From the Department of Pharmacology and
¶ Department of Anesthesiology and Critical Care Medicine,
George Washington University Medical Center, Washington, D. C. 20037 and § The Institute for Genomic Research,
Rockville, Maryland 20850
Received for publication, August 28, 2002, and in revised form, October 9, 2002
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ABSTRACT |
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In mammals, the superfamily of "Cys
loop," ligand-gated ion channels (LGICs), is assembled from a pool of
more than 40 homologous subunits. These subunits have been classified
into four families representing channels that are gated by
acetylcholine, serotonin, Ion channels that are gated by acetylcholine, serotonin,
Most of the known subunits were identified by traditional methods of
cross-hybridization or expression cloning. More recently, data bases of
ESTs have been used to identify more distantly related homologues
through computational searches for characteristic structural motifs
(5-7). However, all of these methods have inherent limitations that
can hinder the identification of a complete gene family. This is
particularly relevant for subunits that are distantly related, that
confer unexpected properties, and that are expressed at low levels in
discrete locations or for short time periods during development. For
such subunits, identification may only be possible through the analysis
of a completely sequenced genome. Previously, during the early stages
of sequencing the human genome, we used the draft sequence data to
identify an elusive subunit of 5-HT3 receptors (8). Here we
have used a similar approach to uncover a very distinctive member of
the subunit superfamily. This subunit cannot be assigned to any of the
known receptor families on the basis of sequence similarity or
functional properties. It appears to represent an old and distinct
branch of the receptor superfamily that displays unique functional
properties. Moreover, it also appears to be the first example of a
subunit from this receptor superfamily that is expressed in human
tissues but has been lost from at least some rodent species.
Isolation of the Human ZAC Subunit cDNA--
A consensus
peptide sequence of subunits for 5HT3 and nACh receptors
was used to search the nr and htgs data bases using the TBLASTN
algorithm (www.ncbi.nlm.nih.gov/blast/). Homologous peptide fragments were identified within the six-frame translation of a human
genomic DNA fragment (GenBankTM accession number AC018665). Oligonucleotide primers were designed from the genomic sequence to
amplify the 5'- and 3'-flanking sequences from fetal brain and spinal
cord cDNA libraries using the Marathon system
(Clontech). Amplification at 95 °C for 45 s, 60 °C for 60 s, and 72 °C for 2 min was performed for 35 cycles using the XL-PCR system (PerkinElmer Life Sciences). Reaction
products were purified from agarose gels and sequenced directly. The
open reading frame of the new subunit cDNA (termed ZAC) was
amplified from a spinal cord cDNA library as described above using
primers containing nucleotides 1-21 (sense) and 1266-1289 (antisense)
of the ZAC subunit cDNA sequence (GenBankTM accession number
AF512521). The cloned product was sequenced over its entire length to
ensure that no mutations had been introduced. Sequence alignments were
generated by the ClustalW program from the MacVector package of
sequence analysis software (Oxford Molecular Group). A cladogram was
constructed using the neighbor-joining method with pairwise distances
measured by absolute differences and gaps ignored. The bootstrap
consensus was generated using 1,000 replications.
Northern Blot Analysis--
Samples of ~2 µg of
poly(A)+ mRNA (Clontech) underwent
electrophoresis on a 1.2% formaldehyde agarose gel, were
transferred to nylon membranes, and were hybridized with an antisense
32P-labeled riboprobe that was derived from the ZAC subunit
cDNA (nucleotides 1-447 of GenBankTM accession number AF512521). The blots were washed at 60 °C in 0.1× SSC, 0.1% SDS before
exposure. The blots were stripped and reprobed with
32P-labeled fragments of the
glyceraldehyde-3-phosphate dehydrogenase cDNA (nucleotides
789-1140; Ref. 9).
