From the Institute of Molecular Physiology, University of Sheffield, Alfred Denny Building, Western Bank, Sheffield S10 2TN, United Kingdom and § Institut de Genetique Humaine, UPR 1142 CNRS, 141 rue de la Cardonille, 34396 Montpellier, France
Received for publication, December 15, 2000, and in revised form, February 6, 2001
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ABSTRACT |
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The first hydrophobic segment of the rat
P2X2 receptor extends from residue Leu29
to Val51. In the rat P2X2 receptor, we mutated
amino acids in this segment and adjoining flanking regions
(Asp15 through Thr60) individually to cysteine
and expressed the constructs in human embryonic kidney cells.
Whole-cell recordings were used to measure membrane currents evoked by
brief (2-s) applications of ATP (0.3-100 µM). Currents
were normal except for Y16C, R34C, Y43C, Y55C, and Q56C (no currents
but normal membrane expression by immunohistochemistry), Q37C (small
currents), and F44C (normal current but increased sensitivity to ATP,
as well as P2X receptors are a family of multimeric membrane proteins that
function as ion channels gated by extracellular ATP. Hydrophobicity plots for P2X receptors suggest that two parts of the protein are
sufficiently long and hydrophobic to cross the plasma membrane (1).
These are the regions, in the P2X2 receptor, of
Leu29 to Val51 and of Ile331 to
Leu353. Considerable experimental evidence now supports the
view that the N and the C termini are intracellular, and the region
between Val51 and Ile331 faces the
extracellular aspect. First, antibodies against N- and C-terminal
epitopes work only in permeabilized cells (2). Second, the proteins can
be glycosylated at both natural and artificially introduced consensus
sequences (NX(S/T)) at several positions in the
extracellular domain from Pro62 to Lys324 (in
the P2X2 receptor), though not at such positions in the N terminus (positions 9, 16, or 26) (2-4). Third, concatenated cDNAs
in which the C terminus of one construct is joined to the N terminus of
a second form functional channels (5).
There is now biochemical evidence that the P2X receptors form channels
as trimers (6, 7). However, the parts of the individual subunits that
contribute to different functions of the receptor are little
understood. Mutations of several positively charged residues have been
shown to decrease the effectiveness of ATP as an agonist at the
P2X1 (8) and P2X2 (9) receptors, and these
residues occupy corresponding positions (e.g.
Lys69, Lys71, Lys188,
Arg290, Arg304, and Lys308 in rat
P2X2 numbering). The region around Lys69 and
Lys71 is of particular interest with regard to a possible
ATP binding site. The P2X2 receptor functions normally when
Ile67 is mutated to cysteine (I67C). However, the
attachment of a negatively charged methanethiosulfonate
((2-sulfonatoethyl) methanethiosulfonate; MTSES1) led to a parallel
rightward shift in the ATP concentration-response curve that was not
seen with neutral (methyl methanethiosulfonate; MTSM) or positively
charged methanethiosulfonate ([2-(trimethylammonium)ethyl] methanethiosulfonate; MTSET). Point mutations that introduced a
negative charge (I67E and I67D), but not those that introduced a
positive charge (I67R and I67K), also caused inhibition of the current
that could be overcome by increasing the ATP concentration. Together
these results provide strong evidence that this region of the
receptor contributes to the ATP binding site (9).
When ATP binds to the P2X receptor the protein undergoes a
conformational change that results in the opening of a cation-permeable channel. The substituted cysteine accessibility method has also been
used to implicate residues in and around the second transmembrane domain in the formation of the ion-conducting pathway (10, 11). In
particular, P2X2-T336C is almost completely blocked by
exposure for 8 min to MTSET and MTSES; both negatively and positively
charged MTS reagents were effective, suggesting that Thr336
is located outside the membrane electric field, but the finding that
outward currents were blocked more rapidly than inward currents indicates that the attached side chain might directly interfere with
permeation (10). Another residue within the second hydrophobic segment
(Asp349) showed prominent block by MTSEA, but not by MTSET
and MTSES. This block did not occur if the MTSEA was applied without
opening, and in view of the fact that MTSEA is quite permeable through the P2X2 receptor channel, this suggests that
Asp349 is situated internal to the "gate" of the
channel (10).
