From the
Department of Physiology and
Pharmacology, School of Biomedical Sciences, University of Queensland,
Brisbane, Queensland 4072, Australia, and ¶St.
Vincent's Institute of Medical Research, 9 Princes St., Fitzroy, Victoria
3065, Australia
Received for publication, January 6, 2003 , and in revised form, April 25, 2003.
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ABSTRACT |
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INTRODUCTION |
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The GlyR is a member of the ligand-gated ion channel (LGIC) receptor
family, which includes the nicotinic acetylcholine receptor cation channel
(nAChR), the -aminobutyric acid type-A and type-C receptor chloride
channels (GABAAR and GABACR), the serotonin receptor
cation channel (5HT3R), and a recently identified zinc-gated cation
channel (10) as well as
invertebrate glutamate and histamine receptors
(11). These receptors are
composed of five structurally similar subunits arranged in a ring to form a
central ion-conducting pore
(12). Adult GlyRs in
vivo are composed of
and
subunits in a 3
/2
stoichiometry (13). Although
subunits readily form homomeric GlyRs in heterologous expression
systems,
subunits form functional receptors only as heteromers with
subunits. The molecular mechanisms of zinc inhibition and potentiation
have recently been investigated in several laboratories
(7,
1416).
Although the molecular basis of potentiation has so far proved elusive, a
strong case has been advanced for a candidate inhibitory site. Harvey et
al. (15) showed that zinc
inhibition of the recombinant
1 GlyR was selectively
abolished by either reducing the pH or by pretreating receptors with a
histidine-specific modifying agent. Since both treatments effectively reduce
the ability of zinc to bind to histidine imidazole rings, this implicated
histidines in the coordination of inhibitory zinc. Mutations of either
His107 or His109 were subsequently shown to abolish zinc
inhibition, strongly suggesting that these residues coordinate zinc at its
inhibitory site (15).
For a pair of histidine side chains to coordinate a zinc ion, the
-carbon atoms need to be within 13 Å of each other
(17). Since histidines 107 and
109 are separated by only one residue, it is certainly feasible that this pair
could coordinate zinc ions within individual
subunits. Indeed,
histidines with this spacing occur in many structurally defined zinc-binding
sites (17), including carbonic
anhydrase II (Protein Data Bank code 1CA2
[PDB]
)
(18), which contains the same
HFH tripeptide as GlyR
1 107109, thereby providing a
plausible model for zinc binding within individual GlyR
1
subunits (15). The recently
resolved crystal structure of a soluble acetylcholine-binding protein (AChBP)
from the snail, Lymnaea stagnalis, has provided a structural model
for the N-terminal ligand-binding domain of LGICs
(19). Modeling of the GlyR
N-terminal domain, based on AChBP, places His107 and
His109 near the lumen of the channel vestibule close to the axis of
symmetry (20). In this
position, His107 and His109 from adjacent
subunits could be close enough to coordinate zinc ions across the subunit
interface, as recently predicted by Laube et al.
(21).
The aim of this study was to use a combination of homology modeling based on the AChBP crystal structure, coupled with functional analysis of GlyRs incorporating site-directed mutations in putative zinc-binding regions, to provide insights into the structure of the inhibitory zinc-binding site.
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EXPERIMENTAL PROCEDURES |
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ElectrophysiologyThe 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 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM 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 1.53 megaohms when filled with the standard pipette solution, which contained 145 mM CsCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM 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 pclamp6 software (Axon Instruments, Foster City, CA). The cells were perfused by a parallel array of microtubular barrels through which solutions were gravity-induced.
Because the subunit can efficiently assemble into functional GlyRs
as either
homomers or
heteromers, it is necessary to
confirm the incorporation of
subunits into functional receptors. This
was achieved in two ways. First, green fluorescent protein expression was used
to identify cells expressing GlyR
subunit protein. Second, picrotoxin
sensitivity was used as a functional assay of the incorporation of
subunits into heteromeric GlyRs
(22). When co-expressed with
subunits in HEK293 cells, incorporation of
subunits increases
the picrotoxin IC50 from around 25 to 500 µM in the
presence of an EC50 glycine concentration
(23). In the present study, we
measured the effect of 1 mM picrotoxin on the magnitude of currents
activated by an EC20 glycine concentration, and cells were assumed
to express
heteromeric GlyRs if 1 mM picrotoxin
inhibited the current by less than 50%.