Cloning of Orthologous Genomic Fragments from Mouse, Rat, and
Dog--
Fragments of genomic DNA were amplified using primer pairs
that correspond to the 3'-ends of genes encoding the mouse, rat, and
dog orthologues of the human galanin receptor 2 and KIAA1067 genes. For
mouse and rat, these were 5'-CACCCGCACTTCCCAACTGCACA and
5'-CTCATCATGGTGCCTGGTCAGGTA (nucleotides 1-23 and 5146-5169 of
GenBankTM accession number AF512522). For dog, they were 5'-CCGACGGTTAATGCGACCTGAGGA and 5'-GGAGCAGGTGGGCGACATGATCGA
(nucleotides 1-24 and 5846-5869 of GenBankTM accession number
AF512523). Aliquots of DNA (50 ng) from mouse, rat
(Clontech), or dog (Novagen) were amplified using
the XL-PCR system (Applied Biosystems). Reaction products were purified
from agarose gels and sequenced directly.
Cell Culture and Transfection--
HEK cells were grown
in Dulbecco's modified Eagle's medium, supplemented with 10% calf
serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. When
cells approached confluence, they were seeded into 35-mm diameter
dishes and transfected with cDNAs encoding the human ZAC subunit
(in pcDNA1.1/amp) and GFP (in pCDM8). Cells were transfected
using calcium phosphate precipitation (6). Cells were used 24-44 h
after transfection.
Electrophysiology--
The whole-cell patch-clamp technique was
used to record currents from HEK cells. The bath was continuously
perfused (5 ml/min) with an extracellular solution containing (in
mM): NaCl, 140; KCl, 4.7; MgCl2, 1.2;
CaCl2, 2.5; glucose, 11; and HEPES, 10 (pH 7.4 with NaOH).
The electrode solution contained (in mM): KCl, 140;
MgCl2, 2.0; EGTA, 11; and HEPES, 10 (pH 7.4 with KOH). The intracellular solution used to characterize the cation permeability of
ZAC channels contained (in mM): KCl, 70;
N-methyl-D-glucamine, 70;
MgCl2, 2.0; EGTA, 11; and HEPES, 10 (pH 7.4 with HCl). The intracellular solution used to determine the contribution of
Cl Acquisition and Analysis of Data--
Currents were amplified
(Axopatch 200A, Axon Instruments), low pass-filtered at 1 kHz, and
digitized (Digidata 1320, Axon Instruments, Foster City, CA) for
acquisition onto the hard drive of a personal computer. Currents were
averaged, superimposed, and measured using pCLAMP software (Axon
Instruments). Zn2+ concentration-response data were
obtained by prolonged (2 s) pressure ejection of randomized agonist
concentrations from low resistance pipettes as described previously
(10). Zn2+-activated currents often exhibited run-up. To
compensate for this, 1 mM Zn2+ was applied
before each concentration of Zn2+. The amplitudes of the
Zn2+-activated currents were subsequently normalized to the
current elicited by the prior application of 1 mM
Zn2+. Graphs of concentration-response relationships were
fitted using the logistic function as described previously (10).
Current-voltage relationships were analyzed by averaging at
least two currents recorded at each holding potential. Individual
current-voltage relationships were plotted, and a linear fit to points
either side of current reversal yielded the equilibrium potential. All data are expressed as the arithmetic mean ± S.E., and statistical comparisons were made using the Student's t test.
Cloning of the Human ZAC Subunit cDNA--
Searches of the
draft human genomic sequence with a consensus sequence of conserved
residues revealed exons of a novel LGIC subunit within unannotated
fragments of a bacterial artificial chromosome clone from
chromosome 17q23. The exon sequences were used to amplify flanking
exons and a contiguous cDNA containing the complete open reading
frame of the subunit. The subunit forms Zn2+-activated
channels (see below) and was termed ZAC. The ZAC subunit cDNA encodes a polypeptide of 411 amino acid residues (Fig.
