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The Journal of Neuroscience, April 15, 1998, 18(8):2944-2953
The Role of an  Subtype M2-M3 His in
Regulating Inhibition of GABAA Receptor Current by Zinc and
Other Divalent Cations
Janet L.
Fisher1 and
Robert L.
Macdonald1, 2
1 Departments of Neurology and
2 Physiology, University of Michigan, Ann Arbor, Michigan
48104-1687
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ABSTRACT |
Sensitivity of GABAA receptors (GABARs) to inhibition
by zinc and other divalent cations is influenced by the  subunit
subtype composition of the receptor. For example, 6 3 2L
receptors are more sensitive to inhibition by zinc than 132L
receptors. We examined the role of a His residue located in the
M2-M3 extracellular domain (rat 6 H273) in
the enhanced zinc sensitivity conferred by the 6 subtype. The 1
subtype contains an Asn (N274) residue in the equivalent location.
GABA-activated whole-cell currents were obtained from L929 fibroblasts
after transient transfection with expression vectors containing
GABAA receptor cDNAs. Mutation of 1
(1(N274H)) or 6 (6(H273N)) subtypes
did not alter the GABA EC50 of 32L receptors.
1(N274H)32L receptor currents were as sensitive to
zinc as 632L receptor currents, although 6(H273N)32L receptor currents had the reduced zinc
sensitivity of 132L receptor currents. We also examined the
activity of other inhibitory divalent cations with varying subtype
dependence: nickel, cadmium, and copper. 632L receptor
currents were more sensitive to nickel, equally sensitive to cadmium,
and less sensitive to copper than 132L receptor currents.
Studies with 1 and 6 chimeric subunits indicated that the
structural dependencies of the activity of some of these cations were
different from zinc. Compared with 632L receptor currents,
6(H273N)32L receptor currents had reduced
sensitivity to cadmium and nickel, but the sensitivity to copper was
unchanged. Compared with 132L receptor currents,
1(N274H)32L receptor currents had increased
sensitivity to nickel, but the sensitivity to cadmium and copper was
unchanged. These findings indicate that H273 of the 6 subtype plays
an important role in determining the sensitivity of recombinant GABARs
to the divalent cations zinc, cadmium, and nickel, but not to copper. Our results also suggest that the extracellular N-terminal domain of
the 1 subunit contributes to a regulatory site(s) for divalent cations, conferring high sensitivity to inhibition by copper and cadmium.
Key words:
GABA; divalent cations; GABA receptor; zinc; cadmium; copper; nickel; recombinant; site-directed mutagenesis;
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INTRODUCTION |
Divalent cations modulate the
activity of many ligand-gated ion channels, including the
GABAA receptor (GABAR). Zinc and copper appear to be
released during synaptic activity and could be important in the
regulation of synaptic transmission (Assaf and Chung, 1984 ; Howell et
al., 1984; Hartter and Barnea, 1988; Kardos et al., 1989; Xie and
Smart, 1991). Other divalent cations may also be involved in
physiological or pathological conditions (Carpenter, 1994). Sensitivity
of native GABARs to inhibition by divalent cations, including zinc, has
regional and developmental dependence (Westbrook and Mayer, 1987; Smart
and Constanti, 1990; Celentano et al., 1991; Legendre and Westbrook,
1991; Smart, 1992; Ma and Narahashi, 1993; Kume et al., 1994; Kumamoto
and Murata, 1995; Trombley and Shepherd, 1996). Sensitivity of GABARs
to zinc also changes with the onset of epilepsy, with decreased
sensitivity after rapid onset of status epilepticus (Kapur and
Macdonald, 1997) and increased sensitivity after chronic
kindling-induced seizure activity (Buhl et al., 1996; Gibbs et al.,
1997). Variations in zinc sensitivity of GABARs may be related to
differences or changes in the subunit subtype composition of these
receptors.