Data AnalysisThe empirical Hill equation, fitted by a nonlinear least squares algorithm (Sigmaplot; Jandel Scientific, San Rafael, CA), was used to calculate the EC50, the IC50, and the Hill coefficients (nH) of excitatory and inhibitory dose-response curves. Statistical significance was determined by a one-way analysis of variance using the Student-Newman-Keuls post hoc test for unpaired data (Sigma Stat; Jandel Scientific), with p < 0.05 representing significance.
Molecular ModelingModels of the extracellular region of the
human GlyR 1 subunit were constructed by homology modeling,
using the crystal structure of AChBP from the snail Lymnaea stagnalis
as a template (Protein Data Bank entry 1I9B
[PDB]
)
(19). Sequence identity
between LGICs and AChBP is only 1524%, in the "twilight
zone" for effective alignment and homology modeling
(24). To improve the
reliability of the alignment, we used ClustalW
(25) to align AChBP with a
large number of Cys loop LGICs, particularly those that are the most similar
to AChBP, such as nAChRs, as described in Ref.
20. Secondary structure
predictions using the PHD prediction algorithm
(26) assisted the alignment in
regions of particularly low homology, such as the N-terminal
-helix.
Swiss-Model (27) was used to
create independent monomer models for each alignment tested. Loop regions that
correspond to gaps in the alignment were modeled by fitting structures from a
loop data base and are the least reliable sections of the models. Acceptable
monomer models were then assembled into pentamers using AChBP as a scaffold.
The assembled pentamer was energy-minimized using INSIGHT II DISCOVER
(Accelrys, San Diego, CA) to eliminate any obvious problems such as steric
clashes. Ramachandran plots and Verify-3D scores
(28) were used to assess the
quality of each model. For regions of a model that scored poorly, other
possible alignments were tested. Four different alignments were tested in the
region in the putative inhibitory zinc-binding region surrounding
His107 and His109
(Fig. 1A). Excluding
the inhibitory zinc-binding region, there are only three residues that fall in
the disallowed region of a Ramachandran plot, Leu83 and
His201, which are in loops corresponding with gaps in the
alignment, and Lys33, which corresponds to Arg23 in
AChBP, which also falls in the disallowed region. Total Verify-3D scores were
above 64 for all models, which is quite acceptable for this type of model
(28). With a sliding window of
21 residues, there are no regions with a negative score, but there are three
regions that score below 0.2. These are the N-terminal helix and the loop
between the second and third
-strands, which are both likely to interact
with residues 110, and the signature Cys loop, which is likely to
interact with the membrane domain. Since our models do not include residues
110 or the membrane domain, they cannot fully represent the native
environment for the three regions mentioned. Finally, a conserved
N-glycosylation site at Asn38 is on the surface of the
model, consistent with it being glycosylated, and the unique disulfide bond of
GlyRs is present in our models.
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RESULTS |
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His107 and His109 from Adjacent Subunits Form the
Inhibitory Zinc-binding SiteTo discriminate experimentally between
intrasubunit and intersubunit models for the inhibitory zinc-binding site, we
examined the zinc sensitivity of GlyRs formed from co-expression of
H107A and
H109A subunits, each of which
individually shows reduced zinc sensitivity. The rationale was that if the
inhibitory zinc-binding site is formed within a subunit, then these mixed
receptors should be zinc-insensitive, since each subunit contains only one of
the two required histidine residues. Therefore, if the resultant recombinant
receptors possess a high affinity zinc inhibitory site, then this site must be
formed at the interface between subunits. It is important to note that this
approach cannot address the question of whether zinc can also be coordinated
within individual subunits. Hence, in all our experiments, it is necessary to
eliminate at least one histidine (His107 or His109) per
subunit to ensure that this does not confound the interpretation of our
results.
The mean glycine EC50, nH, and
Imax values for the WT and all mutant GlyRs employed in
this study are shown in Table
I. As demonstrated previously, the WT GlyR is
highly sensitive to zinc inhibition. As expected from previous studies
employing mutations at these positions
(7,
15,
16,
29), none of these mutations
had dramatic effects on the glycine EC50, nH,
or Imax values. The inhibitory zinc dose responses for the
H107A GlyR, the
H109A GlyR, the double
mutant
H107A,H109A GlyR, and the co-expressed
(
H107A +
H109A) GlyR were measured in the
presence of an EC20 glycine concentration. Zinc inhibition was
quantitated by measuring the steady-state level of the current following
application of zinc and expressing this as a fraction of the peak magnitude of
the zinc-potentiated current
(14). Sample responses to
increasing concentrations of zinc for each of these receptor constructs are
shown in Fig. 2A.