The purpose of the present experiments was to ascertain whether
residues in and around the first hydrophobic segment might also
contribute to the ATP binding site or to the permeation pathway. P2X
receptors are not well conserved in the regions corresponding to the
first 14 amino acids of the P2X2 receptor, and we therefore began our cysteine substitutions at Asp15. We have recently
reported the effects of MTS compounds on the region Asp57
to Lys71 (9); in the present experiments we ended the
cysteine substitutions at Thr60 (see Fig. 1). As a first
approach we used MTSM, a small, neutral methanethiosulfonate, in
conjunction with point mutations to cysteine. We reasoned that this
might provide a picture of cysteines accessible to the aqueous
environment on both the intracellular and extracellular aspects of the
receptor. We followed this with tests of positively and negatively
charged methanethiosulfonates for those positions at which MTSM caused
a large inhibition. In an effort to understand further the mechanism of
the inhibition we studied the effect on the ATP concentration-response
curve and asked whether the inhibition required channel opening.
Finally, we sought to determine whether substituted cysteines in the
two transmembrane domains were sufficiently close to form disulfide bonds.
P2X2 Receptor cDNA and Mutagenesis--
A
P2X2 subunit cDNA carrying a C terminus epitope was
used; its source and the methods used for introducing point mutations were as described previously (10). All mutants were sequenced on both strands.
Electrophysiology--
Transient transfection of human embryonic
kidney 293 cells using Lipofectin or LipofectAMINE 2000 was as
described previously (10, 12-14). Standard whole-cell recordings and
fast-flow agonist applications were made as previously described (10,
12, 13). Internal solution contained (in mM): 145 NaF, 10 EGTA, and 10 HEPES; external solution was (in mM): 147 NaCl, 2 CaCl2, 2 KCl, 1 MgCl2, 13 glucose, and
10 HEPES. Current-voltage relations were obtained by ramp voltages (1-s
duration) from Immunohistochemistry--
Immunohistochemical methods were as
described previously (10). The mouse monoclonal anti-EYMPME
antibody (used at 1:1000 dilution) was obtained from BabCo (Richmond,
CA), and secondary antibody was tetramethyl rhodamine
isothiocyanate-conjugated anti-mouse IgG (1:100 dilution; Sigma).
Effects of Cysteine Substitutions--
We introduced cysteine into
each position individually and studied the actions of ATP on human
embryonic kidney 293 cells expressing the mutated receptors. ATP (30 µM) elicited currents not distinguishable from those in
cells expressing wild-type receptors (1-8 nA) for all the
mutated receptors except Y16C, R34C, Q37C, Y43C, F44C, Y55C, and Q56C.
Cells expressing Y16C, R34C, Y43C, Y55C, and Q56C showed no responses
to ATP (up to 3 or 10 mM); immunohistochemistry showed
staining of the plasma membrane in these cells. For Q37C, ATP-evoked
currents were smaller (0.3 to 2 nA), and the EC50 was about
three times higher than for the wild-type receptor (29 ± 5.7 µM; n = 4). Cells expressing two further
mutations (T18C and L29C) responded well to an initial application of
ATP, but the current declined steeply with repeated applications, and
they could therefore not be usefully studied. At T18C, the current also
declined during the ATP application more rapidly than seen at wild-type
channels; at the end of a 2-s application (30 µM) the
current was 43 ± 7% (n = 5) of its peak for T18C
and 89 ± 2.8% (n = 8) for the wild-type
receptor. This is similar to the finding of Boué-Grabot et
al. (16) for T18A.
At F44C, cysteine substitution caused three distinct changes in the
properties of the receptor. First, ATP-evoked currents returned back to
the baseline level more slowly than normal after a brief (2-s)
application. The times required to return to half the peak current at
the end of ATP application were 0.44 ± 0.04 s (n = 15) for wild-type receptors (30 µM ATP) and 1.2 ± 0.8 (n = 9; 3 µM ATP) or 1.6 ± 0.05 s (n = 29; 30 µM ATP) for F44C.
