From the Division of Neurobiology, Medical Research
Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH,
United Kingdom and the ¶ Department of Biochemistry, Tennis Court
Road, Cambridge CB2 1QW, United Kingdom
Received for publication, October 19, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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The 5-hydroxytryptamine3
(5-HT3) receptor is a member of a superfamily of
ligand-gated ion channels, which includes nicotinic acetylcholine, 5-HT31
receptors are ligand-gated ion channels (LGICs) whose activation
results in membrane depolarization due to the gating of an integral
cation-selective channel (3, 4). 5-HT3 receptors can exist
as homomeric assemblies of 5-HT3A receptor subunits or
heteromeric assemblies of 5-HT3A and 5-HT3B
receptor subunits; these have distinct physical properties (5, 6).
5-HT3 receptors form part of a phylogenetically linked
superfamily of LGIC, which is typified by the nicotinic acetylcholine
(nACh) receptor, also includes the glycine and The occurrence of both anionic and cationic-selective receptors in such
a closely related family provides a basis for the identification of the
molecular determinants of ion selectivity. Since there is overwhelming
evidence to support a major role of the M2 domain in forming the walls
of the ion channel (for a review, see Ref. 10), previous studies have
concentrated on this region of the receptor in the search for amino
acids that play a role in ion conduction. For instance, site-directed
mutagenesis studies on acetylcholine receptors have identified rings of
residues that alter channel gating (11), conductance (12), or the
selectivity among monovalent (12, 13) or divalent (14) cations.
Alignment of the primary amino acid sequences of the M2 regions of the
different members of the LGIC superfamily (a number of representative
sequences are shown in Fig. 1) highlights the striking levels of
conservation found between the M2 domains of both the cationic and
anionic receptors. There are, however, noticeable differences, and it is an evaluation of the differences between the cationic To investigate whether the same structural features are important in
determining the ion selectivity of the 5-HT3 receptor, we
introduced the equivalent mutations into the murine 5-HT3A receptor subunit (Fig. 1), which was then expressed in HEK293 cells and
characterized using the whole-cell configuration of the patch clamp
technique. Our results demonstrate that the mutant 5-HT3A
receptor exhibits a clear anion selectivity.
Cell Culture and Transfection--
Human embryonic kidney (HEK
293) cells stably expressing 5-HT3A receptors were
developed using the eukaryotic expression vector pRc/CMV (InVitrogen,
Abingdon, UK) containing the complete coding sequence for the
5-HT3A(b) subunit from NIE-115 cells as previously described (16). The 5-HT3A-STM, which incorporates three
point mutations, E Electrophysiological Procedures--
In the majority of
experiments, whole-cell currents from single cells were recorded using
an EPC-9 amplifier in conjunction with HEKA software as described
previously (19); however, for voltage ramp experiments, an Axopatch
200B amplifier (Axon Instruments Inc.) controlled using the pClamp7
software suite was used. In most experiments, cells were perfused with
an extracellular solution (E1) of the following composition: 130 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 1.1 mM EGTA, 2 mM
MgCl2, 10 mM HEPES, 30 mM
glucose, pH 7.2, with NaOH. Patch pipettes were made from thin walled
borosilicate glass capillary tubing (Clark Electromedical GC120F-10)
and filled with intracellular solution (I1; 140 mM CsCl, 1 mM MgCl2, 0.1 mM
CaCl2, 10 mM HEPES titrated to pH 7.2 with CsOH). Electrode resistances measured with these solutions were
typically 2-5 megaohms. In ion selectivity experiments, solutions with
modified ionic compositions were used. In our first series of
experiments, the extracellular NaCl concentration was reduced, and the
osmolarity of the resulting solution was maintained by the addition of
mannitol (E2; as E1, except 65 mM NaCl, 130 mM
mannitol). The extracellular chloride concentration was also reduced by
substituting chloride with the anion isethionate (E3; as E1 except 15 mM NaCl, 115 mM sodium isethionate). In further
experiments designed to allow the accurate determination of
permeability ratios
(PCl/PNa or PNa/PCl), we used
simplified versions of the intracellular and extracellular solutions
above, employing Na+ as the predominant cation. The
sodium-based intracellular solution (I2) contained 140 mM
NaCl, 0.1 mM MgCl2, 0.1 mM
CaCl2, 1.1 mM EGTA, 10 mM HEPES
titrated to pH 7.2 with 5 mM NaOH, and the simplified extracellular solution (E4) used contained 140 mM NaCl, 0.1 mM MgCl2, 1 mM CaCl2,
30 mM glucose, 10 mM HEPES titrated to pH 7.2 with 5 mM NaOH. Again, an extracellular solution (E5)
involving isethionate substitution for chloride was employed (as E4
except 126 mM sodium isethionate, 14 mM NaCl).