Averaged zinc inhibitory dose responses are plotted in
Fig. 2B, and the mean
zinc IC50 and nH values for these and all other
recombinant constructs examined in this study are summarized in
Table I. The H107A mutation
reduced the inhibitory potency by a factor of 15, whereas the H109A mutation
almost entirely abolished zinc inhibition
(Table I), consistent with
earlier results (15) and the
proposed role of these residues in zinc coordination. The difference in the
magnitude of the effect of the two mutations indicates that the zinc
coordination is not perfectly symmetric, consistent with the probable
involvement of other residues
(16). As expected, the double
mutant
H107A,H109A GlyR was also insensitive to zinc
inhibition (Fig.
2A).
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When the two single mutant H107A +
H109A subunits were co-expressed, high sensitivity zinc
inhibition was observed (Fig. 2, A
and B). The zinc sensitivity (21 ± 5
µM; Table I) was
not significantly different from that of the
WT GlyR (16
± 8 µM), consistent with it being due to the same
zinc-binding site. This inhibition could not have been mediated by zinc ions
binding within individual subunits, since each subunit contained only one of
the two required histidine residues. Therefore, it must have been due to
intersubunit coordination of zinc ions by His107 and
His109 from different subunits, supporting the hypothesis of an
intersubunit zinc site and the positioning of the inhibitory zinc-binding
region in our initial model (Fig.
1). The two coordinating histidines almost certainly come from
adjacent subunits, since the constraints imposed on the model by the conserved
anchor points make it virtually impossible for His107 and
His109 from nonadjacent subunits to get close enough to coordinate
zinc across the vestibule lumen. The fact that the inhibition seen with the
mixed
H107A +
H109A GlyRs was not seen in
either of the single mutants alone indicates that His107 and
His109 each occupy a specific subunit "face" (either +
or ; see Fig. 1) and are
not flexible to coordinate zinc at either face. Therefore, an intersubunit
zinc site can only occur at one of the two possible +/interfaces,
either H107A/H109A or H109A/H107A. This limits the number of inhibitory zinc
sites in a pentamer of mixed
H107A +
H109A
subunits to a maximum of two (Table
II), demonstrating that two such sites are sufficient for
inhibition by zinc. The relatively low nH for zinc
inhibition in WT GlyRs and our finding that it is unchanged in the mixed
H107A +
H109A GlyRs support the view that
one or two bound zinc molecules may be sufficient for maximal zinc inhibition
in the WT GlyR (Table II).
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Zinc inhibition of the mixed H107A +
H109A GlyRs was incomplete
(Fig. 1, A and
B), however, inhibiting a maximum of 55 ± 5%
(n = 6) of the glycine-activated current. In contrast, a saturating
(1 mM) zinc concentration inhibits the
WT GlyR
current by 100% (Fig.
1B; see also Refs.
7 and
14). The reduced extent of
zinc inhibition is not unexpected, given that each receptor in the mixed
population of mutant receptors contains zero, one, or two zinc-binding sites,
relative to five sites in WT receptors. Assuming the
H109A
and
H107A subunits recombine in a random binomial manner to
form pentameric GlyRs, 6.2% of recombinant GlyRs should have no zinc-binding
sites, 62.4% should have one inhibitory zinc-binding site, and 31.2% should
contain two inhibitory zinc-binding sites per receptor
(Table II). The 55% overall
inhibition could be explained by GlyRs with two zinc sites being inhibited
completely and those with one site being partially inhibited. Although this is
a reasonable explanation, random recombination cannot be assumed, and other
explanations are possible. In summary, the data above show that an
intersubunit zinc-binding site can account for the zinc inhibition observed in
WT
1 homomeric GlyRs but do not rule out an additional role
for intrasubunit binding.
Possible Involvement of Thr112 in Zinc
CoordinationLaube et al.