Second, there was 10-fold increase in sensitivity to ATP
(EC50 0.72 ± 0.1 µM; n = 4). Third, there was a remarkable increase in effectiveness of
Accessibility to MTSM--
We used MTSM for an initial screen of
this segment of the receptor; it is small and uncharged, and we
expected that it would cross the membrane readily and react with
accessible residues on either the cytoplasmic or the extracellular part
of the receptor protein. Fig. 1
illustrates representative ATP-evoked currents prior to and during an
8-min application of MTSM at 1 mM and after washing for 4 min. The effects of MTSM are summarized in Fig. 2. There was no significant effect on
ATP-evoked currents at wild-type receptors. We included T336C as a
control mutation, and found that the inhibition (>80%) was similar to
that previously reported for MTSEA, MTSET, and MTSES (10). At 8 of the
39 cysteine-substituted receptors that responded to ATP, MTSM caused a
large (
Previous studies on the second transmembrane domain found no effects by
MTSEA, MTSET, and MTSES on the positions on the C-terminal side of
D349C (W350C, I351C, L352C, L353C, and T354C). These positions are
clearly at the inner aspect of the second transmembrane domain, and we
re-examined them using MTSM. We found that MTSM (1 mM, 8 min) gave rise to significant inhibition (p < 0.001)
at I351C (76.5 ± 6.2%; n = 3) and L352
(69.8 ± 5.1%; n = 3).
Effects of MTS Compounds with Positively or Negatively Charged Head
Groups--
Val24 and Gly30 are believed to be
on the intracellular aspect of the receptor. The speed of the reaction
of MTSM at V24C (time constant < 2 min; see Fig. 3) and G30C
(
MTSET is not expected to cross the membrane, although it can permeate
the open P2X receptor channel (10). It is therefore a useful probe of
residues accessible to the extracellular aspect or intracellular
residues if ATP has been applied to open the channel while MTSET is in
the extracellular solution (see Ref. 10). MTSES, being negatively
charged, is not expected to enter the cell whether the channel is open
or not. Fig. 3 compares the effects of MTSM with MTSES and MTSET on
ATP-evoked currents in cells expressing these eight mutated receptors.
Val48 is believed to be on the extracellular aspect of the
receptor (see the Introduction). Both MTSET and MTSES caused large
inhibition of the current at V48C but had much lesser effect at any
other position; MTSEA (1 mM; 8 min) also caused inhibition
by 88.8 ± 7.4% (n = 4). The inhibition at V48C
by all four MTS reagents was relatively slow (time constant about 3 min
at 1 mM MTSET; see Fig. 4).
After treatment with MTSET or MTSEA, the ATP-evoked currents did not
return completely to initial holding current. The residual holding
current was 483 ± 80 pA (n = 3) for MTSEA (1 mM) and 411 ± 127 pA (n = 3) for
MTSET (1 mM) when measured 15 s after the first
application of ATP (30 µM; 2 s) in the presence of
methanethiosulfonate. This was in marked contrast to the effect of MTSM
or MTSES, where the currents declined quickly and completely to the
baseline level. It suggests that channel closure is impaired when a
positively charged methanethiosulfonate attaches at
Val48.
Taken together, the results with MTSM, MTSES, and MTSET are consistent
with the topology currently proposed for the P2X2 receptor. Introduction of cysteine at positions Asp15,
Pro19, Val23, Val24, and
Gly30 (before the first hydrophobic segment) and
Ile351 and Leu352 (end of the second
hydrophobic segment) led to significant inhibition by the membrane
permeant MTSM but little or no inhibition by charged MTS derivatives.
Conversely, cysteine substitution at Val48 (at the outer
edge of the first hydrophobic segment) resulted in strong inhibition by
MTSM, MTSES, and MTSET as we have previously described for three
residues (Ile328, Asn333, and
Thr336) at the beginning of the second hydrophobic domain
(10).
Effects of MTSM Modification on the ATP Concentration-response
Curve--
The shape of the concentration-response curve for ATP might
provide information on the mechanism by which the current is inhibited (9, 15). Before treatment with MTSM, the EC50 values for D15C, P19C, V23C, V24C, and V48C were 3.1 ± 0.4 (n = 3), 6.9 ± 0.2 (n = 3),
7.3 ± 0.8 (n = 4), 10.6 ± 1.0 (n = 6), and 3.4 ± 0.2 µM
(n = 3), respectively (Fig. 4). After treatment they
were not different (4.8 ± 0.4, n = 3; 9.3 ± 1.1, n = 3; 10 ± 1.3, n = 4;
9.4 ± 1.4, n = 6; and 4.0 ± 0.4 µM, n = 3, respectively). These values
are close to those for the wild-type receptor (7.9 ± 1.1 µM; n = 8). In other words, MTSM
modification at these positions results in a simple depression of the
maximum current evoked by ATP, with little change in the
EC50. This is similar to the result observed with T336C
(see Fig. 4 and Refs. 9 and 10).