Liquid junction potentials arising at the tip of the patch pipette were
calculated by the method of Barry and Lynch (20), and potential
measurements were corrected post hoc. Drugs were applied in
one of three ways: via a U-tube device capable of complete solution
exchange within 100 ms (19), via a DAD-12 perfusion system (ALA
Scientific Instruments Inc.) (21), or, for voltage ramp experiments,
via a Warner Instruments SF-77B fast solution exchange system (22),
both of which are capable of solution exchange in ~30 ms.
Current-voltage relationships were established by measuring the peak
current evoked by 10 µM 5-HT at holding potentials
between Drugs and Reagents--
All cell culture reagents were from Life
Technologies, Inc. except fetal calf serum, which was from Sigma. 5-HT
hydrochloride was from Research Biochemicals Inc. (St. Albans, UK). All
other reagents were from Sigma.
Functional Characterization of WT and Mutant 5-HT3
Receptors Expressed in HEK293 Cells--
The mutant 5-HT3A
receptor, which we have called the 5-HT3A-STM (for
selectivity triple mutation),
containing the three point mutations defined in Fig.
1 was functionally expressed in HEK293 cells, giving rise to 5-HT-gated whole-cell currents, which were completely blocked by coapplication with the selective
5-HT3 receptor antagonist granisetron (100 nM;
Fig. 2A). Dose-response curves revealed that the 5-HT3A-STM had a 10-fold lower
EC50 for 5-HT (240 ± 30 nM,
n = 5) compared with the WT receptor (Fig.
2C; Table I) (19) with a
reduced Hill coefficient but no significant difference in the maximum
amplitude of the responses (Imax) (Table I). The
5-HT3A-STM receptor showed markedly different activation and desensitization kinetics compared with the WT receptor. In response
to a maximal concentration of 5-HT, 5-HT3A-STM receptor whole-cell currents peaked within 684 ± 117 ms (10-90% rise
time 316 ± 59 ms, n = 8), significantly more
slowly than for the WT receptor (time to peak 198 ± 14 ms,
10-90% rise time 103 ± 9 ms, n = 11; see Fig.
3 and Table I). Furthermore, while both
exhibited desensitization in the continued presence of a maximal
concentration of agonist (30 µM 5-HT), the rate of
desensitization was clearly much slower for 5-HT3A-STM. The
time course of agonist-induced desensitization could be fitted with a
single exponential, giving a time constant of 44.7 ± 3.6 s
(n = 3) for the 5-HT3A-STM, more than
10-fold slower than the WT receptor (3.4 ± 0.5 s,
n = 5; Table I). Surprisingly, shorter duration
applications of agonist (30 µM 5-HT, 5 s) to cells
expressing the 5-HT3A-STM receptor produced whole-cell
responses with a different time course of recovery; receptors appeared
to remain in a conducting state for >20 s after agonist removal (see
Fig. 3). This combination of slower activation, reduced
desensitization, and increased affinity exhibited by the
5-HT3A-STM suggests that this receptor may attain a
desensitized but conducting state (D*) in the presence of agonist, similar to that identified in some mutant acetylcholine receptors (1,
24, 25). Some of these receptors show spontaneous activity (channel
opening), and some are gated by antagonists (1, 26). We therefore
examined the effect of the application of the 5-HT3 receptor antagonists 100 nM granisetron or 100 nM D-tubocurarine alone to the
5-HT3A-STM receptor. Both antagonists had no apparent effect; they did not reduce leak currents (which would be indicative of
spontaneous channel activity) or cause activation of
5-HT3A-STM receptors (data not shown). Experiments to
examine the single channel conductance of the WT and
5-HT3A-STM receptors and hence address the possibility of
the occurrence of a secondary D* state were hampered by the low single
channel conductance of recombinant 5-HT3A receptors (Ref.
