Background
Sequence homology between the serotonin type 3 receptor
(5-HT
3 R), the nicotinic acetylcholine
receptor (nAChR), the GABA
A receptor and the glycine receptor
suggests a large amount of structural similarity within
this superfamily of ligand gated ion channels [ 1 2 3 ] .
This presumed structural homology can be used to guide site
directed mutagenesis studies of particular receptor
subtypes. On a gross level, all members of this superfamily
assemble as pentameric receptors [ 4 ] . In some cases,
receptors can be assembled from a single subunit (5-HT
3A R and nicotinic α7 receptors) [ 1 5 6
7 8 ] . In other cases, at least two different subunits are
required [ 2 4 9 ] . The ligand binding site is thought to
be formed at the junction between two subunits [ 2 4 ]
.
Based on data obtained from affinity labeling and site
directed mutagenesis studies, several regions of the
nicotinic acetylcholine receptor have been implicated in
ligand binding [ 10 ] . Six essential loops appear to
contribute to the binding site with the position of each
loop differing slightly depending on whether the receptor
is heteromeric or homomeric [ 4 ] . For homomeric receptors
such as the α7 nAChR, the loops are identified as A, B, C,
D, E and F [ 4 11 ] . The amino acids that interact with
ligands presumably extend into the binding site from these
loops. Subunits are composed of 2 faces (+ and -) with the
+ face of one subunit forming one side of the binding site
and the - face of another subunit forming the complimentary
side. Some binding loops (A, B and C) are present on the -
face while the remaining loops (D and E) are present on the
+ face. [ 4 ] . The individual amino acids that form
binding interactions with the functional groups present on
a ligand are likely to differ for each receptor subtype,
reflecting the specificity of a particular binding site,
however, the overall structure of the binding domain may be
similar even for binding sites with different ligand
specificities. Identification of ligand specificity
requires identification of both the location or structure
of individual binding site loops and the amino acids
present in a particular receptor subtype.
The purpose of this study is to extend the information
available from other members of this receptor family to the
5-HT
3 R. Sequence homology and a presumed
structural similarity to other ligand gated ion channels
suggests that the E loop region of the 5-HT
3A R forms part of the ligand binding
domain for 5-HT
3 R ligands. This region extends from
Y140 to K153 and is shown in Table 1. The homologous
sequences of other representative members of this family
are also shown. In the center of this region is a critical
glycine residue that is thought to play a role in
establishing a hairpin loop [ 12 ] . Recent x-ray
crystallographic data obtained from an ACh binding protein
(AChBP) shows a loop structure in this region resulting
from a 3 residue turn containing a glycine homologous to
G147 of the 5-HT
3 R. On either side of this putative
turn region are residues that have been identified as
important to receptor binding [ 12 13 14 15 16 17 18 ] .
The formation of this loop structure brings amino acids on
either side of glycine into close proximity and may form a
binding pocket that will accommodate one or more functional
groups. In order to identify the interaction of amino acids
in this binding loop with 5-HT
3 R ligands, we have constructed alanine
mutations of residues throughout this region and evaluated
the alteration in binding affinity of 5 different classes
of 5-HT
3 R ligands (Figure 1). Our data
identifies 3 tyrosine residues that appear to interact
selectively with each structural class and supports the
existence of a loop structure in this region of the
receptor.
Results
All mutant receptors were tested for their ability to
bind the 5-HT
3 R antagonist [ 3H]granisetron. Table
2shows the K
d values for wildtype mouse 5-HT
3AS Rs and the 13 alanine mutations we
evaluated. [ 3H]granisetron is a potent antagonist of the
wt 5-HT
3 R (K
d = 0.98 ± 0.12 nM). This value agrees
with published data for this compound [ 19 20 ] . B
max values range from the 5.5 pmoles/mg
protein observed for E148A to 0.30 pmoles/mg protein for
the K153A mutation, indicating some variability in
expression of the different receptors. In general, however,
receptor expression was similar to that reported by other
laboratories [ 1 20 21 22 ] . No detectable binding was
observed for G147A and V149A mutant receptors. For all
other mutants, decreases in binding affinity (increased K
d ) were observed although the magnitude
of the change was less than 10 fold in all cases. A bar
graph showing the change in K
d value resulting from each alanine
mutation is shown in Figure 2A. The largest decreases in
binding affinity were observed for Y142A (4.6 fold, Figure
3), E148A (5.3 fold) and Q150A - K153A (6 - 8 fold).