Dependence of MTS Inhibition on Channel Opening by ATP--
V24C
was rapidly and completely inhibited by MTSM (Figs. 1, 3, and 5). This
inhibition was essentially the same even when ATP applications were
discontinued during the presence of the MTSM (Fig.
5A, left). On the
other hand, Fig. 5B (left) shows that the
positively charged MTSET produced little or no inhibition of the
currents at V24C unless ATP was repeatedly applied. We interpret this
to indicate that Val24 is situated on the intracellular
aspect of the receptor, but it can be accessed by MTSET entering
through the open channel. This is the same result, and the same
conclusion, as we made previously for inhibition at D349C by MTSEA
(10).
In the case of V48C, inhibition was observed with MTSM, MTSET, and
MTSES (Fig. 3). However, the effectiveness of MTSM and MTSET was
considerably greater when the ATP was repeatedly applied than when it
was not applied during the presence of the MTS derivative (Fig. 5,
A and B, right). This result implies
that conformational changes associated with ATP binding and channel
opening moves V48C into a position in which it is much more readily
accessible to reaction with MTS derivatives. In other words,
Val48 moves as a result of channel opening, and by moving
it becomes more accessible to MTS derivatives.
Disulfide Formation between V48C and I328C--
We have previously
presented evidence that T336C is located in the outer vestibule of the
ionic channel; the evidence for this was that outward currents were
inhibited more rapidly than inward currents as the MTSET reacted with
the cysteine. The present work indicates that Val48 is
situated at the outer edge of the membrane, and we therefore asked
whether these residues were sufficiently close to form disulfides that
altered the properties of the channel. We expressed the double mutants
V48C/I328C, V48C/N333C, and V48C/T336. The current elicited by ATP (30 µM) at the V48C/I328C receptor was much smaller (243 ± 70 pA; n = 11) than wild-type, V48C, or I328C
receptors (Fig. 6). We also observed
relatively large inward currents when the cells were held at
ATP-evoked currents for V48C/N333C and V48C/T336C were similar to those
observed for single cysteine mutants V48C, N333C, or T336C (range 1-8
nA), and application of dithiothreitol at 10 mM for 20 min
had no effect on the currents (see Fig. 6C).
Effects of Introducing Cysteines--
The rat P2X2
receptor was tolerant of cysteine introduced in all but 5 of the 46 positions examined between Asp15 and Thr60
(Figs. 2 and 7). Y16C, Y43C, Y55C, and
Q56C were non-functional; these residues are completely conserved among
all mammalian P2X receptors. R34C also did not express channels;
arginine is found in all subunits except P2X7, where it is
replaced by tryptophan. In four positions the introduction of cysteine
led to an obviously altered phenotype. In the case of T18C and L29C,
the response to ATP declined markedly when ATP was applied more than
once. Thr18 in the P2X2 receptor has been shown
by Boué-Grabot et al. (16) to be phosphorylated by
protein kinase C, and this alters the desensitization kinetics.
Leu29 has not previously been mutated, but we note that it
lies very close to the inner edge of the first hydrophobic domain. Q37C expressed more poorly (smaller maximum currents) than the other mutations and was less sensitive to ATP than wild-type receptors.
The effect of mutating Phe44 to cysteine was surprising in
that it resulted in a 10-fold increase in sensitivity to ATP and an
even larger increase in sensitivity to Effects of Methanethiosulfonates--
We used principally the
methyl, ethyltrimethylammonium, and ethylsulfonate derivatives of
methanethiosulfonates. After modification of a cysteine these would
result in a neutral [-CH2-S-S-CH3], positive
[-CH2-S-S-CH2-CH2-N+(CH3)3],
or negative
[-CH2-S-S-CH2-CH2-SO3
For all the modified cysteines, the reduction in the ATP-evoked current
occurred without change in the EC50 value. In other words,
increasing the ATP concentration could not overcome the inhibition of
the current resulting from methanethiosulfonate application. One can
distinguish broadly between a reduced affinity of the closed channel
for ATP (i.e. binding), an impaired ability of
the channel to open and stay open when ATP is bound (gating), and a
reduced current through the open channel (permeation) (14). Impairment
of binding or gating would usually produce a rightward parallel shift
in the concentration-response curve before the maximum is reduced,
whereas reduction in open channel current would not. It is conceivable
that mutations P19C, V23C, V24C, F44C, and V48C (Fig. 4) all directly
affect permeation, but independent direct measurements would be
required to show this. In no cases was there, for example, any obvious
effect on the rectification of the whole-cell current after cysteine modification.