19; see "Discussion") and were not pursued further.
Ion Selectivity of the WT and 5-HT3A-STM
Receptors--
To compare the ion selectivity of the WT and mutant
receptors, we initially constructed current-voltage relationships in a number of different extracellular solutions, E1 (normal extracellular solution), E2 (NaCl-mannitol), and E3 (NaCl-isethionate), which differ
in their Na+ and/or Cl
To accurately quantify this change in ion selectivity and allow
calculation of the permeability ratio
PCl/PNa for both WT and
5-HT3A-STM receptors, we performed a series of further
experiments using simplified solutions in which Na+ was the
predominant cation (see "Materials and Methods"), and other
monovalent and divalent cations were eliminated or reduced as far as
possible. In these experiments, the current-voltage relationships were
more conveniently established by the use of voltage ramp protocols
( The results presented here show that the introduction of three
point mutations in or near the M2 domain are sufficient to convert the
ion selectivity of homomeric 5-HT3A receptors from cationic
to anionic. Substitution of V13'T in M2 together with the
neutralization of a ring of glutamate residues (E Gating and Desensitization of the
5-HT3A-STM--
Ligand-gated ion channels, exemplified by
the nACh receptor, open rapidly following agonist binding and, in the
continued presence of agonist, may enter an agonist-bound but
nonconducting "desensitized" state (23); this desensitized state of
a receptor possesses a higher affinity for agonist than the closed
resting or open states of the channel. Interestingly,
5-HT3A-STM receptors exhibited a higher apparent affinity
for 5-HT compared with WT, and the whole-cell currents recorded
exhibited a marked reduction in the rate of activation and
desensitization. In fact, the EC50 for 5-HT on the
5-HT3A-STM receptor (240 nM) is similar to the value of 230 nM reported for the EC50 of the
The effects of the 5-HT3A-STM receptor mutations on the
time course of 5-HT-gated whole-cell currents are strikingly similar to
the effects of the equivalent mutations on ACh-gated currents of Ion Selectivity of the 5-HT3A-STM--
Current-voltage
relationships for the WT and 5-HT3A-STM receptors exhibited
similar, inward, rectification in all extracellular solutions used. The
reversal potential for the WT receptor was shifted to a more negative
potential by the reduction in extracellular NaCl concentration (Fig. 4)
and was not significantly altered when extracellular Cl Structural Implications and Location of the Charge Selectivity
Filter--
Perhaps the most striking feature of the STM is the fact
that two of the three amino acids are not technically within the M2
domain at all (as defined in Fig. 1) but instead reside in the M1-M2
loop. This not only highlights the importance of this short linker
segment in normal receptor function but also strongly suggests that
this region of the protein may contribute directly to the ion
conduction pathway. The greatest insight into the individual roles of
the amino acids that make up the STM has been provided by further work
reported by Corringer et al. (26) on the charge selectivity
filter of the
The importance of the arrangement of the polypeptide backbone, rather
than the properties of individual amino acid side chains, in the
determination of the ion selectivity has now been demonstrated for the
voltage-gated K+ channel (36). Based on the structure of
this channel, solved to 3.2-Å resolution, Doyle et al. (36)
suggested that the K+ selectivity results from the specific
coordination of K+ ions by a ring of main chain carbonyl
atoms positioned at the selectivity filter of the channel. Similar
structural features may be important in the selectivity filter of
LGICs. So far, the structure of the ACh receptor, originally solved to
9 Å by electron microscopy (37) has now been refined to 4.6 Å (38),
and future work may yet provide sufficient improvements in resolution
to pinpoint regions of secondary structure within the channel mouth and
gate. Such studies have already allowed insight into the structural reorganization that may occur upon channel gating (39) and may yet
allow insight into the further changes that no doubt occur (40) upon
receptor desensitization.
