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INTRODUCTION |
The ligand-gated ion channel
(LGIC)1 superfamily includes
the nicotinic acetylcholine receptor (nAChR), serotonin type 3 receptor (5HT3R), GABAA receptor (GABAAR),
and glycine receptor (GlyR), as well as invertebrate glutamate and
histidine receptors (1). Functional receptors of this family comprise
five homologous subunits arranged in a ring to form a central
ion-conducting pore. Each subunit is composed of a large extracellular
ligand-binding N-terminal domain, four membrane-spanning segments
(M1-M4), and a large intracellular domain between M3 and M4.
The pore-lining, second transmembrane (M2) domain has an 
-helical
secondary structure that undergoes a conformational change as the
channel is opened (2). To investigate this process in detail,
state-dependent differences in the surface exposure of M2
domain residues can be assayed using the substituted cysteine accessibility method (3). In this technique, residues are mutated individually to cysteines, and changes in their reactivity rates with
soluble cysteine-reactive reagents can identify structural changes
between different functional states. As expected for receptors belonging to the same family, this technique has generally yielded a
good correlation between the open state M2 domain secondary structures
of the nAChR (4-7), GABAAR (8), and 5HT3R (9, 10).
The M2 domain 6' residue, which is a threonine in the GlyR 1 subunit
and the GABAAR 1 and 
1 subunits (see Fig.
1A), lines a critical part of the pore. It is close to the
activation gate (6, 11, 12) and the ionic selectivity filter (13-15)
and forms the main pore blocker binding site (reviewed in Ref. 16). Therefore, structural differences at this level may be expected to have
significant functional consequences. In the homomeric 1T6'C GlyR expressed in a mammalian HEK293 cell line,
Shan et al. (17) concluded that the surface exposure of
introduced 6' cysteines was increased in the channel open state. In
contrast, in the 1T6'C1T6'C
GABAAR expressed in Xenopus oocytes, the 6'
cysteines were found to be exposed in the closed state and rotated to
face the adjacent subunits in the open state (18). Thus, despite having
a high M2 domain amino acid sequence homology (see Fig. 1A)
and a common function in conducting chloride ions, the GlyR and
GABAAR appear to be structurally divergent at this position.
The aim of this study was to conduct a detailed comparative study into
the surface accessibility of the 6' cysteines in the GlyR and
GABAAR when both are expressed recombinantly in a common (HEK293 cell) expression system. The main findings are that the respective pore structures at the 6' positions are significantly different in the closed states but that there appear to be similarities in the mechanisms of channel opening. The results also reveal distinct
differences in the structural and functional properties of
GABAARs depending on whether they are expressed in
Xenopus oocytes or HEK293 cells.
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EXPERIMENTAL PROCEDURES |
Mutagenesis and Expression of GlyR and GABAAR
cDNAs--
The human GlyR 1 subunit cDNA was subcloned into
the pCIS2 plasmid vector, and the rat GABAAR 1 and 1
subunit cDNAs were subcloned into the pIRES2-EGFP plasmid vector
(Clontech, Palo Alto, CA). Site-directed
mutagenesis was performed using the QuikChange mutagenesis kit
(Stratagene, La Jolla, CA), and the successful incorporation of
mutations was confirmed by sequencing the clones. Adenovirus-transformed HEK293 cells (ATCC CRL 1573) were passaged in a
50:50 mixture of minimal essential medium and Dulbecco's modified
Eagle's medium supplemented with 2 mM glutamate, 10% fetal calf serum and the antibiotics, penicillin (at 50 IU/ml), and
streptomycin (at 50 µg/ml). Cells were transfected using a calcium
phosphate precipitation protocol (19). When co-transfecting the
GABAAR 1 and 1 subunits, their respective cDNAs
were combined in a ratio of 1:1. After exposure to transfection
solution for 24 h, cells were washed twice using the culture
medium and used for recording over the following 24-72 h.
Electrophysiology--
The cells were observed using a
fluorescent microscope, and currents were measured using the whole cell
patch-clamp configuration. Cells were perfused by a control solution
that contained the following (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, with the
pH adjusted to 7.4 with NaOH. Patch pipettes were fabricated from
borosilicate hematocrit tubing (Vitrex, Modulohm, Denmark) and heat-polished. Pipettes had a tip resistance of 1.5-3 megohms when
filled with the standard pipette solution, which contained the
following (in mM): 145 CsCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 EGTA, with the pH adjusted to 7.4 with
NaOH. After establishment of the whole cell configuration, cells were
voltage-clamped at 
40 mV, and membrane currents were recorded using
an Axopatch 1D amplifier and pclamp7 software (Axon Instruments, Union
City, CA). The cells were perfused by a parallel array of microtubular barrels through which solutions were gravity-induced. All experiments were conducted at room temperature (19-22 °C).
Methanethiosulfonate ethyltrimethylammonium (MTSET) and
methanethiosulfonate ethylammonium (MTSEA) were obtained from Toronto Research Chemicals (Toronto, Ontario, Canada), whereas all other reagents were obtained from Sigma. MTSET and MTSEA were
dissolved directly into the bath solution at the final concentrations
of 1 and 2.5 mM, respectively, unless indicated otherwise.