Native GABARs are believed to be composed of a pentameric
combination of at least three different subunit families. Many of these
subunit families have multiple subtypes, including (1-6), (1-3), (1-3),  , and  in mammals (Sieghart, 1995; Davies
et al., 1997). Expression of different GABAR subtypes is regulated in
the brain both regionally and developmentally (Laurie et al., 1992a,b;
Wisden et al., 1992). In particular, expression of mRNAs for 1 and
6 subtypes is very different. Whereas 1 subtype mRNA is widely
and highly expressed throughout the brain, 6 mRNA is restricted to
the cerebellum. Recombinant receptors containing 4, 5, or 6
subtypes, along with a and subunit, are more sensitive to zinc
inhibition than those containing an 1 subtype (Burgard et al., 1996;
Knoflach et al., 1996; Saxena and Macdonald, 1996). Both and subunits reduce zinc sensitivity compared with or
receptors, which are highly sensitive to zinc (Draguhn et al., 1990;
Saxena and Macdonald, 1994; Whiting et al., 1997). Sensitivity to other
divalent cations also varies with brain region and developmental stage
of neurons. Cadmium, nickel, copper, lead, and cobalt have been shown
to inhibit GABAR currents with varying affinities and rank
orders of potency depending on the type and developmental stage of the
neuron examined (Draguhn et al., 1990; Ma and Narahashi, 1993;
Narahashi et al., 1994; Kumamoto and Murata, 1995). It has
been suggested that the zinc and cadmium (Celentano et al., 1991;
Kumamoto and Murata, 1995) or zinc and copper sites (Ma and Narahashi,
1993) may interact or overlap. However, these findings vary depending
on the type of neuron preparation. Except for zinc, there is little
information regarding the GABAR subunit subtype dependence of the
actions of divalent cations.
Previous work with rat 1 and 6 subtype chimeras suggested that
the extracellular bridge between the M2 and M3
transmembrane domains might contribute to the difference in zinc
sensitivity between the 1 and 6 subtypes (Fisher et al., 1997).
We focused on a His residue found only in the 4 and 6 subtypes
(Fig. 1). This residue is near the
M2 putative transmembrane domain that may form the lining
of the channel pore. However, consistent with the voltage-independence
of zinc inhibition (Westbrook and Mayer, 1987; Smart and Constanti,
1990), it is probably not within the pore itself (Xu and Akabas, 1996).
Single-point mutations were made in the 1 subtype, converting the
wild-type Asn (N) to either the 6 His (H) or Asp (D), and in the
6 subtype, converting the wild-type His to the 1 Asn or to Asp.
Asp would be expected also to interact with divalent cations, thus
controlling for alterations in the secondary, tertiary, or quaternary
structure of the mutant receptors. We transiently transfected L929
fibroblasts with cDNAs encoding wild-type, mutant, or chimeric subunits, along with 3 and 2L, and determined the role of the
6 H273 in regulating the sensitivity of the receptors to inhibition
by zinc, nickel, cadmium, and copper.
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Figure 1.
Schematic representation of a GABAR subunit and
comparison of the sequence of the M2-M3
extracellular domain of the rat 1 and 6 subtypes. The structure
of the GABAR is believed to consist of a large N-terminal extracellular
domain, four transmembrane domains (hatched boxes), a
large intracellular region between M3 and
M4, and a short extracellular C-terminal domain. The
sequence of the 12 amino acid extracellular link between the
M2 and M3 domains is given (Tyndale et al.,
1995). The sequences of the rat 4 and 6 subtypes are identical in
this region. The splice site in the M1 domain for the
chimeric constructs of the 1 and 6 subtypes is shown by the
arrow (Fisher et al., 1997).
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MATERIALS AND METHODS |
Construction of mutant and chimeric subtype
cDNAs. Point mutations were generated using QuikChange mutagenesis
procedure and products (Stratagene, La Jolla, CA). Rat subunit cDNAs
subcloned into the pCMVneo expression vector (Huggenvik et al., 1991)
were used for creation of the mutants. Chimeras were constructed as described by Fisher et al. (1997). Oligonucleotide primers were synthesized by the University of Michigan DNA synthesis core facility (Ann Arbor, MI). Single amino acid changes were created using two
nucleotide primers, 35 or 36 nucleotides in length, complementary to
one another and encoding the desired amino acid mutation. The 1 N274
mutations were created by replacing the sequence 5'-AAT-3' with
5'-CAT-3' (N274H) or 5'-GAT-3' (N274D). The 6 H273 mutations were
created by replacing the sequence 5'-CAC-3' with 5'-AAC-3' (H273N) or
5'-GAC-3' (H273D). The sequence of the primer region surrounding the
mutations was verified for all constructs with DNA sequencing
(University of Michigan sequencing core).
Transfection of L929 cells. Full-length cDNAs for rat GABAR
1 (Dr. A. Tobin, University of California, Los Angeles), 3 (Dr. D. Pritchett, University of Pennsylvania, Philadelphia), 6, and 2L (F. Tan, University of Michigan) subtypes were subcloned into the
pCMVNeo expression vector and transfected into the mouse fibroblast cell line L929 (American Type Culture Collection, Rockville, MD). Chimeric constructs and mutant subtypes were prepared as described above. For selection of transfected cells, the plasmid pHook-1 (Invitrogen, San Diego, CA) containing cDNA that encodes the surface antibody sFv was also transfected into the cells. L929 cells were maintained in DMEM plus 10% heat-inactivated horse serum, 100 IU/ml
penicillin, and 100 µg/ml streptomycin. Cells were passaged by a 5 min incubation with 0.5% trypsin/0.2% EDTA solution in PBS (10 mM Na2HPO4, 0.15 mM NaCl, pH 7.3).