In conclusion, the results of this study, combined with the findings of
Yakel et al. (9) and Eiselé et al. (8),
which showed that the gating mechanisms of the 5-HT3 and
-aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory
neurotransmission. Using a combination of site-directed mutagenesis and
electrophysiological characterization of homomeric 5-HT3A
receptors expressed in HEK293 cells, we have identified a set of
mutations that convert the ion selectivity of the 5-HT3A
receptor from cationic to anionic; these were substitution of V13'T in
M2 together with neutralization of glutamate residues (E
1'A) and the
adjacent insertion of a proline residue (P
1') in the M1-M2
loop. Mutant receptors showed significant chloride permeability
(PCl/PNa = 12.3, PNa/PCl = 0.08), whereas WT receptors are predominantly permeable to sodium
(PNa/PCl > 20, PCl/PNa < 0.05). Since
the equivalent mutations have previously been shown to convert
7
nicotinic acetylcholine receptors from cationic to anionic (Galzi
J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500-505) and,
recently, the converse mutations have allowed the construction of a
cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the
determinants of ion selectivity represent a conserved feature of the
ligand-gated ion channel superfamily.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid
type A receptors and is thought to have arisen from a common ancestor
over 2000 million years ago (7). All of these receptors are believed to
be constructed from a pseudopentameric arrangement of subunits around a
central water-filled pore. Individual subunits are predicted to contain
a large N-terminal domain and four transmembrane spanning domains
(M1-M4), arranged such that M2 (see Fig. 1 for residue numbering and
lettering in MZ) delineates the wall of the channel. Agonist binding to
the N-terminal domain results in a conformational change that is
communicated to the pore domain, resulting in the opening of the
channel. Evidence suggests that as well as the conservation of
structural features, the underlying molecular mechanisms of operation
of these receptors are also conserved (8, 9). However, once the
channels have opened, one crucial difference becomes clear; whereas the
integral ion channels of the 5-HT3 and nACh receptors are
cation-selective and are primarily concerned with excitatory
neurotransmission, glycine and
-aminobutyric acid receptors are
anion-selective and are consequently involved in inhibitory neurotransmission.
7 nACh and
anionic glycine receptor M2 regions that allowed Galzi et al. (1) to identify the determinants of cation versus
anion selectivity for the
7 nACh receptor. Initially, they
incorporated all of the
1 glycine receptor residues presumed to face
the channel lumen into the
7 nACh receptor and demonstrated that
this resulted in an anionic
7 nACh receptor. Subsequently, the
number of point mutations was reduced until the smallest subset of
determinants of the cationic versus anionic selectivity were
obtained. Three mutations were found to be essential: the substitution
of V13'T together with the neutralization of a ring of glutamate
residues (E
1'A) and the adjacent insertion of the "extra" amino
acid (proline) found in the anionic channels (referred to here as
P
1') in the M1-M2 loop. Subsequently, the "reverse"
mutations in the
1 glycine receptor have been shown to change the
ionic selectivity of this receptor (2). A schematic representation of
these three rings of residues in the channel domain of the receptor is
shown in Fig. 1B.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1'A, the insertion of a proline between -1' and -2' positions (referred to as
1' in this study), and V13'T in the M2
region of the 5-HT3 receptor, was generated by two
successive rounds of oligonucleotide-directed mutagenesis using the
Kunkel method (17) and confirmed by DNA sequencing. The
oligonucleotides used in the reactions were as follows: V13'T,
GATGATGAGGAAAGTACTGTATCCCAGAAG; P
1'/E-1'A:
GAGACTCTCGCGGGCCCACTGTCCG. Cell lines were routinely grown on
90-mm tissue culture plates in a 1:1 mix of Dulbecco's modified
Eagle's medium and F-12 supplemented with 10% fetal calf serum and
500 µg/ml geneticin in a humidified atmosphere at 37 °C and 7%
CO2. For transient transfections, HEK 293 cells at 60-70% confluency (~48 h postpassage) were transfected with WT or mutant plasmid DNA by the calcium phosphate precipitation method (18). For
electrophysiology, experiments cells were grown on 35-mm plates (Falcon), and recordings were performed on cells 1-4 days
post-transfection.