Inhibition binding assays were also conducted. Four test
compounds with structures representative of the major
classes of 5-HT
3 R ligands were chosen: serotonin
(5-HT, the endogenous agonist),
m -chlorophenylbiguanide (
m CPBG, agonist),
d -tubocurarine (
d -tc, antagonist) and lerisetron
(antagonist). The K
i values for inhibition of [
3H]granisetron binding by all four compounds are shown in
Table 3. Little change in K
i value was observed for the majority of
mutations. The values highlighted in bold in Table
3represent the K
i values for inhibition of [
3H]granisetron binding on mutant receptors that increased
over 10 fold compared to the K
i obtained for wildtype receptors. The
bar chart in Figure 2Billustrates the changes in K
i resulting from each mutation on the
test compounds. Ratios of K
i are shown as positive for increases in
K
i on mutant receptors versus wildtype
and negative for decreases. A positive change thus
corresponds to a decrease in binding affinity for the
compound as a result of the mutation. Large decreases in
binding affinity were observed for select compounds only on
the Y140A, Y142A and Y152A mutations.
A more detailed analysis of the competition binding data
obtained for the Y140A, Y142A and Y152A mutations is shown
in Figure 4. For 5-HT, the Y142A mutation produced a 110
fold increase in K
i and Y152A produced a 24 fold increase.
No change in K
i was observed for the Y140A mutation
(Figure 4A). The 5-HT
3 R agonist
m CPBG showed a similar profile for
the changes in K
i values resulting from mutations of the
three tyrosines (Figure 4B). As was observed for 5-HT, the
Y142A mutation produced a large increase in K
i (160 fold) while the Y14 0A and Y152A
mutations produced only 7 and 24 fold changes
respectively.
The K
i value for
d -tc inhibition of [ 3H]granisetron
binding was altered only slightly by the Y142A or Y152A
mutations (6.5 fold and 10 fold changes respectively). The
Y140A mutation, however, produced a 50 fold increase in the
K
i compared to wildtype receptors (Figure
4C).
The K
i value for lerisetron inhibition of [
3H]granisetron binding was increased 160 fold by the Y142A
mutation and 190 fold by the Y152A mutation. Only a 4.6
fold change in K
i resulted from the Y140A mutation.
Lerisetron was the only compound for which a large increase
in the K
i was observed on the Y152A mutation
(Figure 4D). This mutation produced smaller changes in K
i for 5-HT and
m CPBG (24 fold for both) and only a
10 fold change for
d -tc.
In whole cell patch clamp studies, 5-HT perfusion of
cells transfected with Y140A and Y142A cDNA produced no
responses at 5-HT concentrations of up to 1 mM although
specific binding to these receptors was identified in
receptor binding studies. Unlike Y140A and Y142A, Y152A
receptors responded to application of 5-HT. Due to the low
potency of 5-HT on these receptors, only a portion of the
concentration response curve could be determined (up to 1
mM). The EC
50 value was estimated as greater than
370 μM (Figure 5). This value shows a greater than 140 fold
increase in EC
50 compared to wildtype receptors; a
larger change than was observed for the K
i (24 fold). The most dramatic change
observed for whole cell currents was an alteration in the
kinetics of the response elicited by application of 5-HT.
Y152A mutant receptors displayed much slower rise times
compared to wildtype receptors at all concentrations
tested. Peak wt responses were typically obtained in less
than 80 ms while Y152A responses required several seconds
to plateau. Desensitization kinetics were also altered.
While wt receptors desensitized rapidly, mutant receptors
showed no desensitization during the 8 s perfusion
time.
Discussion
The putative E-loop region of the LGIC family of
receptors is homologous to residues Y140 through K153 in
the 5-HT
3 R [ 4 12 13 14 15 16 17 18 ] .
Structure-function studies of this region have been
conducted in several other members of this family of
receptors including GABA
A and nAChR subtypes. In each case,
residues have been identified that alter either the binding
of selective ligands or receptor function [ 4 11 12 13 14
15 16 17 18 ] . In order to determine if this loop also
contains residues critical to the structure or function of
the 5-HT
3 R, we have constructed alanine
mutations of amino acids throughout the homologous region
of the mouse 5-HT
3AS R and investigated the affects on
binding of 5 different structural classes of 5-HT
3 R ligands. We have identified three
tyrosine residues that appear to play a role in binding of
selective ligands to this receptor. In addition, our data
support the existence of a loop structure in this region as
has been hypothesized for the nAChR and identified in a
homologous AChBP [ 12 23 24 ] .