The cells were transfected using a modified calcium phosphate method
(Chen and Okayama, 1987; Angelotti et al., 1993). Plasmids encoding
GABAR subtype cDNAs were added to the cells in 1:1 ratios of 4 µg
each plus 4-8 µg of the plasmid-encoding sFv. After a 4-6 hr
incubation at 3% CO2, the cells were treated with a
15% glycerol solution in BBS buffer [50 mM BES
(N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), 280 mM NaCl, 1.5 mM
Na2HPO4] for 30 sec. The selection procedure
for sFv antibody expression was performed 20-28 hr later as described
by Greenfield et al. (1997). Briefly, the cells were passaged and mixed
with 5 µl of magnetic beads coated with hapten (~7.5 × 105 beads) (Invitrogen). After 30-60 min of
incubation to allow the beads to bind to positively transfected cells,
the beads and bead-coated cells were isolated using a magnetic stand.
The selected cells were resuspended into DMEM, plated onto 35 mm
culture dishes, and used for recording 18-28 hr later.
Electrophysiological recording solutions and techniques. For
whole-cell recording the external solution consisted of (in
mM): 142 NaCl, 8.1 KCl, 6 MgCl2, 1 CaCl2, 10 glucose, 10 HEPES, pH 7.4, and osmolarity
adjusted to 295-305 mOsm. Recording electrodes were filled with an
internal solution of (in mM): 153 KCl, 1 MgCl2, 5 K-EGTA, 10 HEPES, 2 MgATP, pH 7.4, and
osmolarity adjusted to 295-305 mOsm. These solutions provided a
chloride equilibrium potential near 0 mV. Patch pipettes were pulled
from thick-walled borosilicate glass with an internal filament (World
Precision Instruments, Pittsburgh, PA) on a P-87 Flaming Brown puller
(Sutter Instrument Co., San Rafael, CA) and fire-polished to a
resistance of 5-10 M . Series resistance was compensated 75-85%.
Drugs were applied to cells using a modified U-tube delivery system
with a 10-90% rise time of 70-150 msec (Greenfield and
Macdonald, 1996). Currents were recorded with a List EPC-7
(Darmstandt) patch-clamp amplifier and stored on Beta videotape (Sony,
Tokyo, Japan). All experiments were performed at room
temperature.
Analysis of whole-cell currents. Whole-cell currents were
analyzed off-line using the programs Axoscope (Axon Instruments, Foster
City CA) and Prism (Graphpad, San Diego, CA). Normalized concentration-response data for the different isoforms were fit with a
four-parameter logistic equation (Current = Maximum Current/(1 + ([drug]/EC50 or IC50)n),
where n represents the Hill number. All fits were made to
normalized data with the current expressed as a percentage of the
maximum current elicited by saturating GABA concentrations for each
cell for GABA concentration-response curves or, in the case of
modulators, as a percentage of the response to GABA alone. Data are
given as averages of the individual results ± SEM unless noted
otherwise. Statistical tests were performed using the Instat program
(Graphpad). Comparisons of the receptor properties were performed with
one-way ANOVA, Tukey-Kramer multiple comparisons test, and Student's
t test (p = 0.05). For comparisons of
sensitivity, the logs of individual EC50 or
IC50 values were compared.
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RESULTS |
GABA sensitivity of wild-type and mutant 132L and
632L receptors
Wild-type and all four mutant subtypes produced functional
GABARs when cotransfected with 3 and 2L in L929 fibroblasts (Fig.
2A). 132L
receptors were less sensitive to GABA (average GABA EC50 = 10.7 ± 1.8 µM; Hill slope = 1.6 ± 0.1;
n = 5) than were 632L receptors (average GABA
EC50 = 1.8 ± 0.2 µM; Hill slope = 1.4 ± 0.2; n = 6) (Fig. 2B).
The subtype mutations did not affect the sensitivity of the GABARs
to GABA (Fig. 2B). The GABA EC50 values
for receptors containing the 1 mutants were not significantly
different from the EC50 values for receptors containing the
1 wild-type, with average EC50 values of 11.9 ± 2.7 µM (1(N274H)32L, average Hill
slope = 1.3 ± 0.1; n = 4) and 9.1 ± 0.6 µM) (1(N274D)32L, average Hill
slope = 1.2 ± 0.2; n = 4) (data not shown).