The oxidizing reagent, copper-O-phenanthroline (Cu:phen) was
prepared by mixing CuSO4 (stored as 100 mM
stock solution in H20 at 10 °C) and
1,10-phenanthroline (stored as 400 mM stock solution in
ethanol at 10 °C). The final concentrations of copper and
1,10-phenanthroline in the control bathing solution were 100 and 400 µM, respectively. H2O2,
maintained as a 30% stock solution, was diluted to 0.3% in the
control bathing solution. MTSET, MTSEA, Cu:phen, and
H2O2 were used for no longer than 10 min after
being dissolved into the bathing solution at room temperature. The
disulfide-reducing reagent, dithiothreitol (DTT), was prepared daily as
a 1 or 10 mM solution in control bathing solution.
The effects of all sulfhydryl-specific reagents were tested using the
following procedure. After establishment of the recording configuration, two brief applications of agonist at the half-saturating (EC50) concentration were followed by two brief
applications at a saturating (10-20 × EC50)
concentration, all at 30-s intervals. Provided current amplitude
remained constant, the averaged current amplitudes were used as the
control. Following application of sulfhydryl-specific reagents, cells
were washed in control solution for at 1-3 min before the
EC50 and EC100 agonist-activated currents were
measured again.
Data Analysis--
All data were analyzed using Origin 4.0 (Northampton, MA) or Sigmastat 1.0 (Jandel Scientific).
Results are expressed as means ± S.E. of three or more
independent experiments. The empirical Hill equation, fitted by a
non-linear least squares algorithm, was used to calculate the
EC50 and Hill coefficient (nH)
values for glycine and GABA activation. Statistical significance was determined by either linear regression or by one-way analysis of
variance using the Student's-Newmans-Keul post hoc test for unpaired data, with p < 0.05 representing significance.
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RESULTS |
Sulfhydryl Modification of the 1T6'C GlyR--
This
study investigated the surface accessibility of the 6' residues of
the GlyR 1 subunit and the GABAAR 1 and 1
subunits. As shown in Fig. 1, each of the
WT receptor subunits contains a threonine at this position. In this
study the threonines were mutated to cysteines to enable
cysteine-specific reagents to be used as probes of 6' surface
accessibility (3). The GlyR 1 subunit also contained the C41A
mutation, which eliminated the only uncross-linked external cysteine.
The GABAAR 1 and 1 subunits contained no
uncross-linked external cysteines.
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Fig. 1.
Amino acid sequence alignment of the M2
transmembrane segments of human GlyR 1 subunit
and the rat GABAAR 1 and
1 subunits. The residues mutated to cysteine
in this study are indicated in bold and numbered 6'
according to the system of Miller (26), which assigns 1' to the most
intracellular M2 domain residue and 20' to the most extracellular
residue. Arrows denote those residues in the
GABAAR 1 subunit that are exposed to the channel lumen
(8).
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The mean EC50, nH, and
Imax values for glycine-activated currents in
the 1WT and 1T6'C GlyRs are summarized
in Table I. In the absence
of glycine, there was no significant difference in the resting
conductance of cells expressing 1WT and
1T6'C GlyRs, implying that the T6'C mutation did not
induce a steady-state leak conductance through the channels.
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Table I
Glycine and picrotoxin effects at 1 GlyRs incorporating the
indicated amino acid substitutions at the 6' position
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We demonstrated previously that a 1-min application of 1 mM
MTSET had no significant effect on the 1WT GlyR
regardless of whether it was applied in the closed or channel open
states (17, 20). Similarly, MTSET had no effect on the
1T6'C GlyR when applied in the closed channel state
(17). However, when MTSET was applied to the 1T6'C GlyR
in the presence of a saturating (0.5 mM) concentration of
glycine, the channels remained partially activated following the
removal of glycine and MTSET (17). Following the removal of glycine,
the currents declined to 86 ± 2.4% (n = 6) of
the control current magnitude and remained stable at this level until
closed by a 1-min application of 10 mM DTT (e.g.
Fig. 2A). When 0.5 mM glycine was applied to the MTSET-modified GlyRs, it
reversibly activated an additional current component (Fig.
2A). At any given time after the completion of the MTSET
treatment, the total magnitude of the locked-open plus glycine-gated
current was larger than that which could be activated in the same cell
by a continuous application of 0.5 mM glycine alone. This
point is illustrated in Fig 2, A-C. Fig.
2B shows the effect of a long application of 0.5 mM glycine to the same cell as in Fig. 2A, and
both traces are shown superimposed in Fig. 2C. This
experiment was repeated in five cells, and the relative current
magnitudes were quantitated at a common time point 2 min after the
initial application of glycine. It was found that an application of 0.5 mM glycine to the MTSET-modified GlyRs resulted in a net
current magnitude that was 167 ± 6% (n = 5)
larger than that activated in the same cell by a continuous application
of 0.5 mM glycine alone. Together, these observations
indicate that MTSET locked the channels into the open state but did not
lock significant numbers of channels into either the closed or
desensitized states. The MTSET-induced increase in net current
magnitude at late times was most likely because of a reduced transition
rate from the open to the desensitized state.
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Fig. 2.