Representative members of 5 major structural classes of
5-HT
3 R ligands were tested on all mutants
(Figure 1). These ligands include the antagonists [
3H]granisetron,
d -tc and lerisetron, and the
agonists 5-HT and
m CPBG. Most of the mutations tested
produced only minor changes in binding affinity for these
ligands. Large changes in binding are only apparent on
Y140A, Y142A and Y152A receptors for select ligands. The
resulting effects of individual mutations are specific to
particular structural classes of ligands. The Y140A
mutation altered the K
i for
d -tc inhibition, but did not alter
the K
i obtained for any other compound
tested, while Y142A altered the K
i for
m CPBG, 5-HT and lerisetron but had
little if any effect on inhibition by
d -tc. These data indicate the highly
specific nature of the effects introduced by the alanine
mutations and appear to reflect specific changes in
ligand/receptor interaction.
Binding of [ 3H]granisetron is altered only slightly by
the alanine mutations introduced in this study (<10
fold). This result indicates the lack of involvement of
amino acids in this binding loop in the binding of [
3H]granisetron. It is apparent, however, that [
3H]granisetron does occupy the same binding cleft as other
5-HT
3 R ligands as evidenced by the ability
of 5-HT,
m CPBG,
d -tc and lerisetron to displace it
from the binding site. The lack of any large change in
binding of granisetron supports our contention that there
is little global structural perturbation of the binding
site resulting from the introduction of each individual
alanine mutation.
The K
i for inhibition of [ 3H]granisetron
binding by 5-HT was increased 110 fold by the Y142A
mutation. A similar increase was also observed for
m CPBG (160 fold). In contrast, the
Y140A and Y152A mutations produced relatively small changes
in the K
i . Since both compounds are 5-HT
3 R agonists, they are likely to share
the same binding interactions. One of these interactions
appears to be with Y142. Other studies have also identified
binding site interactions for agonists. R91, E106, F107,
W183 and several residues adjacent to the M1 region have
all been demonstrated to alter the action of 5-HT and/or
m CPBG [ 20 21 25 26 ] . Within the
crystal structure of the AChBP, homologous residues are
located in the apparent binding site [ 24 ] . The effects
of agonist on the 5-HT
3 R are mediated by their interaction
with these binding site residues, resulting in stable
receptor conformations, including the channel open state.
Identification of interacting amino acids and their
location in the tertiary structure of the receptor may
provide clues to the mechanism of channel opening. For
example, amino acids homologous to Y142 and W183 (R104 and
W143 respectively) are in close proximity in the AChBP,
although on complementary faces of the receptor subunits [
24 ] . Spier
et. al . have suggested that W183 may
be involved in a cation-π interaction with the amino group
of 5-HT and
m CPBG [ 26 ] . If W183 and Y142 are
located near each other but on opposite faces of the
binding site in the 5-HT
3 R, then they could potentially act in
concert to help stabilize a conformation of the receptor
leading to channel opening.
d -Tubocurarine inhibition was
uniquely altered by the Y140A mutation. An increase in K
i of 50 fold was observed for
d -tc on this mutant. Little if any
change resulted for any other mutation. Thus, while
d -tc may form an interaction with
the receptor at this binding loop, it appears to interact
with Y140 rather than Y142 or Y152. This difference in
binding site interactions of the antagonist
d -tc and the agonists 5-HT and
m CPBG could be the result of a
slightly different positioning of
d -tc in the binding site. The
antagonists
d -tc and [ 3H]granisetron also
interact differently with this region since [
3H]granisetron binding was not altered by mutations at any
of the amino acids tested. While the binding of
d -tc appears to involve some
interaction with this binding loop, the binding of [
3H]granisetron does not. Previous studies have indicated a
point of overlap between [ 3H]granisetron and
d -tc at W89 of the 5-HT
3AS R. [ 20 ] . These studies indicate
that the binding regions for these antagonists are
partially overlapping at W89 but not at Y140. In addition
to Y140 and W89,
d -tc has also been observed to
interact with D206 and several other residues in loop C [
27 ] . The
d -tc binding site appears to involve
interaction with at least three different binding loops (A,
E and C) although additional interacting amino acids may be
found. Data obtained for
d -tc is particularly valuable due to
the rigid nature of this molecule. Determination of
interacting functional groups and their relationship to
individual amino acids could enable
d -tc to be used as a molecular ruler
to determine relative positions of these amino acids.
