1Kresge Hearing Research Laboratory of the South, Department of Otorhinolaryngology and Biocommunication; 2Neuroscience Center, Louisiana State University Health Sciences Center; and 3Tulane Cancer Center and Environmental Health Sciences, Tulane University Medical School, New Orleans, Louisiana 70112
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ABSTRACT |
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Chen, Chu,
Margarett
S. Parker,
Anthony P. Barnes,
Prescott Deininger, and
Richard P. Bobbin.
Functional Expression of Three P2X2 Receptor Splice
Variants From Guinea Pig Cochlea.
J. Neurophysiol. 83: 1502-1509, 2000.
ATP has been suggested to
act as a neurotransmitter or a neuromodulator in the cochlea. The
responses to ATP in different cell types of the cochlea vary in terms
of the rate of desensitization and magnitude, suggesting that there may
be different subtypes of P2X receptors distributed in the cochlea.
Recently three ionotropic P2X2 receptor splice variants,
P2X2-1, P2X2-2, and P2X2-3, were
isolated and sequenced from a guinea pig cochlear cDNA library. To test
the hypothesis that these different splice variants could be expressed
as functional homomeric receptors, the three P2X2 receptor
variants were individually and transiently expressed in human embryonic
kidney cells (HEK293). The biophysical and pharmacological properties
of these receptors were characterized using the whole cell patch-clamp
technique. Extracellular application of ATP induced an inward current
in HEK293 cells containing each of the three splice variants in a
dose-dependent manner indicating the expression of homomeric receptors.
Current-voltage (I-V) relationships for the ATP-gated
current show that the three subtypes of the P2X2 receptor
had a similar reversal potential and an inward rectification index
(I50 mV/I50
mV). However, the ATP-induced currents in cells expressing
P2X2-1 and P2X2-2 variants were large and
desensitized rapidly whereas the current in those cells expressing the
P2X2-3 variant was much smaller and desensitized slower.
The order of potency to ATP agonists was 2-MeSATP > ATP >
,
-MeATP for all three expressed splice variants. The ATP receptor antagonists suramin and PPADS reduced the effects of ATP on
all three variants. Results demonstrate that three P2X2 splice variants from guinea pig cochlea, P2X2-1,
P2X2-2, and P2X2-3, can individually form
nonselective cation receptor channels when these subunits are expressed
in HEK293 cells. The distinct properties of these P2X2
receptor splice variants may contribute to the differences in the
response to ATP observed in native cochlear cells.
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INTRODUCTION |
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Extracellular ATP has been suggested to
serve as a neurotransmitter, neuromodulator, cytotoxin, and mitogen in
various systems (see Bobbin 1996; Bobbin et al.
1998
; Burnstock 1990
; Housley 1997
). Ionotropic (P2X) receptors for ATP are found on cells
throughout the cochlea (Housley et al. 1999
;
Xiang et al. 1999
). The response to ATP differs greatly
among cell types in terms of the biophysics and pharmacology. For
instance, the current response to ATP shows very little desensitization
in outer hair cells (OHCs) (Chen et al. 1997
;
Housley et al. 1992
; Kujawa et al. 1994
;
Nakagawa et al. 1990
) whereas desensitization is
relatively fast in Deiters' cells, pillar cells, and inner hair cells
(Ashmore and Ohmori 1990
; Chen and Bobbin
1998
; Chen et al. 1998
; Skellett et al. 1997
; Sugasawa et al. 1996a
,b
). In addition, the
application of the ATP receptor antagonists also shows a difference in
the blockade of ATP-gated current among the cell types (Chen et
al. 1998
; Skellett et al. 1997
). Such variations
in phenotype between cells may partly be caused by differences in the
subunit composition of the receptors. Furthermore, alternate splicing
of individual subunit mRNA may produce variants with different
properties that contribute to the biophysical differences seen in vivo
(e.g., Partin et al. 1996
).