Movement of Val48 with Channel Opening--
The
inhibition of current observed in V48C closely resembled that which we
have previously found for I328C, N333C, and T336C (10), all of which
are located close to the outer end of the second transmembrane domain.
The finding that all three methanethiosulfonates (positive, neutral,
and negative) cause strong inhibition indicates that this position is
situated outside the membrane electric field. We were surprised
therefore to observe that the reaction at V48C occurred much more
rapidly when ATP was repeatedly applied than when it was not applied
(Fig. 5). This result implies that the cysteine in the position of
Val48 moves to become more accessible when the channel is
opened. A second unique feature of V48C was the finding that MTSET
treatment caused an inward current to persist following the ATP
application. This was not observed for the uncharged reagent MTSM, even
though that caused similar inhibition of the current. We interpret this to indicate that channel closing is inhibited by the attachment of a
positively charged moiety in this position; a similar observation was
made previously for T336C (10). In that earlier work, there were three
positions at the outer edge of the second transmembrane domain that
were reactive with MTSET (Ile328, Asn333, and
Thr336). Because Val48 is situated at the outer
edge of the first membrane-spanning domain, we hypothesized that they
were sufficiently close to form a disulfide bond. This was tested
directly by expressing the doubly mutated receptors V48C/I328C,
V48C/N333C, and V48C/T336C. The latter two combinations resulted in
currents that were not different from wild-type receptors, but the
currents in cells expressing the V48C/I328C combination were very much
reduced in amplitude (Fig. 6). At the same time, these cells exhibited
large steady inward currents at
It is not possible to conclude from the present work whether the
disulfide is formed between V48C and I328C on the same receptor subunit
or on different receptor subunits that contribute to the multimeric
channel. However, the results do put constraints on models for the
channel. In Fig. 7 the two membrane-spanning domains are depicted as
-methylene-ATP). We used methanethiosulfonates of positive, negative, or no charge to test the accessibility of the
substituted cysteines. D15C, P19C, V23C, V24C, G30C, Q37C, F44C, and
V48C were strongly inhibited by neutral, membrane-permeant methanethiosulfonates. Only V48C was also inhibited by positively and
negatively charged methanethiosulfonates, consistent with an
extracellular position; however, accessibility of V48C was increased by
channel opening. V48C could disulfide with I328C, as shown by the large
increase in ATP-evoked current caused by reducing agents. The results
suggest that Val48 at the outer end of the first
hydrophobic segment takes part in the gating movement of channel opening.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
120 to 40 mV. The following MTS reagents used were
obtained from Toronto Research Chemicals (Ontario, Canada):
MTSEA, MTSET, MTSES, MTSM, and butyl methanethiosulfonate
(MTSB). MTSM and MTSB were used from a 1 M stock solution
that was made in Me2SO and kept as frozen aliquots; stock solutions (100 mM) for the other MTS compounds were
made daily by dissolving the solid in control external solution kept at
4 °C. All MTS compounds were diluted to 1 mM immediately
prior (2-5 min) to their application. Numerical estimates of
EC50 values were made for individual cells by least squares
curve fitting as previously described (9), using the function
I/Imax = [ATP]n/(EC50n + [ATP]n), where I is the current as a fraction of the
maximum current (Imax). The figures show this function
fitted to the mean for all cells tested. Results are shown as
means ± S.E. Tests of significance between paired observations
were by Student's t test or non-parametric Mann-Whitney
test; the effect of MTSM at many different positions was compared with
that on the wild-type channel by analysis of variance, followed by
Tukey-Kramer multiple comparison test (InStat software; GraphPad, San
Diego, CA). Results were considered significant for p < 0.05.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
meATP. The wild-type P2X2 receptor is essentially
insensitive to
meATP (13); indeed, we found that 100 and 300 µM
meATP evoked currents in cells expressing
wild-type P2X2 receptors were, respectively, 1.1 ± 0.2 and 8.1 ± 1.3% (n = 5) of the currents evoked by 100 µM ATP. In contrast, at F44C receptors
meATP activated currents with an EC50 value of
10.8 ± 0.5 µM (n = 4), and the maximum current evoked by
meATP (100 µM) (1.7 ± 0.2 nA; n = 5) was similar to that evoked by a
maximal concentration of ATP (3 µM) (1.2 ± 0.2 nA;
n = 5). There was no difference in the holding current
between cells expressing F44C and wild-type receptors.