1A). It has a signal sequence,
a Cys-Cys motif, four predicted transmembrane domains, and several
invariant residues that underpin the conserved secondary structure of
the Cys loop LGIC superfamily (11). However, the sequence of the ZAC
subunit is not closely related to any known subunits of this
superfamily (maximum of 15% amino acid identity with
5-HT3A and nAChR_
A search of ZAC subunit cDNA and genomic sequences against human
ESTs (www.ncbi.nlm.nih.gov/dbEST/index.html) revealed a
surprisingly large number (>150) that contained exons of the ZAC
subunit gene (encoding residues 181-411; Fig. 1A). However,
almost all of these ESTs contain intronic sequences and are derived
from mRNA that is transcribed in the opposite direction to the ZAC
subunit mRNA. These ESTs represent transcripts of the KIAA1067
gene, which is located downstream of the ZAC subunit gene in the
opposite orientation (see below, Fig. 6). The 3'-untranslated region of
the KIAA1067 mRNA is complementary to both protein-coding and
3'-untranslated regions of the ZAC subunit mRNA and terminates
within intron 5 of the ZAC subunit gene. Transcripts of the KIAA1067
gene are ubiquitous and abundant in human tissues. Reverse
transcription/PCR, with primers from the first and last exons of the
ZAC subunit gene, yielded a 1.27-kb ZAC subunit cDNA from prostate,
thyroid, trachea, fetal whole brain, spinal cord, placenta, and stomach but not from adult whole brain, heart, liver, spleen, or kidney cDNA (data not shown). The presence of ZAC subunit mRNA (1.5 kb) in placenta and stomach was confirmed by Northern analysis, using an antisense riboprobe that was derived from the 5'-end of the ZAC
subunit cDNA (Fig. 1C).
ZAC Subunits Form Homomeric Channels That Open
Spontaneously--
After transfection with ZAC and GFP cDNAs, HEK
cells displayed spontaneous currents (mean amplitude =
Agonists at other LGICs (GABA, glycine, glutamate, ATP, 5-HT, and
acetylcholine; all at 100 µM) lacked an ability to
activate recombinant ZAC (n
In the absence of an agonist, we exploited the
Ispont to test antagonists of related LGICs for
negative activity at ZAC. When applied by bath, strychnine (100 nM), bicuculline methiodide (10 µM),
Zn2+ modulates several LGICs, often causing a potent
inhibition of channel activity (13). Surprisingly, the local
application of Zn2+ (1 mM) caused activation of
inward currents in cells expressing ZAC
(Figs. 3-5) but not in cells
expressing GFP alone (n = 4).
Activation of ZAC Channels by
Zn2+--
Zn2+-activated currents
(IZn) had an equilibrium potential
(EZn) of
There was little correlation between the amplitudes of
IZn and Ispont in cells
expressing ZAC. Furthermore, run-up of IZn was
frequently observed during an experiment without a corresponding change in the amplitude of Ispont (Fig.
3B). These observations suggest that Zn2+
activates channels that are closed prior to its application. In cells
expressing ZAC, Zn2+ caused a
concentration-dependent activation of currents, with a
threshold of ~30 µM and an EC50 of 540 ± 9 µM (Fig. 4A). As with the
Ispont, IZn was
insensitive to bath-applied Absence of Functional ZAC Subunit Genes in Rodents--
We were
unable to amplify fragments of an orthologous gene from mouse or rat
genomic DNA using degenerate primers designed from the ZAC amino acid
sequence (data not shown). More surprisingly, searches of the draft
mouse and rat genomic sequences (www.celera.com and
www.ncbi.nlm.nih.gov) failed to yield fragments of orthologous genes.
The human ZAC gene is located between genes encoding the galanin2
receptor and KIAA1067 (Fig.