The 6 mutants also did not affect GABA sensitivity, with average
GABA EC50 values of 1.2 ± 0.4 µM (6(H273N)32L, average Hill slope = 1.1 ± 0.2; n = 5) and 1.1 ± 0.1 µM
(6(H273D)32L, average Hill slope = 1.5 ± 0.2; n = 4) (data not shown).
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Figure 2.
Sensitivity of GABARs to GABA. A,
Representative whole-cell traces from transfected L929 fibroblasts.
Cells transfected with wild-type or mutant subunits produced current
responsive to GABA in a concentration-dependent manner. Varying
concentrations of GABA were applied for 10-15 sec, as indicated, to
cells voltage-clamped to  50 mV. The same time scale applies to all
traces. B, Concentration-response relationships were
constructed by normalizing the peak response to each concentration of
GABA to the maximum current-response for each cell.
Points shown are mean ± SEM. Data were fit with a
four-parameter logistic equation. EC50 values and Hill
slopes for the fits shown are as follows: 132L (10.4 µM; Hill slope = 1.4), 632L (1.9 µM; Hill slope = 1.2),
1(N274H)32L (10.4 µM; Hill
slope = 1.2), and 6(H273N)32L (2.6 µM; Hill slope = 1.2).
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Inhibition of GABAR currents by zinc
Both 132L and 632L receptor currents
were reduced by zinc (Fig.
3A), but 132L
receptor currents were less sensitive to zinc (average IC50 = 151 ± 34 µM; n = 5) than
632L receptor currents (average IC50 = 26 ± 4 µM; n = 7) (Fig. 3B).
However, both receptors were inhibited to the same extent (~80% of
the current) by maximally effective zinc concentrations. The difference in zinc sensitivity between these isoforms, therefore, was in their
affinity for zinc and not in its efficacy. Replacement of H273 in the
6 subtype with the Asn found in the equivalent location in the 1
subtype (N274) reduced the sensitivity of the receptor for zinc
(IC50 of 114 ± 12 µM; n = 4) to near that of the wild-type 132L receptor (Fig.
3A,B). Replacement of N274 in the
1 subtype with the His found in the equivalent location in the 6
subtype (H273) increased the sensitivity of the receptor for zinc
(IC50 = 38 ± 6 µM; n = 4) to that of the wild-type 632L receptor (Fig.
3A,B). Exchanging Asp for either of
these amino acids also produced high sensitivity to zinc, with
IC50 values for zinc of 28 ± 9 µM
(1(N274D)32L, n = 4) and 17 ± 4 µM (6(H273D)32L, n = 4) (data not shown). This was consistent with the
ability of Asp to contribute to binding sites for divalent cations.
These results indicated that replacing the 6 subtype H273 with Asn prevented the higher zinc sensitivity conferred by the 6 subtype and
that a His residue in this location was sufficient to convert the 1
subunit from low to high zinc sensitivity.
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Figure 3.
Sensitivity of GABARs to zinc.
A, Representative whole-cell traces from transfected
L929 fibroblasts. The response to GABA and GABA plus 30 µM zinc is shown for each receptor isoform. GABA
concentrations were near the EC50 value for each receptor:
1 µM for 6 and 6 mutants, or 10 µM
for 1 and 1 mutants. Cells were voltage-clamped to 50 mV.
B, Concentration-response relationships were
constructed by expressing the inhibition by zinc as a percentage of the
response to GABA alone (1 µM or 10 µM) for
each cell. Points shown are mean ± SEM. Data were
fit with a four-parameter logistic equation. IC50 values
and Hill slopes for the fits shown are as follows: 132L (190 µM; Hill slope = 1.0), 632L (25 µM; Hill slope = 1.0),
1(N274H)32L (36 µM; Hill slope = 0.8), and 6(H273N)32L (120 µM;
Hill slope = 1.1).
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Structural dependence of modulation of GABAR currents by other
divalent cations
By using chimeras of 1 and 6 subtypes with a splice site
within the first transmembrane domain (M1), we
demonstrated previously that the increased zinc sensitivity conferred
by the 6 subtype was associated with C-terminal regions, including
the M2-M3 extracellular domain (Fisher et al.,
1997). This finding led us to focus on H273 in the 6 subtype
M2-M3 domain as a potentially important site
for influencing the zinc sensitivity of GABARs. Other divalent cations,
however, also inhibit the activity of GABARs, and it is not known
whether all of these divalent cations act at the same site or whether
multiple allosteric regulatory sites exist. It is also not known
whether all divalent cations show the same subunit subtype
dependence shown by zinc. Therefore, to determine whether there was a
common structural dependence of GABARs for inhibition of currents by
these divalent cations, we measured the responsiveness of the 1/6
chimeras and the His and Asn mutations on the sensitivity of
recombinant receptors to inhibition by nickel, copper, and cadmium. The
1/6 chimera contains 1 sequence in the large extracellular
N-terminal domain through the first half of the M1
transmembrane domain to the splice site (Fig. 1) and 6 sequence for
the remainder of the subunit. The 6/1 chimera is the opposite,
containing 6 sequence in the N terminus and 1 sequence C terminal
to the splice site. A single-point mutation was introduced into the
M1 domain of the 1/6 chimera to create the chimeric
receptors. L258 was converted to the Thr present in the 6 subtype.