Effects of MTSET and MTSEA on the
1T6'C GlyR. A, currents
were activated by 0.5 mM glycine, and MTSET was applied at
a concentration of 100 µM for the period indicated by the
unfilled bar. The channels remained partially activated upon
the withdrawal of glycine, and a subsequent glycine application
reversibly activated an additional current. Channels were closed only
by the application of 10 mM DTT. Scale bars
apply to all traces in A-C. B, effect of a long
application of 0.5 mM glycine to the same cell as in
A. C, superposition of the traces in A
and B reveals the increase in net magnitude of glycine-gated
currents following MTSET modification. D, both traces were
recorded sequentially from the same cell. The left trace
shows the effect of 2.5 mM MTSEA in the closed state. The
right panel shows that MTSEA pre-treatment does not affect
the ability of 100 µM MTSET plus 0.5 mM
glycine to lock the channels in the open state. E, both
traces were recorded sequentially from the same cell. The left
trace shows the lack of effect of 2.5 mM MTSEA when
co-applied with 0.5 mM glycine. The right panel
shows that MTSEA pre-treatment abolishes the ability of 100 µM MTSET plus 0.5 mM glycine to lock the
channels in the open state. All displayed currents in D and
E were recorded from the same cell, and scale
bars apply to all traces.
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Because MTSET induced no current change in the presence of a saturating
glycine concentration, its reaction rate in the fully activated state
could not be measured. However, in the presence of an EC50
(30 µM) concentration of glycine, the reaction proceeded with a time constant of 1.2 ± 0.1 s (n = 4),
indicating a reaction rate of around 830 M
1
s1. This is about 250 times smaller than the rate
constant for the reaction of MTSET with 2-mercaptoethanol in free
solution, the decrease because of electrostatic repulsion, steric
hindrance, or suppressed ionization of the cysteine thiol (3). The
possible contributions of these factors to the reactivity of T6'C are
considered further below.
When applied at a concentration of 10 mM for
60 s, MTSES had no significant effect on either the
1WT or 1T6'C GlyRs regardless of whether
it was applied in the absence or presence of a saturating concentration
of glycine (17). In addition, a prior MTSES application in either
the closed or open state did not significantly attenuate the ability of
MTSET to lock the 1T6'C GlyR into the open state (17).
Thus, MTSES did not react with T6'C.
A 60-s application of 2.5 mM MTSEA also had no significant
effect on the 1WT GlyR regardless of whether it was
applied in the closed or open states (Table
II). Similarly, when applied in the
closed state to the 1T6'C GlyR, 2.5 mM MTSEA
had no significant effect on the magnitude of currents activated by an
EC50 (30 µM) or a saturating (500 µM) concentration of glycine (Table II). In addition,
prior exposure of the 1T6'C GlyR to MTSEA in the closed
state did not significantly affect the ability of a subsequent
application of 100 µM MTSET plus 500 µM glycine to lock the channels open (Fig. 2D). A 60-s
application of MTSEA plus 500 µM glycine also had no
effect on the magnitude of currents activated by either 20 or 500 µM glycine (Table II), although it dramatically
attenuated the effect of a subsequent application of MTSET (Fig.
2E). As shown in Table II, MTSET plus 500 µM
glycine caused 85 ± 3% of channels to be locked into the open
state, while simultaneously reducing the magnitude of the glycine-activable current by 88 ± 2% (both n = 3). Following MTSEA exposure, MTSET plus 500 µM glycine
caused only 16 ± 5% of channels to be locked into the open state
while reducing the magnitude of the glycine-activable current by
17 ± 8% (both n = 4). Both of these values are
significantly different from those obtained without MTSEA
pre-treatment. Taken together, these results provide strong evidence
that MTSEA modifies T6'C in the channel open state but not in the
closed state.
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Table II
Effects of cysteine-modifying reagents on WT and mutant GlyRs and
GABAARs
*, significant relative to the corresponding WT response
(p < 0.05); **, highly significant relative to the
corresponding WT response (p < 0.01); ND, not
determined.
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The effects of MTSEA were likely to have been caused by the covalent
attachment of an ethylammonium group to the 6' cysteine in the open
state. The inability of MTSEA modification to lock the channels open
may have been because of the smaller size of MTSEA relative to MTSET.
On the other hand, the effects of MTSET may have been because of one of
two mechanisms. One possibility is that it directly modified the 6'
cysteines by covalently attaching an ethyltrimethylammonium group. In
this case the reaction would have proceeded only in the open state, and
the resulting cysteine modification would have maintained the pore in
the open state. However, because the methanethiosulfonate (MTS) group
contains a disulfide bond that could directly catalyze the formation of other disulfide bonds, it is also possible that MTSET may have behaved
as an oxidizing agent; MTSET can add thioethyltrimethylammonium to one
cysteine, and a second cysteine can displace this group in a
sulfhydryl-disulfide interchange to generate a cystine-cysteine disulfide. MTSET could thereby induce the formation of disulfide bonds
between subunits, preventing the channels from closing.
To discriminate between these two possibilities, we tested the effects
of oxidizing reagents on the GlyR. We examined the effects of 1-min
applications of 0.3% H2O2 and 100:400
µM Cu:phen on the 1WT and
1T6'C GlyRs. As summarized in Table II, neither reagent
had any effect on either the half-maximal or maximal current magnitudes
of the 1WT or the 1T6'C GlyRs.