Lerisetron inhibition of [ 3H]granisetron binding to
mutant receptors was also investigated. Lerisetron is a
potent 5-HT
3 R antagonist first synthesized by
Orales
et. al. [ 28 ] . As was observed for
5-HT
3 R agonists, lerisetron binding was
altered by the Y142A mutation. This mutation produced an
increase in the K
i of 160 fold compared to wildtype
receptors. This increase in K
i indicates an important interaction of
lerisetron with Y142 and a similarity between the binding
location of lerisetron, 5-HT and
m CPBG. Lerisetron is the only
antagonist tested that shares a binding site interaction in
this region with agonists. In contrast to 5-HT and
m CPBG, however, lerisetron also
interacts with Y152 as indicated by the 190 fold increase
in K
i on Y152A mutant receptors. Since
neither
d -tc nor [ 3H]granisetron interacts
with Y142 or Y152, there appears to be a difference between
the interactions formed by these antagonists compared to
lerisetron. The binding sites of the three antagonists
tested differ with respect to this binding loop. Since the
role of a competitive antagonist is simply to block the
binding of agonists and prevent channel opening, the
specific amino acids that interact with the ligand can vary
for different antagonists. This is less likely to be the
case with agonists since they must produce a conformational
change in the protein to exert their effects.
Only two mutant receptors failed to bind [
3H]granisetron; G147A and V149A. G147 is the conserved
glycine in this putative binding loop. Chirara
et. al. have suggested that the
highly conserved nature of the glycine in this region may
indicate the existence of a loop structure consisting of
either γ or a loose three residue-turn in the nAChR [ 12 ]
. Either of these turns would bring the two putative
β-strands together such that γL109, γY111 and γS115 and
γY117 are all on the same side of an antiparallel β-sheet.
These residues have been identified by affinity labeling,
site-directed mutagenesis or cysteine substitution to lie
on the same surface. A classic 2-residue β-turn would place
these residues on opposite surfaces [ 12 ] . Substitution
of the conserved glycine by alanine may disrupt the
structure of this region and prevent assembly or expression
of the receptor. The recent determination of the crystal
structure of an AChBP supports this hypothesis. The AChBP
displays a large amount of homology to the amino terminal
of LGIC receptors and thus may be similar in structure [ 20
24 ] . The crystal structure of this protein reveals a
loose 3 residue turn incorporating the conserved glycine
residue [ 24 ] . Homologous residues in other LGIC subunits
have also been identified and are shown in Figure 1. The
residues identified in this study as altering binding
affinity of 5-HT
3 R ligands would also be present on the
same surface if this structure is present in the 5-HT
3 R. While Y140 lies somewhat outside
the region identified by Chiara in the nAChR (homologous to
γN107), γL109 and γL119 are homologous to Y142 and Y152 of
the 5-HT
3AS R. The ability of lerisetron to
interact with both Y142 and Y152 also supports the
hypothesis that these two amino acids are present in a loop
structure since the eight intervening residues would
position Y142 and Y152 too far apart to permit them both to
interact with a single ligand even if they were interacting
with functional groups on opposite ends of the molecule. A
loop structure would bring them into closer proximity and
permit interaction with the small molecule lerisetron.
All three tyrosine mutations were investigated using a
whole cell patch clamp assay to determine if functional
changes could be observed. Whole cell responses could not
be obtained for Y140A or Y142A, although specific binding
of [ 3H]granisetron was observed. These data suggest that,
while the receptors do assemble and are capable of binding
[ 3H]granisetron, they are either not transported to the
cell surface or are non-responsive to 5-HT at
concentrations of 1 mM or less.
Y152A does produce functional channels however they
display distinctly altered response kinetics when compared
to wildtype receptors. Y152A responses do not show the
rapid rise times observed in wt receptors. The extremely
slow rise times observed for Y152A receptors may indicate a
change in rate constants preceding channel opening. These
changes in the rate constants for either agonist binding or
channel opening also produce a 140 fold decrease in the
observed EC
50 for 5-HT activation. The slow rise is
followed by a non-desensitizing phase of the response that
is dramatically different from the fast desensitization
observed for wt 5-HT
3AS Rs. Lack of desensitization could
result from either a stabilization of the open state of the
channel or a destabilization of the desensitized state.