(16) showed that a T112A
mutation abolished inhibition by zinc and proposed that Thr112 is
also involved in the coordination of zinc at its inhibitory site. If
Thr112 directly coordinates zinc at an intersubunit inhibitory
site, then co-expression of either the H107A or
H109A subunits with the
T112A subunit
should be able to restore zinc sensitivity, as observed for the two histidines
above. To test this concept, we made the more conservative T112V mutation,
which simply replaces the hydroxyl group of the threonine with a methyl group.
As with the T112A mutation
(16), the T112V mutation had
little effect on glycine sensitivity but completely eliminated sensitivity to
zinc inhibition (Table I). As
expected, the double mutant
H107A,T112V and
H109A,T112V GlyRs were also insensitive to zinc inhibition
(Table I). However, these
experiments provide no information as to whether T112V caused a nonspecific
structural disruption to the zinc coordination scaffold or whether it directly
participated in zinc coordination. To test the possibility of direct
coordination, we co-expressed
H107A +
T112V,
H109A +
T112V, or
H107A +
H109A,T112V subunits. As
summarized in Table I, none of
these mixed receptors was significantly inhibited by 1 mM zinc. The
fact that co-expression did not restore zinc sensitivity provides no evidence
to support a direct role for Thr112 in coordinating zinc. Although
T112V mutant subunits contain both His107 and His109,
the T112V mutation eliminates functional inhibitory zinc-binding sites at both
faces of the subunit, by either 1) disrupting the structure of the
zinc-binding region, either directly or allosterically, or 2) precluding the
formation of GlyRs containing both mutant subunits. These data do not,
however, rule out a direct role for Thr112 in zinc coordination,
since it could have both a coordinating and a more structural role.
Zinc Coordination by Heteromeric
GlyRsTo further characterize the inhibitory zinc-binding site, we
examined its properties in the
1
heteromeric GlyR. As
stated under "Experimental Procedures," the incorporation of
subunits into
heteromers was confirmed for each cell by
measuring the percentage inhibition of the EC20 glycine current
that was inhibited by 1 mM picrotoxin. If the picrotoxin inhibited
this current by no more than 50% (c.f. Refs.
22,
23, and
30), it was assumed that all
recombinant receptors in that cell contained 3
and 2
subunits
(13). Since the arrangement of
the
and
subunits around the pentamer has not been determined,
we considered the possibility that the
subunits may be located either
side-by-side or separated by an
subunit
(Table III).
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Examples of the effects of zinc on heteromeric
WT
WT GlyRs are shown in
Fig. 3A, and the
averaged zinc dose response is plotted in
Fig. 3B. The mean zinc
IC50 and nH values are summarized in
Table I. As previously
demonstrated (7), the zinc
inhibitory potency of these receptors is similar to that of the homomeric
WT GlyR (Table
I). The
subunit contains a histidine at position 132, which
is homologous with His109 in the
subunit. However, it
contains an asparagine (Asn130) at the position homologous with
His107. Since asparagine residues are not known to coordinate zinc
ions (17), it is feasible that
only the
subunit His132, located at the plus side of the
-
subunit interface, may coordinate zinc ions. Given this
possibly, heteromeric
WT
WT GlyRs may
contain a maximum of three intersubunit zinc coordination sites as shown in
Table III (top row).
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Several approaches were employed to determine whether -
subunit interfaces could coordinate zinc ions. First, we incorporated the
subunit H132A mutation to determine its effect on zinc inhibitory
potency. The resultant
WT
H132A GlyRs
contain either one or two
-
subunit interfaces, depending on the
subunit arrangement (Table III,
row 2). As shown in Fig. 3, A and
B, and summarized in
Table I, these receptors were
highly sensitive to zinc inhibition, with a mean IC50 of 11
± 4 µM (n = 3). Thus, heteromeric GlyRs
containing a maximum of two zinc inhibitory sites can be maximally inhibited
by zinc. These results provide no evidence for a contribution of putative
-
interface zinc sites to zinc-induced inhibition of glycine
currents.