60%) inhibition of the current that was significantly
different from the wild-type (p < 0.001); these were
D15C, P19C, V23C, V24C, G30C, Q37C, F44C, and V48C (Fig.
3). This inhibition did not reverse on
washing out the MTSM for up to 10 min (Fig. 1). These effects of MTSM were mimicked closely by another neutral MTS derivative, MTSB. At 1 mM (for 8 min), the inhibitions by MTSB were as follows: V15C, 90 ± 3% (n = 3); P19C, 89 ± 7.8%
(n = 4); V23C, 82 ± 4.1% (n = 3); and V24C, 97 ± 1.1% (n = 4). The inhibition
of the current by MTSM was not obviously dependent on membrane
potential as judged from ramp current-voltage plots.
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Fig. 1.
MTSM inhibition of cysteine-substituted
P2X2 receptors. Currents at wild-type and the
indicated mutated receptors were elicited by ATP (30 µM;
2 s) applied at 2-min intervals. When stable currents were
achieved, MTSM (1 mM) was applied for 8 min and followed by
a 4-min washing.
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Fig. 2.
Cysteine scanning with MTSM in region
Asp15 to Thr60 of the P2X2
receptor. Effect of MTSM (1 mM; 8 min) on currents
evoked by ATP (30 µM) in cells expressing each of the
single point mutations (n = 3-10 for each mutation) is
shown. MTSM was not tested at seven positions at which cysteine
substitution resulted in either non-functional channels (*, Y16C, R34C,
Y43C, Y55C, and Q56C) or channels at which the ATP-evoked currents
declined strongly with repeated applications (**, T18C and L29C).
Black bars indicate those positions at which the effect of
MTSM was significantly different from that at the wild-type
P2X2 receptor (p < 0.001). TM1,
first transmembrane domain.
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Fig. 3.
Effects of charged MTS compounds at
cysteine-substituted P2X2 receptors. At each position,
the effects of MTSES (1 mM) and MTSET (1 mM)
were examined following a similar protocol to that used for MTSM. The
four columns indicate the inhibition observed at 2, 4, 6, and 8 min (n = 3-10 cells for each case).
n.d. indicates not determined.
2 min; see Fig. 3) indicates that MTSM crosses the cell
membrane rapidly. This implies that the slower rates of reaction
observed for some other positions such as P19C indicate a slower
forward reaction rate rather than slower access to the
intracellular soluble compartment.
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Fig. 4.
ATP concentration-response curves are
unaltered by MTSM modification. After obtaining the control ATP
concentration-response curve (open circles), MTSM (1 mM) was applied. Following a 2-min wash, the second ATP
concentration-response curve was obtained (filled circles).
The duration of MTSM application was altered to achieve a significant
but not complete inhibition; it was 0.5 min for V24C, 2 min for P19C,
V23C, and F44C, and 4 min for V48C and T336C. Broken lines
indicate mean EC50 values for ATP before applying MTSM
(derived by fitting to individual cells; see "Experimental
Procedures"; n = 3-6 cells for each case).
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Fig. 5.
V24C is accessible to MTSET when the channel
is open, and V48C is more accessible to MTSET and MTSM when the channel
is opened. A, left, at V24C MTSM (1 mM; 8 min) completely inhibits whether ATP is applied
during the MTSM application (upper panel) or not
(lower panel). Right, in the case of V48C the
MTSM application is fully effective when ATP is applied during the MTSM
application (upper panel) but much less effective when ATP
is not applied (lower panel). B, left,
V24C is sensitive to inhibition by MTSET if ATP is repeatedly applied
(presumably, because MTSET enters the cell through the P2X channel when
it opens). Right, at V48C inhibition by MTSET was much less
when ATP was not applied repeatedly. Calibrations apply to all records.