80 and +40 mV or by the application of voltage ramps (
70 to
+100 mV, in 200 ms) during the plateau phase of an agonist-evoked
response. Dose-response data were fitted with a logistic function using
Kaleidagraph (Abelbeck Software) according to the equation
I = Imax/(1 + (EC50/[L])n), where
Imax is the maximal response, EC50
is the concentration of agonist required for half-maximal effect, [L]
is the concentration of agonist, and n is the Hill
coefficient. Permeability ratios for Na+ and
Cl
were calculated according to the Goldman-Hodgkin-Katz
(voltage) equation (23): Vrev = (RT/F)ln((PNa[Na]o + PCl[Cl]i)/(PNa[Na]i + PCl[Cl]o)), where R,
T, and F have their usual meaning, Vrev is the reversal potential obtained from the
current-voltage relationship, Pion is the
permeability of the ion, and the subscripts i and
o refer to intracellular and extracellular ion
concentrations, respectively. Data were analyzed using Kaleidagraph,
Origin (MicroCal), or Clampex (Axon Instruments) software, and all data
are quoted as the mean ± S.E. for n independent
experiments. Where appropriate, Student's t test for
unpaired data was used, and values of p < 0.05 were
regarded as significant.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Primary amino acid sequences of channel
lining regions of members of the ligand-gated ion channel
superfamily. A, an alignment of the channel lining
regions of representative amino acid sequences of the
5-HT3A receptor subunit and those from related LGIC are
shown. The region illustrated includes the M2 transmembrane domain with
individual amino acids being referred to by the single letter code
devised by Miller (41). Boxes indicate amino acids that are
highly conserved among cationic and anionic receptors. The
arrows indicate amino acids that differ between anionic and
cationic receptors and that have been mutated in this study. The
5-HT3A-STM mutant illustrated contains the E 1'A and V13'T
point mutations and the insertion of a proline residue between G
2'
and E
1', which is referred to as P
1" in this study. The position of
the cytoplasmic (-4'), intermediate (-1'), and extracellular (20')
rings of charged residues bordering M2 in the cationic channels (12)
are also indicated. B, a schematic representation of the
channel domain of a LGIC showing the rings of amino acids that have
been mutated in this study.
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Fig. 2.
Characterization of 5-HT3A-STM
receptors expressed in HEK 293 cells. A,
5-HT3A-STM receptor currents were reversibly antagonized by
the selective 5-HT3 receptor antagonist granisetron (100 nM). The antagonist was preapplied for 20 s before
being coapplied with 5-HT and had no effect alone (data not shown).
B, typical whole-cell currents from experiments used to
establish the dose-response relationship for 5-HT3A-STM
receptor activation by 5-HT. C, graph illustrating the
pooled, normalized, concentration response data determined from
experiments similar to those illustrated in A. The
parameters determined from curve fits according to the Hill equation
are given in Table I. All traces shown are from single experiments but
are representative of at least three others
Activation and desensitization properties of WT and 5-HT3A-STM
receptors
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Fig. 3.
Activation and desensitization of WT and
5-HT3A-STM receptors. A-C, example of
whole-cell current traces of 5-HT-gated currents in HEK 293 cells
stably expressing WT or 5-HT3A-STM receptors. Currents were
evoked by the application of 30 µM 5-HT for the duration
indicated by the solid bars.
5-HT3A-STM responses were slower to activate than WT
receptors and desensitized more slowly in the continuous presence of
agonist (see Table I). Shorter duration applications of 5-HT resulted
in a different time course of recovery of 5-HT3A-STM
receptor currents. The vertical calibration bar corresponds to 200 pA.
D, the activation time course for WT and
5-HT3A-STM receptors is shown on an expanded time scale.
E, the differences in the time course of recovery of the
5-HT3A-STM receptor due to different agonist application
durations are highlighted by overlay of the traces shown in
B and C. All traces shown are from single
experiments but are representative of at least three others.
concentrations. Sample
traces illustrating the WT and 5-HT3A-STM receptor currents
recorded at the range of holding potentials studied to allow
construction of a steady state current-voltage relationship are shown
(Fig. 4, A and
B). The resulting current-voltage relationships
obtained from such data sets are given in Fig. 4C, and the reversal potentials estimated from the current-voltage data are
given in Table II. The reversal potential
of the WT receptor in normal extracellular solution (E1) was
9.0 ± 1.6 mV. The data show that reduction of the extracellular NaCl
concentration causes a leftward shift in the reversal potential from
9.0 ± 1.6 mV to
19.5 ± 2.0 mV
(
Vrev =
10.5 mV), indicative of a
cation-selective channel. The reversal potential of the WT receptor in
NaCl-isethionate solution (E3) was
7.1 ± 1.3 mV. This value is
not significantly different from that determined in solution E1
(
Vrev ~ 0 mV) and indicates that chloride
ions make no significant contribution to the ion current passing
through the WT receptor. In the equivalent I-V experiments
conducted for the 5-HT3A-STM receptor, the reversal potential in normal extracellular solution (E1) was 0.1 ± 1.8 mV,
and reduction of the extracellular NaCl concentration caused a
rightward shift in the reversal potential to 21.8 ± 0.9 mV
(
Vrev = +21.7 mV). Reduction of the
extracellular Cl
concentration also caused a rightward
shift in the reversal potential from 0.1 ± 1.8 mV to 26.9 ± 2.9 mV (
Vrev = +26.8 mV). Both of these
findings indicate that a significant component of the ion current
passing through the 5-HT3A-STM receptor is carried by Cl
ions. These data therefore suggest that the
cation-selective WT 5-HT3A receptor has been converted to
an anion-selective receptor by the introduction of the three point
mutations.