The second approach involved removing all -
subunit zinc
sites via the H109A mutation. The resultant heteromeric GlyRs would then have
contained either one or two
-
interface sites, depending on the
subunit arrangement (Table III,
row 3). Since the resultant
H109A
WT GlyRs
receptors were completely insensitive to zinc inhibition
(Fig. 3, A and
B, and Table
I), whereas heteromers containing a similar number of sites at
-
subunit interfaces were highly zinc-sensitive, it is concluded
that inhibitory zinc-binding sites are not formed at
-
interfaces
or at
-
interfaces. In a final experiment, we investigated the
zinc sensitivity of the
H107A,H109A
N130H
GlyR. Although experiments described above suggest that this construct would
not be zinc-sensitive, it was considered worth trying, since a high zinc
sensitivity would have been evidence for a side-by-side arrangement of
subunits (Table III, row 4). As
anticipated, however, the construct was completely insensitive to zinc
inhibition (Fig. 3, A and
B, and Table
I). In summary, we found no evidence for a direct contribution of
the
subunit to inhibitory zinc-binding sites.
Possible Conformations of the Inhibitory Zinc-binding
RegionIn our initial model of the GlyR extracellular domain, the
inhibitory zinc-binding region is anchored by the flanking sequences, which
have strong homology to AChBP, but the zinc-binding region itself is not
reliably modeled because of weak homology and the inclusion of 3 extra
residues relative to AChBP. It is now worth reconsidering models of possible
conformations of this region in terms of how well they fit our experimental
evidence for intersubunit inhibitory zinc-binding sites. Since subunits
do not appear to contribute directly to these sites, we have restricted the
modeling to
1 homopentamers. To sample possible
conformations of this region, we built a series of models (AD), each
based on a different alignment in the 104115 region as shown in
Fig. 1A. To maintain
as much of the AChBP secondary structure as possible, the gaps in the
alignment with the zinc-binding region were placed at either end of the short
-strand in AChBP. Each model was built independently from scratch, as
described under "Experimental Procedures." Models were then
assessed in terms of how well they fit the experimental data and their quality
as protein structures.
We have shown here that intersubunit coordination of zinc by
His107 and His109 can account for essentially all the
properties of zinc inhibition. Therefore, we examined each of the models to
see whether they were consistent with such coordination. The constraints of
zinc coordination by histidine imidazole nitrogens
(17) place the zinc less than
6 Å from the histidine -carbon atom. Consequently, the
-carbon atoms of two histidines coordinating the same zinc must be
within 12 Å of each other. For tetrahedral geometry, the most common
coordination geometry for zinc, the
-carbons must be within 10 Å
of each other. The intersubunit distance between
-carbons of
His107 and His109 is <10 Å for model C (making
tetrahedral coordination possible), <12 Å for models A and D
(suggesting that coordination is possible), but >15 Å for model B
(essentially ruling this model out as a viable model for an intersubunit zinc
site). Given appropriate distance constraints, side chain orientation and
surrounding structure also determine the feasibility of zinc coordination. To
examine the feasibility of zinc binding in each model, we tested whether a
reasonable zinc site could be produced by manually adjusting the side chain
position of the two histidines within allowed rotamers while keeping the
backbone fixed. Model C was able to most closely approach ideal tetrahedral
geometry, but models A and D could also approach a reasonable zinc-binding
site. The best positions achieved are shown in
Fig. 4, together with a
possible location for bound zinc in models A, C, and D. Interestingly, for
models C and D, coordination by His109 and His107 was
from the minus and plus faces, respectively, whereas this was reversed in
model A.
We have also shown that Thr112 appears to have a structural role in the zinc-biding site but may have an additional direct role in zinc coordination. None of our models are consistent with Thr112 directly coordinating zinc, together with His107 and His109. Models C and D do, however, indicate that the side chain hydroxyl, which is removed in both the T112V (this study) and T112A (16) mutants that abolish zinc inhibition, could form hydrogen bonds with other parts of the structure. These potential hydrogen bonds could be determinants of a structural role for Thr112. Although we have not tested its role here, an E110A mutation has been shown to reduce sensitivity to zinc inhibition by 16-fold (16), indicating that it could be a third ligand for coordinating zinc. Of the models that could coordinate zinc by the two histidines, only in models C and D is Glu110 close enough to play a role in coordinating zinc, either directly or through a water molecule. Ramachandran plots and Verify-3D scoring both measure how well a model structure fits the characteristics of real protein structures, independent of the modeling process. Ramachandran plots for our models reveal that the inhibitory zinc-binding region of model C has no residues in the disallowed regions, whereas the other models have one or two residues each in the disallowed regions. Model C also gave a markedly higher Verify-3D score than the other three models, indicating that it can be objectively considered the most reasonable protein structure of the four models. In summary, whereas models A and D are possible models of an intersubunit inhibitory zinc-binding site, model C is our preferred working model.