Mutations of homologous or nearby residues in both the
nAChR and GABA
A receptors have also been demonstrated
to alter the agonist response. Mutation of the homologous
residue in the GABA
A receptor γ-subunit (T142) to serine
altered the efficacy of the agonist Flumazenil, converting
it to a partial agonist [ 29 ] . In the nAChR, mutation of
mouse and rat εP121 to leucine altered both the binding of
acetylcholine and the stability of the open state of the
channel. εP121 is homologous to P154 in the 5-HT
3 R and is only two residues away from
Y152. The authors of this study concluded that this portion
of the acetylcholine binding site was closely linked to the
channel opening region of the receptor [ 30 ] . It is
reasonable to conclude that the homologous region in the
5-HT
3 R may perform a similar function. The
link between an agonist binding domain and a conformational
change leading to channel opening is not unexpected since
the two must obviously be linked. If binding to this region
of the receptor is shown to be a critical step between the
binding of agonists and the opening of the channel, further
investigation of the amino acids in this loop may provide
valuable clues to molecular basis of this process.
Conclusions
Our data indicate an important role for this putative
binding site loop in the interaction of the 5-HT
3 R with different ligands and
illustrate the difference in binding of different
structural classes of ligands. Each structural class shows
different patterns of interaction with amino acids in this
region of the receptor. [ 3H]granisetron does not appear to
interact with any of the amino acids tested while
d -tc interacts with only Y140,
m CPBG and 5-HT with Y142 and
lerisetron with both Y142 and Y152. Similar selective
effects have been observed on other residues including
W183, W89, F107 and E106 among others [ 20 21 25 26 ] . Our
data in conjunction with those of other laboratories
indicates the differences in orientation of different
ligands within the same binding cleft. These differences in
orientation result in different amino acid/functional group
interactions. As the structural detail of these
interactions emerges, these differences could potentially
be exploited to produce more potent and specific ligands.
For example construction of a "hybrid" ligand that combines
the interactions of [ 3H]granisetron with those of
d -tc, 5-HT or
m CPBG could produce an antagonist
with increased affinity due to the additional binding
energy of these interactions and a greater specificity
since it would utilize more structural features of the
binding site.
The requirement of a glycine at position 147 and the
ability of lerisetron to interact with both Y142 and Y152
also support the hypothesis that the secondary structure in
this region of the receptor is formed by a loop structure.
If the loose 3 residue turn proposed by Chiara
et. al. and shown for the AChBP is
present in the 5-HT
3 R, then Y140, Y142 and Y152 would be
present on the same side of the sheet and all three would
be capable of extending into the binding site. A similar
observation has been made for the nAChR where γS111, γY117,
γL119, δR113 and δT119 of the mouse nAChR receptor and
γL109 and γY111 of the torpedo nAChR are all thought to be
present in the binding site [ 12 13 14 15 16 17 18 ] .
Materials and Methods
Materials
[ 3H]granisetron was purchased from New England
Nuclear, 5-HT from Spectrum, and
m CPBG and
d -tc from Research Biochemical
International. Lerisetron was provided by Dr. Karen
Kirschbaum at The University of Louisiana at Monroe,
Monroe, LA. All other reagents were obtained from
commercial sources.
Site directed mutagenesis
Wild type 5-HT
3AS mouse receptor cDNA was obtained
from Dr. Michael White [ 20 ] . Mutant receptors were
constructed using either the Quick Change Mutagenesis kit
(Stratagene) or the Altered Sites Mutagenesis kit
(Promega). All mutations were confirmed by commercial DNA
sequencing.
Cell culture and transfection
tsA201 cells, a derivative of HEK293 cells, were
seeded at a density of 5 × 10 6cells/100 mm dish. Cells
were grown in DMEM medium containing 10% FBS, 100
units/ml penicillin/streptomycin for nine hours in 5% CO
2 and transfected with 10 μg mouse
5-HT
3AS R cDNA per 100 mm dish using the
calcium phosphate technique (New Life Technologies, NY).
Media was changed 12-14 hrs after transfection. The cells
were allowed to grow for another 24 hours and then
harvested.
For whole cell patch clamp experiments, tsA201 cells
were seeded to a density of 0.25 × 10 6cells/60 mm dish.