This mutation alone did not affect the properties of the 1 subtype
(Fisher et al., 1997).
Inhibition of GABAR currents by nickel
Both 132L and 632L receptor currents
were reduced by nickel (Fig.
4A), and as with zinc,
632L receptor currents (average IC50 = 108 ± 9 µM; n = 5) were more sensitive to
inhibition by nickel than 132L receptor currents (average
IC50 = 1.3 ± 0.3 mM; n = 6) (Fig. 4B).
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Figure 4.
Sensitivity of GABARs to nickel. A,
Representative whole-cell traces from transfected L929 fibroblasts. The
responses to GABA and GABA plus 300 µM nickel are shown
for each receptor isoform. GABA concentrations were near the
EC50 for each receptor: 1 µM for 6 and
6 mutants, or 10 µM for 1 and 1 mutants. Cells
were voltage-clamped to 50 mV. B,
Concentration-response relationships were constructed by expressing
the inhibition by nickel as a percentage of the response to GABA alone
(1 µM or 10 µM) for each cell.
Points shown are mean ± SEM. Data were fit with a
four-parameter logistic equation. IC50 values for the fits
shown are as follows: 132L (1.1 mM), 632L
(102 µM), 1(N274H)32L (142 µM), and 6(H273N)32L (208 µM). C, Sensitivity of the chimeric
constructs of the 1 and 6 subtypes to 600 µM
nickel. GABA concentration was near the EC50 for each
receptor (60 µM for 1/632L and 0.3 µM for 6/132L). The inhibition by nickel was
normalized to the response to GABA for each cell. Error bars represent
mean + SEM for four cells.
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To localize the subtype functional domain that determined the
sensitivity of the receptors to inhibition by nickel, we compared the
extent of inhibition by 600 µM nickel of currents from
32L receptors containing wild-type or chimeric subtypes
(Fig. 4C). For each GABAR isoform, currents were evoked by
EC50 GABA concentrations. Wild-type 132L receptor
currents were less inhibited by nickel than wild-type 632L
receptor currents. The extent of inhibition by 600 µM
nickel of 1/6 chimeric subunit receptor currents was not
significantly different from that of wild-type 632L receptor currents (Fig. 4C). The extent of inhibition by 600 µM nickel of 6/1 chimeric receptor currents was not
significantly different from that of wild-type 132L receptor
currents. This pattern was comparable to that of zinc, suggesting that
high nickel sensitivity was associated with domains of the 6 subtype
C terminal to the M1 domain.
To determine whether the 6 H273 was responsible for the higher
sensitivity to nickel of 632L receptors, we examined the nickel sensitivity of the mutant 6(H273N)32L and
1(N274H)32L receptor currents (Fig.
4A,B). In contrast to the result
obtained for zinc, the sensitivity of 6(H273N)32L
receptor currents to nickel (average IC50 = 212 ± 16 µM; n = 5) was only slightly but
significantly reduced compared with wild-type 632L currents. This indicated that this His residue was not required for high sensitivity to nickel but that it might contribute to or influence the
sensitivity. The 1(N274H) mutant subtype increased the
sensitivity to nickel (average IC50 of 142 ± 21 µM; n = 5) compared with the wild-type
132L receptor (Fig. 4B). The degree of
inhibition by 300 µM nickel of the
1(N274H)32L receptor was significantly different
from either of the wild-type receptors, again suggesting that a His in
this location contributed to but was not solely responsible for the
higher sensitivity to nickel associated with the 6 subtype.
Inhibition of GABAR currents by cadmium
Both 132L and 632L receptor currents were
reduced by cadmium (Fig. 5A).
However, unlike the difference in sensitivity seen with zinc,
132L and 632L receptor currents had similar sensitivity to inhibition by cadmium, with average IC50
values of 102.6 ± 34.4 µM (n = 5)
and 134.1 ± 19.3 µM (n = 5),
respectively (Fig. 5B). Although previous work suggested
that the zinc and cadmium binding sites might overlap or
interact (Celentano et al., 1991; Kumamoto and Murata, 1995), these
data suggested that the structural dependence of cadmium sensitivity
might be different from that of zinc.
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Figure 5.