Furthermore, neither reagent was able to mimic the effect of MTSET in
maintaining the 1T6'C GlyR in the open state
(n = 3 for each reagent). An example of such an
experiment on the 1T6'C GlyR is shown in Fig.
3. Although Cu:phen induced a weak
transient inhibition, it had no irreversible effects (Fig.
3B). The H109A mutation, which eliminates zinc inhibition
(21), had no effect on this transient inhibitory action of copper (data
not shown).
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Fig. 3.
Effect of Cu:phen on the
1T6'C GlyR. Cu:phen had no
irreversible effect on the 1T6'C GlyR, regardless of
whether it was applied in the channel closed state (A) or
open state (B). Glycine was applied at concentrations of 50 µM (EC50) and 0.5 mM (saturating;
sat.), as indicated.
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Cysteine reactivity with thiol-containing compounds is determined by
the local electrostatic potential, the sulfhydryl ionization state, and
steric accessibility of the MTS reagent to the sulfhydryl group (3).
Unfortunately, it was not possible to determine the contribution of
electrostatic potential changes as the only available soluble,
negatively charged MTS derivative, MTSES, had no measurable effect
(17). However, it is unlikely that electrostatic potential changes
alone would have been able to account for the infinitely large observed
reaction rate difference (see Ref. 5). Thus, the reaction rate was
likely to have been dominated by the sulfhydryl ionization state or
steric accessibility. Because the MTS reaction rate increases
dramatically with thiol ionization (22), and thiol ionization is
suppressed in a hydrophobic environment, one possibility is that the 6'
cysteines exist in a hydrophobic environment in the closed state
(perhaps by facing the protein interior) and increase their exposure to
the aqueous environment in the open state. An equally plausible
alternative is that the 6' cysteines remain in an aqueous environment
in the closed state but that access of the externally applied MTS
reagents in the closed state is precluded by either an electrostatic
impediment or pore constriction external to the 6' position. In either
scenario, the access of MTSET to the 6' cysteines is increased in the
open state, and MTSET holds the channel open by covalently
attaching a positively charged ethyltrimethylammonium group to
T6'C.
Sulfhydryl Modification of the
1T6'C
1T6'C GABAAR--
Both
of the above models contrast dramatically with results obtained
recently on the structurally and functionally homologous GABAAR by Horenstein et al. (18). That study
investigated the state-dependent reactivity changes of the
T6'C residues in the rat 1T6'C1T6'C
GABAAR expressed recombinantly in Xenopus
oocytes. They concluded that the T6'C residues are exposed to the
external aqueous environment in the closed state and rotate to face
the adjacent subunit when the channel is opened. Furthermore, when
applied in the open state, Cu:phen promotes the formation of an
intersubunit disulfide bond between adjacent 1 subunits that locks
the channel in the open state (18). We examined the effects of
cysteine-reactive reagents on the rat
1WT1WT and 1T6'C1T6'C GABAARs expressed
recombinantly in mammalian HEK293 cells.
The mean EC50, nH, and
Imax values for GABA-activated currents in the
WT and mutant GABAARs are summarized in Table
III. We were surprised to find that
incorporation of the T6'C mutations into both the 1 and 1
subunits resulted in a dramatic increase in the rate of desensitization
(e.g. Fig. 4A). In
the presence of a saturating 20 µM (10 × EC50) GABA concentration, the
1WT1WT GABAAR desensitized
with a time constant of 1370 ± 280 ms (n = 4)
whereas in the presence of 100 µM (20 × EC50) GABA, the 1T6'C1T6'C GABAAR desensitized with a time constant of 87 ± 2 ms
(n = 4). This rapid desensitization rate made it
difficult to apply cysteine-modifying reagents with a high degree
of confidence to the channel open state. In the absence of GABA, there
was no significant difference in the resting conductance of cells
expressing 1WT1WT and 1T6'C1T6'C GABAARs, implying
that the mutations did not induce a steady-state leak conductance
through the receptors.
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Fig. 4.
Effect of 10 mM DTT on
GABAAR current magnitude. A, comparative
responses of the 1WT1WT
GABAAR and the 1T6'C1T6'C
GABAAR to a long application of 100 µM GABA.
B, in the 1WT1WT
GABAAR, DTT has a weak effect on the magnitude of currents
activated by a saturating (20 µM) GABA concentration.
C, In the 1T6'C1T6'C
GABAAR, DTT induces a dramatic increase in the magnitude of
currents activated by a saturating (100 µM) GABA
concentration.
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When activated by 20 µM GABA, the
1WT1WT GABAAR was weakly but
significantly potentiated by a 2-min application of 10 mM DTT (see Fig. 4B and Table II). Upon removal of DTT,
currents gradually returned to the control magnitude over the following 3-5 min. This effect is similar to that observed when the same receptors are expressed in Xenopus oocytes (18). In contrast to this relatively modest effect, a 10 mM application of
DTT caused a dramatic potentiation of the
1T6'C1T6'C GABAAR when
activated by 100 µM GABA (see Fig. 4C and
Table II). It appears that the T6'C residues of both the 1 and 1
subunits contributed to this effect as DTT had a similar effect on the
1WT1T6'C GABAAR and the
1T6'C1WT GABAAR (Table II).