Seven subunits of the P2X receptors (i.e., P2X1,
P2X2, P2X3, P2X4, P2X5,
P2X6, and P2X7) have been isolated
(Barnard et al. 1997). To date P2X2 is the
only ATP receptor type that has been identified in the inner ear
(Brandle et al. 1997
; Housley et al. 1995
; Parker et al. 1998
;
Troyanovskaya and Wackym 1998
). In addition, several
splice variants of P2X2 have been identified in different tissues (Brandle et al. 1997
; Housley et al.
1995
; Koshimizu et al. 1998
; Parker et
al. 1999
; Simon et al. 1997
;
Troyanovskaya and Wackym 1998
). Heterologous expression
of the wild-type P2X2 (or P2X2-1 or
P2X2a) receptor isoform and the P2X2-2 (or
P2X2b) splice variant obtained from rats resulted in the
formation of functional homomeric receptors with different
characteristics (Brandle et al. 1997
; Koshimizu
et al. 1998
; Simon et al. 1997
). Parker
et al. (1998)
recently sequenced three splice variants of the
P2X2 receptor from a guinea pig organ of Corti cDNA
library: P2X2-1, P2X2-2, and
P2X2-3 (or P2X2?). The sequences of the
P2X2-1 and P2X2-2 variants are similar to
those previously isolated from the rat. The P2X2-3 has not
been characterized functionally and is unusual in that a significantly long sequence of amino acids is included in the putative extracellular domain. Therefore the purpose of this study was to examine whether these three variants, especially the P2X2-3, could be
expressed as functional homomeric receptors. For this purpose the three splice variants were individually and transiently expressed in human
embryonic kidney cells (HEK293). The properties of the three P2X2 receptor variants were characterized using the whole
cell voltage-clamp technique.
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METHODS |
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Construction of recombinant plasmids
The recombinant plasmids
pEGFP-N1-P2X2-1,
pEGFP-N1-P2X2-2, and
pEGFP-N1-P2X2-3, which carry the entire protein coding sequence of the guinea pig P2X2 splice variants, were
constructed as follows. Previously described cDNA clones (Parker
et al. 1998) isolated from a guinea pig organ of Corti library
(Wilcox and Fex 1992
) were digested with Bam
HI and Hin dIII and subcloned into a eukaryotic expression
vector (Enhanced Green Fluorescent Protein, pEGFP-N1, Clontech, Palo
Alto, CA). The insertion of the cDNA eliminated the translation of the
green fluorescent protein encoded downstream of the cloning site in
pEGFP-N1 (data not shown). Transfection quality DNA was obtained by
Qiagen Maxiprep column purification from One Shot competent cells
(Inv
F, Invitrogen, Carlsbad, CA) bearing the respective expression
plasmid. Verification of each variant inserted into the expression
vector was confirmed by restriction mapping and by sequencing each
variant at the 5' and 3' ends.
Functional expression in HEK293 cells
Cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS) at 37°C in 5% CO2. Approximately 103 cells were seeded onto sterile coverslips within 35-mm culture dishes before transfection with the appropriate cDNA expression plasmid and the native pEGFP vector as a fluorescent reporter. Twenty-four hours after plating, cells were transfected using Lipofectin (Life Technologies, Gaithersburg, MD). For transfection, 1 µg of pEGFP-N1 and 1 µg of cDNA construct were mixed with 97 µL of OptiMEM (serum free; Life Technologies, Gaithersburg, MD) and incubated at room temperature for 30 min. Ten µL of Lipofectin and 90 µL of OptiMEM were also incubated at room temperature for 30 min. These reagents were combined and incubated for 10 min at room temperature before addition of 800 µL of OptiMEM to make the transfection medium. The culture medium was removed from the cells and replaced with transfection medium. Cells were then incubated for 3-5 h at 37°C and 5% CO2. The transfection medium was removed and replaced with MEM + 10% FCS. Twelve to twenty-four hours later, cells expressing green fluorescence were analyzed for electrophysiological properties.