6A). Consequently, we
amplified and sequenced the region between the orthologues of these two genes from mouse, rat, and dog genomic DNA. For all four species, there
is detectable homology between the DNA sequences of this region. This
is most apparent where the human sequence represents an exon of the ZAC
gene (Fig. 6B). However, for mouse and rat, most of the
fragments that are homologous to exons of the human ZAC subunit gene
contain numerous insertions, deletions, and substitutions that
introduce in-frame stop codons (Fig. 6B). Only a footprint of the ZAC gene remains in these species, and they are no longer capable of expressing a functional ZAC subunit. In contrast, dog genomic DNA from this region encodes a full-length ZAC subunit that
displays 84% amino acid conservation with the human ZAC subunit (Fig.
6C).
The ZAC subunit displays all of the structural motifs that are
common to the superfamily of Cys loop LGICs. However, its distinctive sequence and function prevent its classification within any of the four
established receptor families. Sequence analysis indicates that a
common ancestral gene gave rise to the present day ZAC, nACh, and
5-HT3 receptor subunit genes. Since they diverged, a functional ZAC subunit gene has been retained by some mammals (e.g. human and dog) but has been lost by others
(e.g. mouse and rat). Most human genes, including those for
all previously identified LGIC subunits, have rodent orthologues.
However, there is evidence for a differential loss of a limited number
of genes from either rodent or human genomes (e.g. Refs. 14
and 15). With respect to ZAC, this raises the possibility that humans
and rodents differ in some fundamental cell signaling mechanisms. To
date, the properties of endogenous LGICs have been studied
predominantly in rodent cells. An absence of ZAC from mice and rats may
explain why its unusual properties have not been described previously.
ZAC exhibits TC-sensitive spontaneous activity. Some nACh and
GABAA receptor subtypes are also spontaneously active,
either in their natural environment (16) or when expressed in
recombinant systems (17). Persistent GABAA channel
openings attenuate cellular excitability and can contribute to the
maintenance of resting membrane potential (18). Spontaneous nACh
channel activity may participate in embryonic muscle development (16).
The physiological role of spontaneous ZAC remains to be determined.
However, the ability of TC to block this activity will be useful for
characterizing the properties of native ZAC in tissue preparations.
Zn2+ causes a concentration-dependent
activation of ZAC. Zn2+ can also modulate the activity of
related LGICs, although, in most cases, it inhibits channel function
(19-22). However, at the glycine receptor, its effect is biphasic,
potentiating glycine-evoked responses at low concentrations and
inhibiting them at higher concentrations (20). Zn2+ may
activate ZAC through an action at the putative N-terminal loop B
ligand-binding domain. Subunits of each LGIC family display characteristic loop B residues that may reflect a role in agonist selectivity (11). Notably, the relevant loop B residues in the ZAC
subunit (Leu-177 and Asn-179) are distinct from the corresponding residues of related subunits. This also suggests that ZAC represents a
distinct class of Cys loop LGICs. It is also possible that
Zn2+ activates ZAC through an allosteric site that is
unrelated to the loop B motif. This would be analogous to the direct
activation of GABAA receptors by general anesthetics
(23).
A relatively high concentration of Zn2+ (>30
µM) is required for activation of ZAC. Zn2+
is concentrated in various tissues, including the forebrain, testes,
and neuroendocrine cells (24, 25). In the hippocampus, pituitary, and
pancreatic It is possible that, in its native environment, the affinity of ZAC for
Zn2+ is increased by a cofactor or by post-translational
modification. The observed run-up of IZn
indicates an increased ability of Zn2+ to activate the
channel during the course of an experiment. Future experiments will
examine whether this phenomenon is caused by post-translational
regulation. It is also possible that Zn2+ is not the
physiological agonist for ZAC. However, with the knowledge that
Zn2+ behaves as an agonist and TC as an inhibitor of
spontaneous gating, these agents will be useful tools for
characterizing the properties of native ZAC receptors in tissues from
human and other non-rodent species.