The DTT-potentiated currents in the
1T6'C1T6'C GABAAR declined
progressively when the cell was perfused in DTT-free bathing solution
(Fig. 4C). The potentation observed in both the WT and
mutant receptors may have been because of either the reduction of
endogenous disulfide bonds or a pharmacological effect of DTT at the
alcohol or anesthetic binding site (23). To discriminate between these
two possible modes of action, we investigated the effect of 200 mM ethanol in the presence of a saturating (100 µM) GABA concentration on both the
1WT1WT GABAAR and the
1T6'C1T6'C GABAAR. As
summarized in Table II, ethanol had no significant effect on either
receptor, indicating that DTT was acting by reducing endogenous
disulfide bonds.
When applied in the closed channel state, Cu:phen had no effect on the
1WT1WT GABAAR (Table II, Fig.
5A, left panel). However, in the 1T6'C1T6'C
GABAAR, the rate of current reduction upon removal of DTT
was accelerated dramatically by Cu:phen (Fig. 5B). Following
the removal of DTT, the GABA-activated current reduced to 76 ± 3% (n = 3) after 20 s in the standard bathing
solution. However, in the presence of Cu:phen, the GABA-activated current magnitude reduced to 3.3 ± 2% (n = 3) of
control magnitude after 20 s. When combined with the results
obtained using DTT, these results indicate that disulfide bonds form
spontaneously, but relatively slowly, in the closed state in the
1T6'C1T6'C GABAAR. Because
this slow rate of disulfide bond formation complicated investigations
into the reactivity of the 6' cysteines, all subsequent experiments on
1T6'C1T6'C GABAARs in the
closed state were performed immediately following a 2-min exposure to 10 mM DTT to ensure that all 6' cysteines were in the
reduced state. Then, the effects of subsequent pharmacological
manipulations were compared with the effects of spontaneous disulfide
formation in the same cell.
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Fig. 5.
Effects of 100:400
µM Cu:phen and 0.3%
H2O2 on currents activated by saturating GABA
concentrations in WT and mutant GABAARs. All
recordings shown in this figure were commenced immediately after the
completion of a 1-min cell exposure to 10 mM DTT.
A, in the 1WT1WT
GABAAR, Cu:phen has no effect on the saturating (20 µM) GABA-activated current magnitude, regardless of
whether it was applied in the closed or open states. B, both
traces were recorded from the same cell expressing
1T6'C1T6'C GABAARs. In the
left panel, a gradual reduction in the magnitude of currents
activated by 100 µM GABA is observed upon switching the
bath solution to the standard control solution. In the right
panel, the current reduction rate was greatly accelerated by
100:400 µM Cu:phen and reversed by a subsequent
application of 10 mM DTT. C, when applied
together with 100 µM GABA in the desensitized state,
Cu:phen reversibly reopens the channels. However, a subsequent GABA
application reveals a dramatic current reduction that is reversed by 10 mM DTT, implying the formation of disulfide bonds in the
closed or desensitized states. D, results of a similar
experiment to C, but using 0.3%
H2O2 in place of Cu:phen.
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When applied in the presence of 20 µM GABA,
Cu:phen had no effect on the 1WT1WT
GABAAR (Table II, Fig. 5A, right
panel). However, when Cu:phen was applied to the
1T6'C1T6'C GABAAR in the
presence of 100 µM GABA, it had two distinct effects. First, it reversibly reopened the channel from the desensitized state
(Fig. 5C). Second, following the removal of Cu:phen, the peak magnitude of GABA-activated currents was decreased dramatically (see Table II and Fig. 5C). This reduction in current was
not spontaneously reversible but was reversed by a 30-60-s application of 10 mM DTT (see Table II and Fig. 5C).
We were surprised by the ability of GABA + Cu:phen to reopen the
channels and investigated this phenomenon further. The reopening effect
was found to require the simultaneous presence of GABA and Cu:phen. If
either reagent was removed, the receptor immediately resumed a
non-conducting configuration (n = 5 for each
condition). Application of 100 µM CuSO4 in
the presence of GABA caused no detectable current activation
(n = 3 cells), thus eliminating a putative
pharmacological action of copper. Furthermore, in the continuous
presence of GABA, a second application of Cu:phen elicited a current of
similar magnitude to the first (n = 3 cells). This last
observation eliminated the possibility that the formation of disulfide
bonds following the first application of Cu:phen may have closed the
channels and prevented Cu:phen from subsequently reopening them.
Finally, H2O2 also caused a dramatic 87 ± 3% (n = 4) reduction in the magnitude of the
GABA-activable current that was reversed by 10 mM DTT (Fig.
5D). However, H2O2 did not activate
the receptors convincingly. Although Cu:phen activated a current with a
magnitude of 28 ± 3% (n = 3) of the saturating GABA-activated current magnitude, H2O2
activated a current of only 7 ± 2% (n = 4) of
the saturating GABA current magnitude. This difference was significant
(p < 0.05) using a one-way analysis of variance.
MTSET was used to further investigate the state-dependent
surface accessibility of the 6' cysteines. MTSET had no significant effect on the 1WT1WT GABAAR
regardless of whether it was applied in the absence or presence of GABA
(see Fig. 6A and Table II). However, when MTSET was applied to the
1T6'C1T6'C GABAAR in the
closed channel state, its effects closely resembled those of Cu:phen.