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Fig. 4.
Current-voltage relationships for WT and
5-HT3A-STM receptors. A and
B, sample traces illustrating experiments used to
determine the current-voltage relationships for the WT and
5-HT3A-STM receptors. 5-HT-evoked (10 µM,
2 s) current responses were recorded at the holding potentials
shown. These traces are from experiments conducted in E2
(NaCl-mannitol; see "Materials and Methods") extracellular solution
and are representative of five similar experiments.
C, current-voltage relationships determined from the
data like that shown in A and B for WT and
5-HT3A-STM receptors in E1 (normal extracellular), E2
(NaCl-mannitol) and E3 (NaCl-isethionate) solutions are shown. The
resulting current-voltage relationships were corrected for liquid
junction potentials post hoc according to the method of
Barry and Lynch (20), and the mean reversal potentials determined from
these data sets are given in Table II.
Reversal potential data for WT and 5-HT3A-STM receptors
70 to +100 mV, in 200 ms) applied during the plateau phase of an
agonist-induced response. The resulting data obtained for the WT
receptor shows the expected inward rectification profile for the
5-HT3A receptor and a reversal potential close to 0 mV
(
0.6 ± 0.3 mV) in symmetrical NaCl (Fig.
5). Substitution of 90% of the
extracellular chloride with isethionate did not have any significant
effect on the reversal potential of the WT receptor
(
Vrev = 1.2 ± 1.2 mV), consistent with
it being essentially impermeable to Cl
ions. In fact, a
shift in the reversal potential (
Vrev) of
less than 1 mV under such conditions is consistent with a
PCl/PNa of <0.05 or a
PNa/PCl of >20.
Equivalent experiments on the 5-HT3A-STM receptor showed a
reversal potential close to 0 mV in symmetrical NaCl, and this was
shifted to 41.0 ± 2.5 mV by the substitution of of 90% of the
extracellular chloride with isethionate. The shift in the reversal
potential (
Vrev) of 43.7 ± 3.5 mV is
consistent with a PCl/PNa
of 12.3 or a PNa/PCl of
0.08 and convincingly demonstrates that the 5-HT3A-STM
receptor is selective for chloride and relatively impermeable to
sodium.
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Fig. 5.
Determination of the ion selectivity of the
WT and 5-HT3A-STM receptors. Data obtained from
voltage ramp protocols for WT and 5-HT3A-STM receptors in
symmetrical NaCl solutions (A, solution E4; see "Materials
and Methods") and upon substitution of 90% of the extracellular
chloride by isethionate (B, solution E5). Voltage ramps
( 70 to +100 mV in 200 ms) were applied during the plateau phase of an
agonist-induced response (10 µM 5-HT) and under control
conditions (no 5-HT) The current-voltage curves shown represent the net
5-HT-evoked current after any leak currents (control ramps) have been
subtracted. Individual data sets were normalized to the current
recorded at
70 mV and then averaged. Occasional error
bars from the averaging process are shown. The resulting
current-voltage relationships were corrected for liquid junction
potentials post hoc according to the method of Barry and
Lynch (20). The reversal potentials were determined individually for
each current-voltage relationship before averaging, and the mean values
obtained for each solution are shown below the relevant
current-voltage curve. The shift in reversal potential as a result of
the reduction in extracellular chloride
Vrev
is also shown. This was not significant for the WT receptor, consistent
with its known cation selectivity. In contrast, a large
Vrev was seen for 5-HT3A-STM,
consistent with the construction of a chloride-selective
receptor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1'A) and the
adjacent insertion of the "extra" amino acid (proline) found in the
anionic channels (referred to here as P
1") in the M1-M2 loop
(Fig. 1) lead to the formation of an anion-selective 5-HT3A receptor. These mutations also had a significant effect on other properties of the 5-HT3A receptor, and these are discussed below.