-aminobutyric acid, or glycine. By
searching anonymous genomic sequence data for exons that encode
characteristic motifs of the channel subunits, we have identified a
novel LGIC that defines a fifth family member. Putative exons were used
to isolate a full-length cDNA that encodes a protein of 411 amino
acid residues. This protein (ZAC) contains all of the motifs that are
characteristic of Cys loop channel subunits but cannot be assigned to
any of the four established families on the basis of sequence
similarity. Genes for ZAC are present in human and dog but appear to
have been lost from mouse and rat genomes. Transcripts of ZAC subunits
were detected in human placenta, trachea, spinal cord, stomach, and
fetal brain. Transfection of human embryonic kidney cells with ZAC
subunit cDNA caused expression of spontaneous current. By screening
with a broad range of potential agonists and antagonists, we determined that tubocurarine inhibits the spontaneous current whereas
Zn2+ activates the expressed receptors. The absence
of Zn2+-activated channels in rats and mice may explain why
this fifth member of the LGIC superfamily has evaded detection until now.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyrate, and glycine are a superfamily of homologous
neurotransmitter receptors
(nACh,1 5-HT3,
GABAA/C, and glycine receptors, respectively). Each of these four receptor families is composed of multiple receptor subtypes
derived from distinctive assemblies of five homologous subunits (1, 2).
Although a wide variety of subunit genes has been identified in
mammalian genomes (42 in human), each subunit can be assigned to one of
the four receptor families on the basis of sequence similarity and, in
most cases, functional activity. Many of these subunits confer unique
properties to recombinant receptors in which they assemble, properties
that have often been poorly defined in prior studies of native tissues
(3, 4). Consequently, the identification of novel receptor subunits
continues to reveal unexpected properties of this receptor superfamily.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to the ZAC currents contained (in mM):
KCl, 70; K+-gluconate, 70; MgCl2, 2.0; EGTA,
11; and HEPES, 10 (pH 7.4 with KOH). Junction potentials were nulled
prior to each experiment. Their inappropriate compensation was ignored
in graphs of current-voltage relationships, but equilibrium potential
values in the text were corrected. Cells were clamped at
60 mV unless
otherwise stated. Drugs were applied either by pressure ejection from
modified micropipettes or by bath perfusion. Experiments were performed
at 22-24 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 subunits; Fig. 1A).
This is much lower than the level of identity between known
subunits of 5-HT3 receptors (30-40%) or nACh
receptors (25-90%). Consequently, the ZAC subunit appears to
represent a distinct species that diverged from the common ancestral
subunit of the present day nACh and 5-HT3 receptors (Fig.
1B).
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Fig. 1.
Comparative sequence analysis and mRNA
expression for the human ZAC subunit. A,
alignment of human nAChR_ 7, 5-HT3A, and ZAC
subunit sequences. Conserved residues are boxed and
highlighted for identity (heavy
shading) or chemical similarity (light
shading). The potential signal sequence (S) and
four putative transmembrane domains (M1-M4) are indicated
by lines over the corresponding sequences. B, a
cladogram of a ClustalW alignment of human subunit sequences that are
homologous to residues 27-319 of the nACh receptor
subunit
(nAChR_
). The distance matrix employed neighbor joining (12) with
bootstrapping (1000 repetitions). The rooting outgroup was the
GABAA
1 subunit (GABA_
1). Branch points predict the
order of divergence from a common ancestral gene. C,
hybridization of poly(A)+ mRNA from small intestine
(lane 1), placenta (lane 2), lung (lane
3), peripheral blood leukocytes (lane 4), and stomach
(lane 5) with a 32P-labeled riboprobe for the
ZAC subunit (top panels). The same blots were
probed with 32P-labeled fragments of the human
glyceraldehyde phosphate dehydrogenase cDNA (bottom
panels).
180 ± 76 pA) immediately after achieving the whole-cell configuration at
60 mV. These were not seen in recordings from cells expressing GFP
alone (Fig. 2A). This suggests
that ZAC subunits form channels that can open in the absence of
agonist. The existence of spontaneous current (Ispont) enabled us to screen for compounds that
have either positive or negative intrinsic activity.
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Fig. 2.