Following the removal of DTT, the GABA-activated current reduced to
74 ± 6% (n = 4) after 20 s in the standard bathing solution (e.g. Fig. 6B, left
panel). However, in the presence of MTSET, the GABA-activated
current reduced to 14 ± 3% (n = 4) of control
magnitude after 20 s.
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Fig. 6.
Effect of 1 mM MTSET and 2.5 mM MTSEA on GABAARs. All recordings shown
in this figure were commenced immediately after the completion of a
1-min cell exposure to 10 mM DTT. A, MTSET had
no significant effect on the 1WT1WT
GABAAR regardless of whether applied in the absence
(left panel) or presence (right panel) of a
saturating (20 µM) GABA concentration. B, both
traces were recorded from the same cell expressing
1T6'C1T6'C GABAARs. GABA was
applied at a saturating (100 µM) concentration
throughout. The left panel shows the effect of exposure to
standard bathing solution immediately following removal of DTT. In the
right panel, the current reduction rate was greatly
accelerated by MTSET and reversed by a subsequent application of 10 mM DTT. C, when applied to together with 100 µM GABA in the desensitized state, MTSET reopens the
channels and locks them in the open state after the removal of GABA.
This effect is reversed by 10 mM DTT, and a subsequent GABA
application activates the original control current magnitude.
D, application of 2.5 mM MTSEA in the closed
state induces partial irreversible activation of the
1T6'C1T6'C GABAARs and a
decrease in magnitude of a subsequent application of 100 µM GABA. The channels were returned to the closed state
by 10 mM DTT. E, when applied together with 100 µM GABA in the desensitized state, MTSET reopens the
channels and locks them in the open state after the removal of GABA.
This effect is reversed by 10 mM DTT.
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The effect of MTSET on the 1T6'C1T6'C
GABAAR was also examined in the desensitized state. In this
experiment, GABA was applied 2 s before MTSET to ensure that
>90% of receptors were in the desensitized state. MTSET was found to
re-open the channels from this state (Fig. 6C). This
reaction proceeded with an average time constant of 35 ± 9 s
(n = 4), indicating a mean reaction rate of 29 M1s1. Upon removal of both
MTSET and GABA, the current magnitude reduced to a steady-state level
of 31 ± 5% (n = 4) of the peak MTSET-induced current magnitude (Fig. 6C), indicating that around
one-third of the channels were held in the open state. MTSET
modification also strongly reduced the magnitude of the current that
was available for activation by GABA (Fig. 6C), indicating
that the remainder of the channels were returned to the closed
desensitized state. The MTSET-modified receptors were returned
efficiently to the closed state by DTT, and a subsequent application of
GABA activated the currents with a peak magnitude similar to the
original control (Fig. 6C). Similar results were observed in
each of four cells.
These results indicate that the effects of MTSET on the
1T6'C1T6'C GABAAR depend on
whether it is applied in the closed or desensitized states. Because it
is unlikely that both actions could have been mediated by covalent
attachment of the same ethyltrimethylammonium group, it is possible
that at least one of the actions may have been mediated by MTSET acting as an oxidizing reagent or by reacting with a non-identical set of subunits.
MTSEA, applied in either the closed and open states, has been shown
previously to irreversibly reduce the magnitude of currents in
Xenopus oocyte-expressed 1T6'C1
2
GABAARs (8). In this study, we investigated the effect of
2.5 mM MTSEA on the 1WT1WT
GABAAR and the 1T6'C1T6'C
GABAAR expressed in HEK293 cells. As summarized in Table
II, MTSEA had no effect on the 1WT1WT
GABAAR in either the absence or presence of a saturating
GABA concentration. However, when applied in the closed state to the
1T6'C1T6'C GABAAR, it
irreversibly activated the channels to 30 ± 5%
(n = 3) of the peak current magnitude while
simultaneously reducing the magnitude of the current activated by a
saturating (20 µM) concentration of GABA (see Fig. 6D and Table II). A 10 mM DTT application
efficiently closed the channels and restored the original magnitude of
the GABA-activated current. When applied together with 20 µM GABA in the channel-desensitized state, MTSEA mimicked
the effect of MTSET in returning the channels to the open state (see
Fig. 6E and Table II).
Effect of 6' Mutagenesis on GlyR Function--
To further probe
the relationship between the physicochemical properties of the 6'
residue and the function of the receptor, we introduced a series of
mutations at the 6' position of the GlyR 1 subunit. The identity of
these mutations and their effects on Imax,
EC50, and nH values of glycine-gated
currents in 1 homomeric receptors are summarized in Table I. This
table also shows the effect of each mutation on the picrotoxin
sensitivity of currents activated by the EC50 glycine
concentrations as indicated. Note that GlyRs incorporating serine,
glutamine, glutamic acid, and lysine mutations did not yield measurable
currents. Interestingly, glutamine, glutamic acid, and lysine were the
most polar amino acids tested.
The EC50 is a measure of the free energy input required to
activate the receptor. If channel opening is accompanied by a movement of the 6' residue toward an increasingly hydrophilic environment, it
might be expected that the ease of activating the receptor should be a
function of the hydropathy of the introduced amino acid. This was
investigated by plotting the glycine EC50 values against
some properties of the substituted amino acids (Fig.