7-2 mutant nACh receptor for acetylcholine (1) and the estimated
affinity of 5-HT for the desensitized state of the 5-HT3
receptor (50 nM) (27). The relevance of the lower Hill
coefficient noted for the 5-HT3A-STM receptor (1.45),
compared with WT is not clear but may reflect an alteration to the
normal gating mechanism of the receptor. For instance, large variation
in Hill coefficients (1.6-3.4) were noted for L9' substitutions
of the 5-HT3 receptor reported by Yakel et al.
(9), and Hill coefficients ranging between 1.4 and 2.8 were reported by
Galzi et al. (1) for various channel lining mutants in the
7 nACh receptor.
7
nACh receptors (1). In this, and other studies (1, 24, 25), the
combination of slower activation, reduced desensitization and increased
affinity has led to the suggestion that the mutant receptor may attain
a desensitized but conducting state (D*) in the presence of agonist.
The slow decay of 5-HT3A-STM receptor whole-cell current
responses could therefore be due to a similar phenomenon. If the
mutations have changed one (or more) of the desensitized states of the
receptor into a conducting one, then the reduced rate of current decay
may reflect passage from the open to a conducting desensitized state
before subsequent entry into a further (nonconducting) desensitized
state. Such a model may also explain the long duration open times
observed even in responses to short pulses of agonist (Fig.
3E), where the receptor may remain in a high affinity
desensitized (conducting) state until agonist unbinding occurs. Further
work, including single channel recording, has established unequivocally
that the mutant nACh receptor can indeed attain a desensitized
conducting state; it shows spontaneous channel activity and can even be
gated by the competitive nACh receptor antagonist
dihydro-
-erythroidine (1, 26). Attempts to gather similar evidence
for the existence of a desensitized conducting state of the
5-HT3A receptor were hampered by the low single channel
conductance of the homomeric receptor. Although we have previously been
able to use fluctuation noise analysis to study the unitary conductance
of the WT and mutant 5-HT3 receptors (19), preliminary
studies on the 5-HT3A-STM receptor failed to detect
any resolvable channel noise (alternating current
signal).2 These findings may
therefore reflect a reduced single channel conductance of the receptor,
but further studies would be required to support this conclusion. It
might, however, be anticipated that the conductance of the mutant
receptor would be low, since we have mutated one of the rings of
charged residues that Imoto et al. (12) has shown affect
conductance in the nACh receptor. In addition, it is likely that the
inner and outer vestibules of the channel play a role in concentrating
the appropriately charged ionic species (28); since we did not modify
these regions, the 5-HT3A-STM receptor may inadvertently be
designed to effectively concentrate ions of the wrong charge. Indeed,
similar observations reported for the converse glycine receptor STM
described by Keramidas et al. (2) make this an attractive
hypothesis. We also investigated if D-tubocurarine or
granisetron, both of which are antagonists of the 5-HT3
receptor, could activate 5HT3A-STM, but both compounds were
inactive in this respect. Furthermore, the use of
D-tubocurarine or granisetron did not provide any evidence
for the occurrence of spontaneous channel activity, since they had no
effect on the small resting leak currents observed in whole-cell
recordings on the 5-HT3A-STM receptor. Therefore, whether
or not the 5-HT3 and
7 nACh receptors share an ability
to attain a D* state is still not resolved and warrants further
investigation. The recent identification of 5-HT3B, which
can give rise to 5-HT3A/B heteromers with increased single
channel conductance (~16 pS) compared with the 5-HT3A
homomer (5) may prove useful in this respect. Furthermore, the
likelihood of
7 nACh receptor mutants exhibiting spontaneous activity appears to be linked to the V13' substitution (26); alternative mutations to V13'T within the 5-HT3 receptor
may therefore be an interesting starting point for further work and may
provide evidence for subtly different gating mechanisms between the two receptors. In fact, a recent report describing a 5-HT3A
receptor V13'S mutation supports this idea, since this receptor
has a similar EC50 and slowly desensitizing phenotype to
the STM described here and yet also exhibits clear spontaneous gating
behavior (29).
