of structurally diverse ligands
(Fig. 1 B), including agonists that potentiate GABA-mediated
Cl
current (I
GABA
) [positive modulators such as flurazepam
(FZM) and zolpidem (ZPM)], inverse agonists that inhibit I
GABA
[negative modulators such as 3-carbomethoxy-4-ethyl-6,7-
dimethoxy- -carboline (DMCM)], and antagonists that bind at
the BZD site but have no effect on I
GABA
(zero modulators such as
Ro15-1788). The structural elements that couple BZD binding to
modulation of I
GABA
are only beginning to be defined, and the
mechanisms by which certain BZD ligands potentiate I
GABA
,
whereas others inhibit (i.e., BZD efficacy), remain unknown. Be-
cause the therapeutic value of BZDs depends on their ability to
modulate I
GABA
, we sought to map the structural elements un-
derlying BZD efficacy.
Previously, using
2
/
1
chimeric subunits, we identified resi-
dues located near the channel domain of the
2
subunit (in the
pre-M1 region, the extracellular end of M2, and the M2-M3 ex-
tracellular loop) that are required for modulation of I
GABA
by
BZD-positive modulators (Boileau et al., 1998; Boileau and Cza-
jkowski, 1999). Interestingly, the identified
2
subunit residues
were not critical for inhibition of I
GABA
by the BZD site inverse
agonist DMCM (Boileau and Czajkowski, 1999), suggesting neg-
ative allosteric modulation is governed by structural elements
distinct from that of positive allosteric modulation. In the
2
subunit, a stretch of
20 residues called Loop F links the BZD
Received Oct. 3, 2007; revised Jan. 31, 2008; accepted Feb. 20, 2008.
This work was supported by National Institutes of Health Grants F32 MH082504 (S.M.H.), NS34727, and
MH66406 (C.C.). We thank Dr. Andrew Boileau and Srinivasan Venkatachalan for careful reading of this manuscript
and helpful discussions.
Correspondence should be addressed to Cynthia Czajkowski, Department of Physiology, University of Wisconsin
Madison, 601 Science Drive, Madison, WI 53711. E-mail: czajkowski@physiology.wisc.edu.
DOI:10.1523/JNEUROSCI.5727-07.2008
Copyright © 2008 Society for Neuroscience 0270-6474/08/283490-10$15.00/0
3490 The Journal of Neuroscience, March 26, 2008 28(13):3490 3499 binding site to the beginning of -strand 9 near the transmem-
brane channel gating domain (Fig. 1 A) and, thus, is in an ideal
position to transduce BZD binding site movements to move-
ments near the channel domain. Previous studies examining the
related acetylcholine binding protein (Gao et al., 2005; Hibbs et
al., 2006), the nicotinic acetylcholine receptor (Leite et al., 2003;
Lyford et al., 2003), the serotonin type-3A receptor (Thompson
et al., 2006), and the GABA
A
R agonist binding site interface
(Newell and Czajkowski, 2003) support the idea that the Loop F
region is dynamic. Here, we used the substituted cysteine acces-
sibility method (SCAM) to test the idea that
2
Loop F plays a role
in differentiating the efficacy of allosteric modulation and that
positive and negative allosteric modulator binding to the BZD
site initiates distinct conformational movements within this
region.
Materials and Methods
Site-directed mutagenesis. Rat cDNA encoding
1
,
2
, and
2L
receptor
subunits in the pUNIV vector (Venkatachalan et al., 2007) were used for
all molecular cloning and functional studies. This vector [kindly pro-
vided by S. Venkatachalan and A. Boileau (Department of Physiology,
University of Wisconsin-Madison, Madison, WI)] is advantageous, be-
cause it can be used for expression in Xenopus oocytes and mammalian
human embryonic kidney 293 (HEK293) cells without further subclon-
ing. All
2L
cysteine mutants were made by recombinant PCR and veri-
fied by double-stranded DNA sequencing.
Expression in Xenopus laevis oocytes. Capped cRNA was transcribed in
vitro from NotI-linearized cDNA using the mMessage mMachine T7 kit
(Ambion, Austin, TX). Oocytes were harvested from X. laevis and pre-
pared as described previously (Boileau et al., 1998). Oocytes were in-
jected within 24 h of treatment with 27 nl (115 pg/nl/subunit) in the
ratio 1:1:10 ( : : ) (Boileau et al., 2002) and stored at 16°C in ND96
buffer (in m
M
: 96 NaCl, 2 KCl, 1 MgCl
2
, 1.8 CaCl
2
, 5 HEPES, pH 7.2)
supplemented with 100 g/ml gentamycin and 100 g/ml BSA until used
for electrophysiological recordings.
Two-electrode voltage clamp. Oocytes were perfused continuously (5
ml/min) with ND96 while held under two-electrode voltage clamp at
80 mV in a bath volume of 200 l. Borosilicate glass electrodes (0.4 1.0
M ) (Warner Instruments, Hamden, CT) used for recordings were filled
with 3
M
KCl. Electrophysiological data were collected using GeneClamp
500 (Molecular Devices, Sunnyvale, CA) interfaced to a computer with a
Digidata 1200 A/D device (Molecular Devices) and were recorded using
the Whole Cell Program, v.3.6.7 (kindly provided by J. Dempster, Uni-
versity of Strathclyde, Glasgow, UK).
Concentrationresponse analysis. Six to ten concentrations of GABA
were used for each determination of GABA EC
50
value. Each response
was scaled to a low, non-desensitizing concentration of GABA (EC
15
)
applied just before the test concentration to correct for any drift in I
GABA
responsiveness over the course of the experiment. All concentration
response data were fit by the following equation: I
I
max
/[1
(EC
50
/
[A]
n
)], where I is the peak response to a given drug concentration, I
max
is
the maximum amplitude of current, EC
50
is the drug concentration that
produces a half-maximal response, [A] is drug concentration, and n is the
Hill coefficient using Prism v.4.0 (GraphPad Software, San Diego, CA).
BZD modulation was defined as follows: [(I
GABA
BZD
/I
GABA
)
1],
where I
GABA
BZD
is the current response in the presence of GABA and
BZD, and I
GABA
is the current evoked by GABA alone. BZD modulation
(6 7 different concentrations) was measured at 1
M
GABA (EC
25
). The
reported values for maximum potentiation represent I
GABA
potentiation
in the presence of 3
M
FZM and 10
M
ZPM, respectively.
Methanethiosulfonate modification. Four derivatives of methanethio-
sulfonate