Spontaneous currents mediated by ZAC are
blocked by TC. A, Ispont
recorded from an HEK cell expressing recombinant ZAC appeared rapidly
upon establishing the whole-cell configuration at 60 mV.
Uncompensated capacitive currents appear during whole-cell recording in
response to
5 mV steps. Leak currents were negligible in cells
expressing GFP alone. B, TC (100 µM), applied
with pressure for 2 s, reduced
Ispont in cells expressing ZAC but had no effect
on cells expressing GFP alone. C, the TC-inhibited current
exhibits rectification. The TC-inhibited current amplitude was plotted
against holding potential. Data points are average current amplitudes
from three recordings normalized to the amplitude of currents recorded
at
60 mV. Vertical bars represent ± S.E.
5). We also examined
agonists of metabotropic receptors that are inactive at known mammalian
LGICs (galanin, epinephrine, dopamine, histamine, neuropeptide Y,
oxytocin, morphine, somatostatin, angiotensin II; all at 1 µM). In addition, relatively nonspecific modulators of
glutamate and GABAA receptors were tested (glutathione (100 µM), ketamine (100 µM), allopregnanolone (1 µM), and propofol (10 µM)). None of these
compounds affected membrane currents in cells expressing ZAC
(n
3).
-bungarotoxin (10 µg/ml), mecamylamine (10 µM), and
ondansetron (1 nM) had no effect on
Ispont (n
2). By contrast, TC
(100 µM), a relatively nonselective inhibitor of nACh and
5-HT3 receptors, inhibited Ispont
when applied to the bath or by local pressure ejection
(n = 6, Fig. 2B). The
Ispont that was blocked by TC had an equilibrium
potential of
2 ± 3 mV (n = 3, Fig.
2C). Currents were larger at positive potentials than
at corresponding negative potentials, indicating that
Ispont is outwardly rectifying (Fig. 2C).
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Fig. 3.
Zn2+ activates ZAC.
A, leak subtracted Zn2+ (1 mM)-activated currents, recorded from a cell expressing
ZAC, with intracellular and extracellular solutions containing equal
K+ and Na+ concentrations (~140
mM), respectively. Traces are averages of two currents
recorded at each potential. The graph illustrates that
IZn exhibited pronounced outward rectification
with equilibrium potentials dependent on the concentration of
intracellular K+. IZn were recorded
from cells expressing ZAC with electrode solutions containing either
140 mM K+ (open circles) or in which
K+ was substituted by equimolar NMDG+ (70 mM; filled circles). B, a graph
illustrating the time-dependent run-up of Zn2+
(1 mM for 2 s)-activated currents. Data points fitted
with a sigmoid function were IZn amplitudes at
each time point normalized to the maximum IZn
recorded from each cell (n = 9). The inset
graph illustrates a lack of Ispont run-up
recorded from the same cells. Ispont amplitudes
were normalized to the maximum Ispont recorded
from each cell. Representative current traces recorded from a single
cell are shown in the right panel.
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Fig. 4.
Concentration-dependent
activation of ZAC by Zn2+. A, exemplar
Zn2+-activated currents were recorded from the same cell
and superimposed. Each trace is the average of three currents. A
logistics fit to the Zn2+ concentration-response
relationship yielded an EC50 of 540 ± 9 µM (n = 5). Current amplitudes are
expressed as percentage of those activated by 1 mM
Zn2+ in each cell. Vertical bars represent ± S.E. B, spontaneous and Zn2+ (1 mM)-evoked currents were unaffected by ondansetron (1 nM), mecamylamine (10 µM), -bungarotoxin
(10 µg/ml), and ketamine (100 µM). Each trace
represents three currents averaged under control conditions or in the
presence of the drugs indicated.
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Fig. 5.
TC inhibits ZAC activity. TC
caused a concentration-dependent inhibition of
IZn amplitude recorded from cells expressing
ZAC. Exemplar Zn2+ (1 mM)-activated currents
recorded from the same cell under control conditions and in the
presence of TC are illustrated. Each trace is the average of three
currents. A logistics fit to the TC (30 nM-100
µM) concentration-response relationship yielded an
IC50 of 6.6 ± 0.8 µM (n = 4). Current amplitudes are expressed as percentage of control.