Sensitivity of GABARs to cadmium.
A, Representative whole-cell traces from transfected
L929 fibroblasts. Responses to GABA and GABA plus 100 µM
cadmium are shown for each receptor isoform. GABA concentrations were
near the EC50 for each receptor: 1 µM for
6 and 6 mutants, or 10 µM for 1 and 1
mutants. Cells were voltage-clamped to 50 mV. B,
Concentration-response relationships were constructed by expressing
the inhibition by cadmium as a percentage of the response to GABA alone
(1 µM or 10 µM) for each cell.
Points shown are mean ± SEM. Data were fit with a
four-parameter logistic equation. IC50 values for the fits
shown are as follows: 132L (64 µM),
632L (103 µM), 1(N274H)32L
(52 µM), and 6(H273N)32L (425 µM). C, Sensitivity of the chimeric
constructs of the 1 and 6 subtypes to 100 µM
cadmium. GABA concentration was near the EC50 for each
receptor (60 µM for 1/632L and 0.3 µM for 6/132L). The inhibition by cadmium was
normalized to the response to GABA for each cell. Error bars represent
mean + SEM for n  four cells.
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To localize the subtype functional domain that determined the
sensitivity of the receptors to inhibition by cadmium, we compared the
extent of inhibition by 100 µM cadmium of currents from
32L receptors containing wild-type or chimeric subtypes (Fig. 5C). For each GABAR isoform, currents were evoked by
EC50 GABA concentrations. Wild-type 132L receptor
currents were inhibited by cadmium to the same extent as wild-type
632L receptor currents. Inhibition by cadmium of currents from
receptors containing the 1/6 chimeric subunit (n = 5) was not significantly different from inhibition of currents from
the wild-type receptors. However, 6/132L receptor currents
(average IC50 for cadmium of 696 ± 203 µM; n = 7) were significantly less
sensitive to cadmium inhibition than wild-type receptor currents. These
data suggested that regions of the 6 subtype C terminal to the first
transmembrane domain and residue(s) in the N-terminal extracellular
domain of the 1 subtype were required for cadmium sensitivity.
To determine whether the 6 H273 was responsible for the sensitivity
to cadmium of 632L receptors, we examined the cadmium sensitivity of the mutant 6(H273N)32L and
1(N274H)32L receptors (Fig.
5A,B). The 6(H273N)
mutation decreased the sensitivity of the receptor for cadmium, with an
IC50 of 432.4 ± 55.8 µM
(n = 4), suggesting that this His was important for
cadmium sensitivity as well as for zinc sensitivity. Because the H273N
mutation accounted for only part of the loss of sensitivity
compared with the chimeric subunit, other residues might also
contribute to cadmium sensitivity. The 1(N274H) mutation
did not alter the inhibition by cadmium compared with the wild-type
132L receptor, with an IC50 of 54.7 ± 15.3 µM (n = 4). Consistent with the findings
from the chimeric receptors, these results suggested that although
1- and 6-containing receptors were equally sensitive to cadmium, the structural determinants of the properties of inhibition of these
subtypes were different. It is interesting that the 1/6 chimera
did not confer significantly greater sensitivity to cadmium than the
wild-type subtypes, although it presumably contained the domains
responsible for cadmium sensitivity for both the 1 and the 6
subtypes. This suggests that these sites are not additive or that one
of the sites was not functional.
Inhibition of GABAR currents by copper
Both 132L and 632L receptor currents were
reduced by copper (Fig.
6A), but unlike the
other divalent cations that we examined, 1132L receptor
currents were more sensitive than 632L receptor currents to
inhibition by copper (Fig. 6B). The concentration-response curves were fitted best with a two-population logistic equation. The IC50 values (and relative
contributions) ± SE of the fitting parameters for the 132L
receptor were 9.0 ± 2.6 µM (51.9 ± 5.7%) and
1.89 ± 0.81 mM (48.1 ± 4.8%)
(n = 3). For the 632L receptor the data were
fit with IC50 values (and relative contributions) of
13.3 ± 2.1 µM (16.3 ± 5.7%) and 1.73 ± 0.25 mM (83.7 ± 5.2%) (n = 5). The
difference in sensitivity of the isoforms appeared to be attributable
primarily to the greater contribution of the higher affinity site for
the 1-containing receptors.
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Figure 6.
Sensitivity of GABARs to copper. A,
Representative whole-cell traces from transfected L929 fibroblasts.
Responses to GABA and GABA plus 100 µM copper are shown
for each receptor isoform. GABA concentrations were near the
EC50 for each receptor: 1 µM for 6 and
6 mutants, or 10 µM for 1 and 1 mutants. Cells
were voltage-clamped to 50 mV. B,
Concentration-response relationships were constructed by expressing
the inhibition by copper as a percentage of the response to GABA alone
(1 µM or 10 µM) for each cell.