7). This figure reveals that there was no
significant correlation between glycine EC50 and side-chain
volume, hydrophilicity, hydrophobicity, or hydropathy. We conclude that
the relationship between the channel gating energy and the
physicochemical properties of the introduced residues is complex.
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Fig. 7.
Correlation between the mean glycine
EC50 and the physicochemical properties of the introduced
amino acids at the GlyR 6' position. The log (EC50)
for glycine was plotted against the amino acid volume (27),
hydrophobicity (28), hydrophilicity (29), and hydropathy (30). The
p value refers to the probability that the linear
coefficient R value was zero.
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DISCUSSION |
GlyR in the Closed and Open States--
When applied in the
absence of glycine, MTSET has no effect, but when applied in the
presence of glycine, MTSET locks the 1T6'C GlyR in the
open state (17). Because this action is not mimicked by oxidizing
reagents, MTSET must act by adding a polar quaternary ammonium group to
one or more 6' cysteines in the open state only. This attached group
prevents the channel from closing either by steric hindrance because of
its size or by biasing the conformational equilibrium toward the open
state because of its affinity with the aqueous pore environment. The smaller hydrophilic cysteine-specific reagent, MTSEA, also modified T6'C in the open state only. However, MTSEA-modified GlyRs
closed readily upon removal of glycine. Together, these
observations indicate that GlyR channel opening is accompanied by an
increase in the exposure of the 6' cysteines to the external aqueous
environment. This may arise because of either 1) an increase in the
ionization state of the cysteines because of a transition from a
hydrophobic (protein interior) to a hydrophilic (pore-lining)
environment or 2) the removal of a barrier impeding the accessibility
of the cysteines to externally applied MTS reagents.
Limited support for the former alternative is provided by the
mutagenesis experiments summarized in Fig. 7 and Table I. In particular, the three most polar substitutions, glutamic acid, glutamine, and lysine, did not yield functional receptors. It is
possible that these residues could not tolerate being buried in a
hydrophobic environment in the closed state and induced a conformational change that disrupted receptor function. Apart from
these three residues, there was a poor correlation between amino acid
physicochemical properties and glycine EC50 values, implying a complex effect of the T6' substitutions on GlyR activation energetics.
The GABAAR in the Closed State--
When expressed in
HEK293 cells, DTT induced a large (~ 400%), reversible current
increase in the 1T6'C1T6'C GABAAR. Conversely, Cu:phen or MTSET caused a dramatic
decrease in current magnitude. This current reduction was not
spontaneously reversible but was reversed by a further application of
DTT. The most likely explanation is that Cu:phen promoted the formation of disulfide bonds in the channel closed state, thereby preventing the
channels from opening. The ambient dissolved oxygen in the control
bathing solution may have been sufficient to catalyze the formation of
these disulfides at a slow rate. By reducing these bonds, DTT would
have increased the number of receptors available for activation. MTSET
appeared to be acting as an oxidizing reagent as its effect in the
channel closed state mimicked that of Cu:phen but differed drastically
from its effect in the channel-desensitized state. As discussed below,
MTSET directly modified the 6' cysteines in the desensitized state. In
the closed state it is likely that MTSET either modified the 6'
cysteines on the other (non-identical) subunit or indeed behaved as an
oxidizing agent.
When applied in the closed state, MTSEA had two effects on the
DTT-reduced 1T6'C1T6'C
GABAARs. First, it locked the receptors into a partially
open state, and second, it reduced the magnitude of the GABA-activated
current (Fig. 6D). Both effects were reversed by DTT.
Because MTSEA had virtually identical effects when applied in the
desensitized state (Fig. 6E), both effects were most likely
to have been the result of direct MTSEA modification of the 6'
cysteines. These results agree in part with those of Xu and Akabas (8).
They found that the 6' cysteines of Xenopus oocyte-expressed
1T6'C1WT GABAARs also reacted
with MTSEA in the closed and open states. However, they found that
MTSEA reduced current flux but did not lock the receptors into the
partially open state.
Biochemical cross-linking experiments on the same GABAAR
subunits expressed in HEK293 cells show that intersubunit dimers do not
form in the presence of Cu:phen in the closed state (18). Because both
the 1 and 1 subunits contain endogenous cysteines in
membrane-spanning domains, the disulfide bond formation is therefore
likely to occur between the 6' and endogenous cysteines within a single
subunit. Following their reduction by DTT in the closed state, the 6'
cysteines remain inaccessible to direct covalent modification by MTSET
but accessible to modification by the smaller MTSEA.
The GABAAR in the Open and Desensitized States--
In
Xenopus oocyte-expressed
1T6'C1T6'C GABAARs, the
co-application of Cu-phen with a saturating concentration of GABA
locked the channels in the open state (18). In contrast, when the same
1T6'C1T6'C GABAARs were
expressed in HEK293 cells, the GABA-gated currents desensitized too
rapidly to reliably apply cysteine-reactive reagents in the open state. Following the application of H2O2 or Cu:phen
with GABA in the channel-desensitized state, the current magnitude was
reduced dramatically. Because this effect was reversed by DTT, it is
concluded that disulfide bond formation locked the channels in the
desensitized state. However, it is important to note that
desensitization is not necessarily accompanied by disulfide bond formation.