Vertical bars represent ± S.E.
5 ± 1 mV (n = 6) and outward rectification similar to that of
Ispont (Figs. 2C and 3A).
We used alternative electrode solutions to examine the ionic
selectivity of ZAC. Substitution of half the intracellular
K+ with N-methyl-D-glucamine (70 mM), caused a right shift in the current-voltage
relationship (Fig. 3A), producing a positive
Zn2+ equilibrium potential (2 ± 3 mV,
n = 3). This demonstrates a role for intracellular
K+ ions in IZn. Substitution of half
of the intracellular K+Cl
with
K+-gluconate (70 mM) had no significant effect
on Zn2+ equilibrium potential (n = 4),
demonstrating that the channels have negligible Cl
permeability.
-bungarotoxin (10 µg/ml), mecamylamine
(10 µM), ondansetron (1 nM), and ketamine (100 µM; Fig. 4B). By contrast, TC caused a
concentration-dependent inhibition of
IZn (Fig. 5) with an IC50 of
6.6 ± 0.8 µM (n = 4).
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Fig. 6.
A functional ZAC gene is absent from
orthologous loci in rodents. A, the region of human
chromosome 17q23 encoding the galanin2 receptor (GALR2), ZAC
subunit, and the human orthologue of Exo70 (KIAA1067) was
aligned with the orthologous region of mouse chromosome 11. Exons are
indicated by black rectangles. Mouse exons that are
homologous to human exons, but which contain insertions and deletions
that generate in-frame stop codons, are indicated in gray.
B, the sequence of exon 5 from the human ZAC subunit gene is
aligned with orthologous sequences from mouse, rat, and dog genomic
DNA. For the human sequence, exon 5 is in uppercase, inside
brackets. Nucleotides that are conserved between
human and other species are shaded. C, alignment
of the deduced amino acid sequences for the human and dog ZAC subunits
(hZAC subunit and dZAC subunit, respectively). Conserved residues are
shaded.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells, Zn2+ is concentrated in vesicles at
high concentrations (>1 mM; Ref. 25). During vesicular
release, it is possible that micromolar concentrations of
Zn2+ are present within the synaptic cleft. However, high
affinity binding to extracellular albumin is likely to reduce free
Zn2+ concentrations to the low nanomolar range within a
short distance from its source. Native ZAC may be located close to a
vesicular source of free Zn2+. Indeed, fetal brain is among
a variety of human tissues that express transcripts of the ZAC subunit.
Expression of ZAC subunit mRNA was detectable in several other
human tissues. However, the absence of a functional ZAC subunit gene in
rats and mice precludes the use of these rodents for more detailed
localization studies. To resolve the distribution of ZAC expression at
the cellular level, it will be necessary to perform such studies on
tissues from human, dog, or other non-rodent species.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF512521-AF512523.
To whom correspondence should be addressed: The Institute for
Genomic Research, 9712 Medical Center Dr., Rockville, MD 20850. Tel.:
301-838-3536; Fax: 301-838-0208; E-mail: ekirknes@tigr.org.
Published, JBC Papers in Press, October 14, 2002, DOI 10.1074/jbc.M208814200
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ABBREVIATIONS |
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The abbreviations used are:
nACh, nicotinic
acetylcholine;
GABA, -aminobutyric acid;
5-HT, 5-hydroxytryptamine;
EST, expressed sequence tag;
TC, tubocurarine;
LGIC, ligand-gated ion
channel;
HEK, human embryonic kidney;
GFP, green fluorescent
protein.