Cells were grown in DMEM culture medium containing 10%
FBS and 100 units/ml penicillin/streptomycin for 12 hours
prior to transfection. Transfections were performed using
Superfect Transfection Reagent (Qiagen, CA). Ten μg of
cDNA were mixed with DMEM medium containing no serum or
antibiotics in a volume of 150 μl. Twenty μl of Superfect
reagent were then added and the mixture incubated at room
temperature for 15 min. The reaction was terminated by
adding 1 ml of DMEM medium containing 10% FBS and 100
units/ml penicillin/streptomycin and the entire mixture
added to cells in the 60 mm dish. Cells were exposed to
Superfect Reagent for 3 hours. At that time, the reagent
was replaced with DMEM medium containing 10% FBS and 100
units/ml penicillin/streptomycin and incubated for an
additional 24 hours prior to use.
Binding assays
Transfected cells were scraped from the dishes, washed
twice with Dulbecco's PBS (New Life Technologies, NY),
then resuspended in 1.0 ml ice cold PBS/100 mm dish.
Cells were either used fresh or frozen at this step until
needed. Immediately prior to use, cells were homogenized
on ice in PBS using a glass tissue homogenizer then
centrifuged at 35 000 × g for 30 minutes in a Beckman
JA20 rotor (4°C). Membranes were washed once more with
PBS at 4°C then resuspended in 1 ml PBS/100 mm dish.
Protein content was determined using a Lowry assay
(Sigma. Diagnostics, St. Louis, MO). Membranes were
initially prepared and B
max and K
d values determined in the presence
and absence of a cocktail of protease inhibitors
(Complete Protease Inhibitor Cocktail, Roche Diagnostics,
Mannheim Germany). No change in B
max was observed as a result of
omitting the protease inhibitor (B
max (+ protease inhibitor)= 3.1 ± 0.11
pmoles/mg protein vs B
max (- protease inhibitor) = 3.2 ±
0.13 pmoles/mg protein.) hence all assays were performed
in the absence of the cocktail. Binding assays were
performed in PBS.
For K
d determinations, 100 μl of homogenate
was incubated at 37°C for 1 hour with varying
concentrations of [ 3H]granisetron (NEN, MA). Specific
binding of [ 3H]granisetron was determined as the bound [
3H]granisetron not displaced by a saturating
concentration of a competing ligand. K
d values were determined by fitting
the binding data to the following equation using Graphpad
PRISM (San Diego CA): B = B
max [L] n/ ([L] n+ K n), where B is
bound ligand, B
max is the maximum binding at
equilibrium L is the free ligand concentration and n is
the Hill coefficient.
K
i determinations, 100 μl of homogenate
was incubated at 37°C for 2 hours with varying
concentrations of inhibitor and [ 3H]granisetron (NEN,
MA). Binding was terminated by rapid filtration onto a
GF/B filters. The IC
50 values were calculated by fitting
the data to the following equation using Graphpad PRISM
(San Diego CA): θ = 1/ (1+(L/IC
50 )), where θ is the fractional
amount of [ 3H]granisetron bound in the presence of
inhibitor at concentration L as compared to the amount of
[ 3H]granisetron bound in the absence of inhibitor. IC
50 is the concentration at which θ =
0.5. The K
i is calculated from the IC
50 value using the Cheng-Prusoff
equation.
Electrophysiological Recordings
Transfected cells were transferred to a recording
chamber containing extracellular solution (140 mM NaCl,
1.7 mM MgCl
2 , 5 mM KCl, 1.8 mM CaCl
2 , 25 mM HEPES pH 7.4). Patch
electrodes of resistance 2.5-3.0 MΩ were filled with
filtered intracellular solution containing 145 mM KCl, 2
mM MgCl
2 , 1 mM EGTA, 25 mM HEPES (pH 7.4).
Cells were clamped in whole cell configuration at a
holding potential of -60 mV. A continuous extracellular
solution flow (0.8 ml/min) was maintained throughout the
recording procedure. 5-HT was dissolved in extracellular
solution and delivered to cells using a rapid perfusion
system (Warner Instruments, Hamden, CT) at a rate
matching the extracellular solution flow rate. The drug
perfusions lasted for a period varying from 4 to 8
seconds. Currents elicited by agonist application were
measured using an Axopatch 200 B amplifier (Foster City,
CA). The data were plotted and analyzed by non-linear
curve fitting (Graphpad PRISM, San Diego CA) according to
the following equation: I = 1/(1 +(EC
50 / [C]) n), where is the normalized
current at 5-HT concentration [C], EC
50 is the concentration of 5-HT needed
to obtain half maximal activation and n is the apparent
Hill coefficient.