was largely substituted with isethionate. This, in agreement with other
findings (30-35), indicates that the current passing through the WT
receptor is predominantly carried by Na+ ions. In contrast,
equivalent experiments on the 5-HT3A-STM receptors showed
reversal potential shifts to positive potentials, consistent with an
appreciable chloride permeability of the receptor. Use of simplified,
symmetrical NaCl conditions to allow accurate determination of the
chloride permeability of the WT and 5-HT3A-STM receptors further supported these conclusions, showing that the insignificant chloride permeability of the cation-selective WT receptor
(PCl/PNa < 0.05) was
replaced by a highly chloride permeable channel in the
5-HT3A-STM receptor
(PCl/PNa = 12.3;
PNa/PCl = 0.08). The three mutations introduced into the WT receptor are therefore sufficient to switch the selectivity of the 5-HT3 receptor
from cationic to anionic. These results complement those from the
7 nACh and glycine receptor studies (1, 2) in which the same three
mutations were capable of producing similarly dramatic changes in ion
selectivity. For instance, in the case of the glycine receptor study
the chloride permeable WT receptor
(PNa/PCl = 0.04;
PCl/PNa = 24.6) was
converted to a sodium-selective channel with a
PNa/PCl of 3.7 (PCl/PNa = 0.27) by the
"reverse" set of mutations. The properties of the three amino acids
that make up the STM and their location in or near M2 therefore have
important implications for the understanding of channel gating and ion
selectivity of the entire LGIC superfamily.
7 nACh receptor, which demonstrates an essential role
of the inserted proline residue in the resultant anionic phenotype. In
their model, the E
1'A and V13'T mutations are considered to be
"permissive" rather than "essential" for the conversion in ion
selectivity; the E
1'A/V13'T double mutant does not change the ion
selectivity, but these changes combined with the addition of the
proline result in the receptor becoming anion-selective (26). Thus, the
critical difference between the cationic and anionic channels is the
introduction of this proline residue in the M1-M2 intracellular loop
and is consistent with the idea that a large structural rearrangement
is likely to underlie the dramatic change in ion selectivity of the
channel rather than the addition or removal of charged amino acid side chains. What is not clear at present is over what distance such a
structural rearrangement may occur. Is it just at the level of the
intracellular mouth of the channel, or does it extend to a
rearrangement of part or all of the M2 domain as well? Further mutagenesis experiments have been designed to address this point and do
show that the inserted proline residue can generate functional anionic
receptors even when inserted at the
3' or
1' positions (26);
however, insertion at
4' resulted in cationic receptors, and
insertion at 0' resulted in nonfunctional receptors. Clearly, a higher
resolution structure of the channel region of the protein is now almost
a prerequisite for an interpretation of the effects of such mutations.
7 nACh receptors were likely to be conserved, highlight the striking
level of conservation of the molecular mechanisms underlying the
functioning of these and related members of the LGIC superfamily.
Additionally, now that an equivalent study has been reported for the
glycine receptor (2), it is clear that the determinants of cation
versus anion selectivity identified here represent a
conserved feature of the LGIC superfamily.
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ACKNOWLEDGEMENTS |
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We thank Andrew Randall, Elizabeth Fletcher, John Peters, and Nigel Unwin for advice or comment on various aspects of the study and Graham Smith for technical assistance.
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FOOTNOTES |
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* A preliminary report of these findings has been published in abstract form (15).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.
§ Recipient of a Medical Research Council studentship. Present address: Neuroscience Dept., SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North), Harlow, Essex CM19 5AW, United Kingdom.
A Wellcome Trust Senior Research Fellow in Basic Biomedical
Science. To whom correspondence should be addressed: Dept. of Biochemistry, Tennis Court Rd., Cambridge CB2 1QT, United Kingdom. Tel.: 44 1223 766047; Fax: 44 1223 402310; E-mail: s.lummis@mole.bio. cam.ac.uk.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M009575200
2 M. J. Gunthorpe and J. A. Peters, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: 5-HT3, 5-hydroxytryptamine3; LGIC, ligand-gated ion channel; nACh, nicotinic acetylcholine; STM, selectivity triple mutation; WT, wild type.
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