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DISCUSSION |
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The potency of zinc inhibition is increased as the glycine concentration is
reduced (7). In addition, zinc
inhibition reaches a steady-state level much faster in the absence of glycine
(14). A single channel study
found that high (50 µM) zinc concentrations reduced
1 GlyR open probability by reducing mean channel open time
and the relative abundance of long channel bursts
(16). It concluded that zinc
increased the rate at which the channel exits from the open state. Together,
these results suggest that glycine-induced activation is accompanied by a
structural change at the zinc inhibitory site and that zinc acts to prevent
this by stabilizing the closed conformation. Based on our models, the
inhibitory zinc-binding site is located in close proximity to the
agonist-binding site, and allosteric interaction between the two could occur
via relatively minor local movements. Alternatively, since both these sites
are at subunit interfaces, they could interact via global movements of
subunits relative to one another. Consistent with this idea of intersubunit
movements, our models place Asp80, which has been implicated in the
potentiating zinc-binding site, at the subunit interface on the outside of the
pentamer. Although this position is quite distant from the inhibitory
zinc-binding site, mutations of His109 affect zinc potentiation as
well as inhibition (15).
A model of receptor activation involving global intersubunit movements has been proposed to account for a large body of functional data obtained mainly from the nAChR (reviewed in Refs. 31 and 32). This idea is also supported by recent structural evidence (33). An essential feature of this model is that the largest displacements occur at the subunit interfaces. The GlyR inhibitory zinc-binding site is located in an ideal position to hinder these movements by locking adjacent subunits into a fixed, closed position.
Zinc Sites in Other LGICsAlthough zinc affects most LGIC
members (10,
3437),
to date putative zinc-binding sites have been identified only in the
GABAAR and in the GABACR. In the GABAAR
5 subunit, zinc sensitivity is reduced by a H195D mutation
in the extracellular domain, immediately preceding
-strand 9, close to
the membrane domain (38). In
the GABAAR
subunits, zinc sensitivity is reduced by mutating
a histidine in the second transmembrane domain
(39,
40) or at the position
homologous to His109 in the GlyR
1 subunit
(41). This latter observation
is the only evidence so far that the GlyR zinc inhibitory site characterized
in the present study may also be functional in other LGIC members. However, it
is of interest to note that the recently identified zinc-activated cation
channel (10) contains aspartic
acid residues at positions homologous to 107 and 110 in the GlyR
1 subunit, which could contribute to a channel-activating
zinc site. In GABACR
1 homomers, zinc inhibition is
abolished by mutating a histidine residue at the subunit interface, close to
the ligand-binding site (42).
A recent paper has identified an inhibitory zinc-binding site at the interface
between
1 and
3 subunits of the
GABAAR, on the outside of the extracellular domain close to the
membrane (43), a site that is
quite distinct from the GlyR site characterized here. Finally, there is strong
structural and functional evidence for a potentiating calcium binding site at
the interface between adjacent nAChR
7 subunits
(44). In contrast with the
present study, the high affinity site is proposed to be associated with the
channel open state. Intersubunit binding sites appear to be emerging as a
common means for modulation of LGICs by divalent cations, although the
location of the site may vary. There is some sense to this, given the
intersubunit location of agonist binding sites and the evidence for
intersubunit movement upon agonist binding
(33).
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FOOTNOTES |
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These authors contributed equally to this work.
|| Supported by the National Health and Medical Research Council of
Australia.
** Research in the laboratory of this author is funded by the Australian Research Council. To whom correspondence should be addressed. Tel.: 617-3365-3157; Fax: 617-3365-1766; E-mail: j.lynch{at}mailbox.uq.edu.au.
1 The abbreviations used are: GlyR, glycine receptor; LGIC, ligand-gated ion
channel; AChBP, acetylcholine-binding protein; nAChR, nicotinic acetylcholine
receptor; 5HT3R, serotonin type 3 receptor; GABAAR, GABA
type A receptor; GABACR, GABA type C receptor;
Imax, maximum (saturating) current magnitude; WT, wild
type.
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REFERENCES |
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