Points shown are mean ± SEM. Data are fit with a
two-population logistic equation. C, Sensitivity of the
chimeric constructs of the 1 and 6 subtypes to 100 µM copper. GABA concentration was near the
EC50 for each receptor (60 µM for
1/632L and 0.3 µM for 6/132L).
The inhibition by copper was normalized to the response to GABA for
each cell. Error bars represent mean + SEM for n three cells.
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To localize the subtype functional domain responsible for the
sensitivity to inhibition by copper, we compared the inhibition by 100 µM copper of currents from 32L receptors
containing wild-type or chimeric subtypes (Fig. 6C). For
each GABAR isoform, currents were evoked by EC50 GABA
concentrations. The extent of inhibition by 100 µM copper
of 1/6 chimeric receptor currents (n = 4) was not
significantly different from that of wild-type 132L receptor
currents. The extent of inhibition by copper of 6/1 chimeric
subunit receptor currents (n = 4) was not significantly different from that of wild-type 632L receptor currents. This suggested that regions in the N-terminal extracellular domain of the
1 subtype were responsible for the higher copper sensitivity.
As expected from the chimera data, the C-terminal mutations in
the 1 and 6 subtypes had no effect on the sensitivity or the
receptor currents to copper inhibition (Fig.
6A,B). The
1(N274H)32L receptor data were fit with two
populations with IC50 values (and relative contributions)
of 9.7 ± 2.2 µM (57.8 ± 5.8%) and 2.78 ± 1.3 mM (43.2 ± 4.6%), whereas the fits of the
6(H273N)32L receptor data were 3.8 ± 1.12 µM (15.3 ± 13.6%) and 1.94 ± 0.30 mM (84.7 ± 0.44%). The degree of inhibition by 100 µM copper of the mutant receptors was not significantly
different from the inhibition of wild-type receptors for either of the
mutations, indicating that H273 of 6 and N274 of 1 did not
influence copper inhibition of GABAR current. These data were
consistent with data from the chimeric subunits, indicating that high
sensitivity to inhibition of current by copper is associated with the
extracellular N-terminal domain of the 1 subtype.
| |
DISCUSSION |
We examined the role of H273 of the rat 6 subtype in the
sensitivity of recombinant GABARs to inhibition by divalent cations. Previous studies of 1/6 chimeric subunits in our laboratory suggested that the extracellular domain between the second and third
transmembrane domains in which this His is located may be important in
the higher sensitivity to zinc of 632L receptors compared with
132L receptors (Fisher et al., 1997). GABARs containing the
mutant receptors showed wild-type responsiveness to GABA, indicating
that the GABA binding sites and transduction pathways were not
substantially affected. Mutation of H273 to the Asn found in the
homologous location in the 1 subtype reduced the zinc sensitivity,
whereas exchanging a His for N274 in the 1 subtype produced
6-like sensitivity. This His could influence zinc sensitivity
through several different mechanisms: it may contribute to the binding
site for zinc, remotely influence the properties of the binding site by
changing the structure of the receptor, or modify the transduction
pathway through which zinc binding reduces the GABAR current.
Substitution of Asp for the His in 6 or the Asn in 1 also
produced high sensitivity to zinc. Because Asp is structurally
unrelated to His but shares the ability to participate in zinc binding,
this suggested that the residue in this location may contribute to the
zinc binding site. However, because receptors containing subunits
that lacked the His residue were still sensitive to zinc but with
higher IC50 values, this His was not required for zinc
binding but instead apparently increased the attractiveness of the
receptor for zinc. Our results do not rule out participation of other
residues in the subunits in zinc binding, and it is possible that
the residues that contributed to the binding of other divalent cations
could also contribute to one or more zinc sites. Additionally, although
our findings may explain the higher sensitivity of 4- and
6-containing receptors to zinc, the 5 subtype also confers
relatively high sensitivity to zinc (Burgard et al., 1996), but like
1 contains an Asn residue in this location. Because 4 and 6
subtypes share this His residue, the identical mutation of the 4
subtype would probably also reduce zinc sensitivity. However, it is
possible that other residues in the 4 subtype influence zinc binding
and that this His residue plays a role only in the zinc sensitivity of
the 6 subtype.
A His residue responsible for inhibition by divalent cations has also
been identified in the structurally related  1 subunit. subunits
are highly expressed in the retina and are believed to form the
GABAC class of receptors (Tyndale et al., 1995). Homomeric 1 receptors are highly sensitive to block by zinc, nickel, and cadmium, and a His has been shown to be responsible for this inhibition (Wang et al., 1995). However, the location of this residue in the large
N-terminal extracellular domain does not correlate with the location of
the 6 His we have identified. This suggests that although His
residues in both GABAA and GABAC receptors
influence the sensitivity to inhibition by divalent cations, the
structural domains responsible for the inhibition are different.