Biochemical cross-linking experiments on the same GABAARs
expressed in HEK293 cells show that 1 subunits dimerized only in the
presence of both GABA and Cu:phen (18). When taken in isolation, this
experiment does not resolve whether the dimerization occurred in the
open or desensitized states. However, when taken together with the
electrophysiological data presented here, the results strongly suggest
that 1 subunit dimerization occurs in the desensitized state.
We were surprised to find that the co-application of GABA and Cu:phen
reopened the channels from the desensitized state. Even more surprising
was the observation that a second application of Cu:phen activated a
current with similar magnitude to the first, as this implies that
Cu:phen can open dimerized channels. Although we do not understand the
mechanism by which this occurred, it was unlikely to have been an
effect of oxidation as it was not replicated by
H2O2, and it was not a pharmacological effect
of copper.
When MTSET or MTSEA were applied in the desensitized state, they locked
around 30% of the channels into the open state with the remainder
being returned to the desensitized state. Because this effect was not
mimicked by Cu:phen or H2O2 but was reversed by
DTT, it must have been because of the direct covalent modification of
the 6' cysteines. The extremely slow reaction rate implies that
access to the 6' cysteines in the desensitized state was limited by
steric hindrance, a non-polar environment, electrostatic repulsion, or
a combination of these factors. One possibility is that the reaction
could occur only during rare spontaneous transitions from the
desensitized to the open state (24). In this case, the MTSET or MTSEA
modification may have sterically prevented the channel from re-closing.
Alternatively, the reaction may have proceeded slowly in the
desensitized state. In this case, the increased hydrophilicity of the
attached group may have opened the channels by favoring a conformation
where the 6' side chain had increased exposure to the aqueous pore. The
difference in 6' cysteine reactivity with MTSET between the closed and
desensitized states provides strong evidence for a pore structural
difference between these configurations. This is consistent with a
recent study on the nAChR that also showed a different pore structure between the closed and desensitized states (25). Interestingly, the
nAChR 6' cysteine was accessible to MTSEA in the closed state but not
in the desensitized state (25), implying that the structural basis of
desensitization is not identical to that observed here for the
1T6'C1T6'C GABAAR.
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CONCLUSIONS |
The closed state reactivity of 6' cysteines in the GlyR and the
GABAAR differ in two respects. First, the
GABAAR 6' cysteines spontaneously form disulfide bonds in
the closed state, whereas those of the GlyR do not. Second, the
GABAAR 6' cysteines are accessible to externally applied
MTSEA whereas the GlyR 6' cysteines are not. Although it is not
possible to define the structural basis for these differences, these
results provide evidence for divergent pore structures in the closed
channel state. Closed state structural differences have been identified
previously in cationic members of the LGIC family. Although the nAChR
pore was shown to admit externally applied MTSEA and MTSET as far
as the 2' residue (4-6), access of the same compounds in the
5HT3R pore was impeded near the 14' residue (10). Thus,
closed state pore structures show considerable variation in both
anionic and cationic members of the LGIC family.
On the other hand, substituted cysteine accessibility studies reveal
that cationic LGIC family members have remarkably similar patterns of
M2 domain residue exposure in the channel open state (4-7, 9, 10). Of
particular relevance to the present study, MTSET modification of 6'
cysteines irreversibly inhibited current in both the nAChR and
5HT3R, whereas MTSES had no effect on either receptor
(4-7, 9, 10). The present study could not directly compare 6' cysteine
accessibility in the open states of the 1T6'C GlyR and
1T6'C1T6'C GABAAR because of
the fast desensitization rate of the
1T6'C1T6'C GABAAR. The
observation that MTSET locked both receptors into the partially open
state provides strong evidence for a common activation mechanism in
this part of the pore. However, the pore structures are unlikely to be
identical as MTSEA also locked the
1T6'C1T6'C GABAAR in the open
state but had no such effect on the 1T6'C GlyR.
The present study reveals distinct differences in the properties of
GABAARs expressed in Xenopus oocytes and HEK293
cells. When expressed in HEK293 cells, the 6' cysteines can form
disulfide bonds in the closed state. However, this does not occur when
the same receptors are expressed in Xenopus oocytes
(18). Furthermore, when expressed in HEK293 cells, the
GABAAR is locked in the desensitized state by Cu:phen, but
when expressed in Xenopus oocytes, it is locked in the open
state by Cu:phen (18). Together, these results indicate the surface
orientation of the GABAAR 6' cysteines varies dramatically
depending on the expression system. Moreover,
1T6'C1T6'C GABAARs expressed
in HEK293 cells desensitize at a much faster rate than they do when
expressed in Xenopus oocytes. These structural and
functional differences could be because of expression system-specific differences in subunit folding and assembly, post-translational modifications, or membrane lipid composition. Regardless of their origin, the results indicate that caution should be applied when comparing results obtained using the two expression systems.
,
TOP
ABSTRACT
INTRODUCTION
Supported by an Ernest Singer postgraduate scholarship awarded by
the University of Queensland. Current address: Cardiovascular Research
Inst., University of California, San Francisco, CA 94143-0130.