C, summary of experiments shown in A and
B. *, p < 0.05; **, p < 0.01 (n = 3-10 in each case).
60 mV;
it normally required less than
50 pA to hold a human embryonic kidney
293 cell at
60 mV, but for V48C/I328C this was
235 ± 52 pA
(n = 8). This suggested that the P2X2
receptor channel was constitutively open in this mutated receptor.
Dithiothreitol (10 mM) greatly increased the amplitude of
the current evoked by ATP (about 6-fold) over 20 min and progressively reduced the sustained holding current in the absence of ATP (Fig. 6,
A and C). The ATP-evoked current increased
exponentially with time constant (
) of 5.9 ± 0.8 min
(n = 8) with 10 mM dithiothreitol, whereas
the sustained inward holding current declined rather more slowly
(
= 19.3 ± 7.4 min; n = 6) and had
reached
76 ± 41 pA at 20 min. A further reducing agent,
bismercaptoethanol (5 mM), also potentiated the ATP-evoked
currents (Fig. 6, B and C). Its action was
somewhat more rapid than that of dithiothreitol (
= 1.2 ± 0.5 min; n = 4). Bismercaptoethanol also reduced
the inward holding current from
299 ± 115 to
48 ± 17 pA
(n = 4) during a 20-min application (
= 4.3 ± 1.6 min; n = 3).
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Fig. 6.
V48C/I328C disulfide alters channel
opening. A, P2X2 receptor with double
mutation V48C/I328C showed very small responses to ATP, but these
increased 6-fold after applying dithiothreitol (10 mM;
solid bar). The effect declined when dithiothreitol
application was discontinued, and was repeatable. ATP (30 µM; 2 s) was applied at the times indicated at the
top of each current trace (min). B,
bismercaptoethanol (5 mM) had a similar effect.
C, summary of potentiation of ATP-evoked current in
P2X2-V48C/I328C by dithiothreitol (filled
diamonds) and bismercaptoethanol (open squares). Also
shown are the lack of any effect of dithiothreitol of wild-type
(open circles), V48C (filled triangles), I328C
(filled triangles), and V48C/T336C (filled
squares) receptors. All currents were normalized to that measured
prior to application of dithiothreitol or bismercaptoethanol
(n = 3-8 cells for each case). BMS,
bismercaptoethanol.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Schematic summary of results with
methanethiosulfonates on cysteine-substituted rat P2X2
receptor. All the residues shown have been substituted by cysteine
and tested with methanethiosulfonates. Shaded residues
indicate positions at which cysteine substitution results in strong
inhibition. Open letters indicate positions where cysteine
substitution results in non-functioning channels. Bold
circles indicate two positions at which functional channels were
expressed, but methanethiosulfonates could not be studied because of
profound run-down of response when ATP application was repeated.
Val48 and Ile328 can be disulfided. The present
work reports results from Asp15 to Thr60 and
Trp350 to Thr354. Other results included are
from Refs. 9 and 10.
meATP. Insensitivity to
meATP has come to be regarded as a major distinguishing feature among the different subtypes of P2X receptor, with those containing P2X1 and P2X3 subunits sensitive
(P2X1 and P2X3 homomers and P2X2/3 and P2X1/5 heteromers) and those not containing these
subunits several hundred-fold less sensitive (17). There have not been previous reports of point mutations conferring sensitivity to
meATP in P2X receptors of the insensitive subclasses
(P2X2, P2X4, P2X5, and
P2X7). Phenylalanine is found in this position in the
P2X2 and P2X3 subunits, but in the others the
residue is leucine, valine, or isoleucine. One interpretation of the
increased effectiveness is that this position contributes to the ATP
binding site. This seems somewhat unlikely in view of the fact that it is situated well within the first hydrophobic domain. A more likely explanation might be that
meATP normally can bind to the P2X receptor in much the same way as ATP but that it has very low efficacy
to induce the conformational change leading to channel opening. From
the results for the wild-type channel, the EC50 for
meATP can be very crudely estimated as around 1 mM,
but this is difficult to verify experimentally because of doubts that the
meATP might contain small amounts of ATP. In F44C, the
EC50 value for ATP shifted about 10-fold (from 8 to 0.7 µM); the EC50 value for
meATP must then
have shifted about 100-fold (from about 1000 to 10 µM). A
direct effect of this mutation on channel gating was also indicated by
the observation that the currents evoked by ATP took longer to decline
to the baseline in F44C than in wild-type channels; this is consistent
with slowed channel closing. Further analysis of this position with
other substitutions, and single channel recordings, is likely to
provide insight into the mechanisms of gating. One corollary of this
interpretation is that
meATP should act as a competitive
antagonist of ATP at wild-type P2X2 receptors. Although
this has not been reported, there is evidence that
meATP is an
antagonist of ATP action at P2X receptors in sympathetic neurons
(18).