Contributions to zinc inhibition from other subunits
The GABAR has a complex structure, and native GABARs are believed
to consist of a pentameric combination of two , two , and one
, , or subunits. The subunit alone clearly does not
determine all the properties of zinc inhibition. Contributions from the
, , , and subunits also influence these properties. Because the His residue in the M2-M3
extracellular bridge appears to be important in the subunit
contribution, it is possible that this region plays a role in the other
subunits as well. At the equivalent location in subunit is a Glu
that is conserved among all subtypes. Glu would be capable of
participating in zinc binding, consistent with the high sensitivity of
heterodimers to inhibition by zinc (Draguhn et al., 1990). In
addition, in all subtypes there is a His that is only three amino
acids N terminal to the Glu (H267 in 3) that has recently been shown to regulate zinc sensitivity in 3 homomers and 13 heterodimers expressed in Xenopus oocytes (Wooltorton et al., 1997). Both
Glu and His residues may contribute to subtype regulation of zinc inhibition. The (1-3) and subunits all contain a lysine residue at the M2-M3 location. The positive charge of
this residue would repel cation binding, consistent with the reduced
sensitivity to zinc of - and -containing GABARs (Draguhn et al.,
1990; Whiting et al., 1997). The subunit has a serine residue that
would not be expected to influence zinc binding, and
receptors have an intermediate sensitivity to zinc between the
heterodimers and the heterotrimers (Saxena and Macdonald,
1996). Although a zinc binding pocket(s) could be formed by residues
from many different regions of the subunits rather than a single
homologous domain, it is interesting that this location in the
M2-M3 extracellular bridge appears to be a
location of heterogeneity among subunits and that characteristics of
the residues are consistent with their contributions to zinc
sensitivity of the receptor.
Multiple sites for divalent cations
We also examined the role of H273 of the 6 subtype in the
sensitivity of GABARs to three other divalent cations, nickel, cadmium,
and copper, to determine whether they shared a common structural
dependence for activity with zinc. Although only copper and zinc are
believed to play physiological roles in regulating synaptic activity,
the effects of other divalent cations can be important in understanding
their neurotoxicity, as well as helpful in understanding the structural
contributions of the different GABAR subunits to the actions of
divalent cations. Replacement of the His residue in 6 with Asn
reduced the sensitivity to both cadmium and nickel, although not to the
same level seen with the chimeric receptor. This suggests that although
this His contributes to these sites, other residues also in the
C-terminal extracellular domains significantly influence the
sensitivity to cadmium and nickel. The 6 subtype confers a
relatively low sensitivity to copper, and replacement of the H273 with
Asn did not affect inhibition by copper.
The 1 subtype also appears to have a distinct site(s) for divalent
cation binding, conferring high sensitivity to inhibition by copper and
cadmium. The sensitivity to copper, and probably to cadmium, was
associated with the large N-terminal extracellular domain of the
subunit. This is in contrast to the 6 subtype in which all high
sensitivity to zinc, cadmium, and nickel was associated with regions C
terminal to this domain. The N-terminal extracellular domain has a high
degree of sequence variability among the subtypes, and there are
numerous divergent amino acids in the 1 subtype compared with the
6 subtype, including His, Glu, and Asp residues that could
contribute to high sensitivity to divalent cations. Further work may
indicate which of these amino acid differences are responsible for the
higher copper and cadmium sensitivity of the 132L
receptors.
| |
FOOTNOTES |
Received Dec. 4, 1997; revised Jan. 29, 1998; accepted Feb. 4, 1998.
This work was supported by National Institutes of Health Grant
RO1-NS33300 (R.L.M.) and National Institute on Drug Abuse Training Grant 5T32-DA07268 (J.L.F.). We acknowledge the assistance of Dr. Naomi
Nagaya.
Correspondence should be addressed to Dr. Robert L. Macdonald, 1103 East Huron Street, Neuroscience Lab Building, Ann Arbor, MI
48104-1687.
Dr. Fisher's present address is Baylor College of Medicine, Division
of Neuroscience, One Baylor Plaza, Houston, TX
77030-3498.
| |
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T. Defazio and J. J. Hablitz
Zinc and Zolpidem Modulate mIPSCs in Rat Neocortical Pyramidal Neurons
J Neurophysiol,
October 1, 1998;
80(4):
1670 - 1677.
[Abstract]
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Abstract
Introduction