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1. | Karlin, A. (2002) Nat. Rev. Neurosci. 3, 102-114[CrossRef][Medline] [Order article via Infotrieve] |
2. | Reeves, D. C., and Lummis, S. C. (2002) Mol. Membr. Biol. 19, 11-26[CrossRef][Medline] [Order article via Infotrieve] |
3. | Paterson, D., and Nordberg, A. (2000) Prog. Neurobiol. 61, 75-111[CrossRef][Medline] [Order article via Infotrieve] |
4. | Mohler, H., Crestani, F., and Rudolph, U. (2001) Curr. Opin. Pharmacol. 1, 22-25[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Hedblom, E.,
and Kirkness, E. F.
(1997)
J. Biol. Chem.
272,
15346-15350 |
6. | Davies, P. A., Hanna, M. C., Hales, T. G., and Kirkness, E. F. (1997) Nature 385, 820-823[CrossRef][Medline] [Order article via Infotrieve] |
7. | Lustig, L. R., Peng, H., Hiel, H., Yamamoto, T., and Fuchs, P. A. (2001) Genomics 73, 272-283[CrossRef][Medline] [Order article via Infotrieve] |
8. | Davies, P. A., Pistis, M., Hanna, M. C., Peters, J. A., Lambert, J. J., Hales, T. G., and Kirkness, E. F. (1999) Nature 397, 359-363[CrossRef][Medline] [Order article via Infotrieve] |
9. | Tokunaga, K., Nakamura, Y., Sakata, K., Fujimori, K., Ohkubo, M., Sawada, K., and Sakiyama, S. (1987) Cancer Res. 47, 5616-5619[Abstract] |
10. | Adodra, S., and Hales, T. G. (1995) Br. J. Pharmacol. 115, 953-960[Abstract] |
11. | Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., and Sixma, T. K. (2001) Nature 411, 269-276[CrossRef][Medline] [Order article via Infotrieve] |
12. | Saitou, N., and Nei, M. (1987) Mol. Biol. Evol. 4, 406-425[Abstract] |
13. | Harrison, N. L., and Gibbons, S. J. (1994) Neuropharmacology 33, 935-952[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Mural, R. J.,
Adams, M. D.,
Myers, E. W.,
Smith, H. O.,
Miklos, G. L.,
Wides, R.,
Halpern, A., Li, P. W.,
Sutton, G. G.,
Nadeau, J.,
et al..
(2002)
Science
296,
1661-1671 |
15. | Morinaga, T., Nakakoshi, M., Hirao, A., Imai, M., and Ishibashi, K. (2002) Biochem. Biophys. Res. Commun. 294, 630-634[CrossRef][Medline] [Order article via Infotrieve] |
16. | Jackson, M. B. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3901-3904[Abstract] |
17. |
Davies, P. A.,
Kirkness, E. F.,
and Hales, T. G.
(2001)
J. Physiol. (Lond.)
537,
101-113 |
18. | Brickley, S. G., Revilla, V., Cull-Candy, S. G., Wisden, W., and Farrant, M. (2001) Nature 409, 88-92[CrossRef][Medline] [Order article via Infotrieve] |
19. | Smart, T. G., and Constanti, A. (1990) Br. J. Pharmacol. 99, 643-654[Abstract] |
20. | Bloomenthal, A. B., Goldwater, E., Pritchett, D. B., and Harrison, N. L. (1994) Mol. Pharmacol. 46, 1156-1159[Abstract] |
21. | Gill, C. H., Peters, J. A., and Lambert, J. J. (1995) Br. J. Pharmacol. 114, 1211-1221[Abstract] |
22. |
Palma, E.,
Maggi, L.,
Miledi, R.,
and Eusebi, F.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10246-10250 |
23. | Amin, J., and Weiss, D. S. (1993) Nature 366, 565-569[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Vallee, B. L.,
and Falchuk, K. H.
(1993)
Physiol. Rev.
73,
79-118 |
25. | Frederickson, C. J., Suh, S. W., Silva, D., Frederickson, C. J., and Thompson, R. B. (2000) J. Nutr. 130 (suppl.), 1471-1483 |