]
side chain on the receptor protein. MTSM had no significant effect on
the wild-type P2X2 receptors but gave strong inhibition of
ATP-evoked currents in eight of the cysteine-substituted receptors (Figs. 2 and 7). Where MTSM has no effect, we cannot say whether the
cysteine is not accessible to an aqueous solution or whether the
cysteine is modified, but this modification does not change the channel
properties in any way that we have studied. The rapid rate at which
MTSM reacted with some cysteines at intracellular locations
(e.g. G30C and V24C) indicates that there is
little obstacle to its passage across the plasma membrane. Other
methanethiosulfonates did not have significant effects on cysteines at
intracellular positions in the N-terminal region of the receptor,
except for V24C. In this case, the action of MTSET was dependent on ATP
application, suggesting that it entered the cell through the open
channel. We have shown previously that MTSET is about 16% as permeable as sodium through P2X2 receptors (10); we have not used
MTSEA, because we (10) and others (19) have found that it can enter the
cell quite readily, presumably in its uncharged state. Cysteine substitutions at positions at the inner end of the second transmembrane domain (Trp350 to Thr355) have previously been
shown to be unreactive to MTSET; the present work showed that two of
them were accessible to MTSM, and this is consistent with an
intracellular location. In general, the results with MTSM and MTSET are
as would be expected on the basis of the topological models currently
proposed for the receptor (Fig. 7).
60 mV. Application of either
dithiothreitol or bismercaptoethanol greatly increased the ATP-induced
current and concomitantly decreased the persistent inward current.
Taken together, these observations suggest that a disulfide formed
between V48C and I328C results in a channel that is constitutively open and cannot be opened by applying ATP. The small currents that we
observed prior to adding the reducing agent might indicate that the
disulfide bond had not formed in all the channels or that the channels
could operate, though poorly, with the disulfide bond in place. The
impairment of channel opening by the V48C/I328C disulfide is quite
consistent with the conclusion reached above that Val48
moves during channel opening. Moreover, the finding that the V48C
channel does not open when this position is "tethered" to I328C
suggests that the movement of Val48 is not "fortuitous"
at some incidental part of the protein, but is a necessary component of
the gating mechanism.
-helices. There is no evidence for this, except to say that it is
strongly favored by secondary structure prediction algorithms (20, 21).
The accessible residues in the first transmembrane domain are located
at one side of the helix. The proximity of Val48 and
Ile328 indicated by the present results is fully consistent
with this structure. For both transmembrane segments virtually all of
the accessible residues mapped by cysteine scanning can be aligned along one face of an
-helix (Fig. 7). Key residues involved in gating the mechanosensitive channel of Escherichia
coli (which also has two membrane-spanning domains per subunit)
have a similar relative orientation (22). The proposed model suggests
several opportunities for future experiments to increase our
understanding of the modus operandi of P2X receptors.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Jean-Christophe Gelly, Daniele Estoppey, Gareth Evans, and Jayne Bailey for cell biology and molecular biology assistance.
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FOOTNOTES |
---|
* This work was supported by The Wellcome Trust (to R. A. N. and A. S.), The Royal Society (to R. A. N. and F. R.), CNRS, and La Fondation de la Recherche Medicale (to F. R.).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.
Contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 44 114 222 4668; Fax: 44 114 222 2360; E-mail: R.A.North@shef.ac.uk.
Published, JBC Papers in Press, February 6, 2001, DOI 10.1074/jbc.M011327200
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ABBREVIATIONS |
---|
The abbreviations used are:
MTSES, (2-sulfonatoethyl) methanethiosulfonate;
meATP,
-methylene-ATP;
MTS, methanethiosulfonate;
MTSEA, (2-aminoethyl) methanethiosulfonate;
MTSET, (2-(trimethylammonium)ethyl) methanethiosulfonate;
MTSB, butyl
methanethiosulfonate;
MTSM, methyl methanethiosulfonate;
EC50, concentration evoking half-maximal
response.
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