Electrophysiological recordings
Single HEK293 cells demonstrating fluorescence were
voltage-clamped using the whole cell variant of the patch-clamp
technique as described before (Chen and Bobbin 1998).
Membrane currents were filtered at 5 KHz (
3dB) using a four-pole
low-pass Bessel filter digitized with a 12-bit A/D converter and stored
for offline analysis using a microcomputer. Voltage paradigms were
generated from a 12-bit D/A converter using pClamp software. After the
whole cell configuration had been established, series resistance and cell capacitance compensation were carried out before the recording, with 90% series resistance compensation normally applied. All experiments were conducted at room temperature (22-24°C).
Solutions and drugs
The modified Hank's Balanced Salt solution (HBS) utilized
for transfected cells and perfusion bath contained (in mM) 140 NaCl, 5.4 KCl, 2.5 CaCl2, 0.5 MgCl2, 10 HEPES, and 10 glucose. The internal solution contained (in mM) 140 CsCl, 1.0 MgCl2, 5 HEPES, 11 EGTA, and 0.5 CaCl2. Internal and
external solutions were adjusted to a pH of 7.4 and had osmolalities of
300 and 287 mOsM, respectively. The drugs tested were freshly prepared
from powder or from stocks at desired concentrations in the HBS
external solution. All the chemicals and drugs including ATP were
purchased from Sigma (St. Louis, MO) except for 2-methylthioATP
(2-MeSATP), ,
-methyleneATP (
,
-MeATP),
pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and
suramin, which were from Research Biochemicals International (Natick,
MA). All the drug solutions were delivered from a U-tubing system that
applied drug solution around the cell for a select period of time while
the bath was continuously exchanged with a perfusion system separate
from the U-tubing system as described previously (Chen and
Bobbin 1998
; Chen et al. 1997
). The solution surrounding a cell can be exchanged within 20 to 30 ms (Murase et al. 1989
).
All the data are reported as means ± SE. Student's t-test or analysis of variance (ANOVA) were applied for statistical comparison when appropriate, and differences were considered significant when P < 0.05.
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RESULTS |
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Current-voltage relationship of the ATP-gated receptor channels
When HEK293 cells were individually and transiently transfected
with the P2X2 splice variants, a homomeric
ATP-gated ion channel was formed as evidenced by the inward currents
that were evoked at negative holding potentials in response to the
application of ATP to the HEK293 cells expressing the splice variants
(Fig. 1, A-C). No ATP-induced
current was elicited in nontransfected cells (data not shown). To
determine the current-voltage (I-V) relationships of the
ATP-gated current in cells expressing individual subunits, ATP (30 µM
for the P2X2-1, 10 µM for the
P2X2-2, and 1 mM for the
P2X2-3) induced current responses were recorded
at various holding potentials from 100 to +20 mV (Fig. 1D). The reversal potential of the ATP-gated currents was
1.2 ± 0.4 (SE) mV (n = 4) for the
P2X2-1,
0.5 ± 0.3 mV (n = 6) for the P2X2-2, and
1.5 ± 0.6 mV
(n = 5) for the P2X2-3, suggesting that the P2X receptors in HEK293 cells form nonselective cation channels. The I-V relationship (Fig. 1D)
for the current exhibited inward rectification. This rectification was
quantified by calculating a ratio of amplitudes of the ATP-gated
current at 50 mV and
50 mV (I50
mV/I
50 mV) as described
by Evans et al. (1996)
and Chen and Bobbin
(1998)
. The rectification indices for the three subunits were
similar (P2X2-1: 0.13 ± 0.01, n = 5; P2X2-2: 0.11 ± 0.01, n = 4; P2X2-3: 0.14 ± 0.02, n = 5) and suggested a strong inward
rectification of three P2X2 splice variants. This
index was smaller than that of cloned P2X1 and
P2X2 (Evans et al. 1996
) but
similar to the ATP-induced current recorded in Deiters' cells
(Chen and Bobbin 1998
).
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ATP-induced dose-response relationship
Figure 2 shows that ATP-gated
current responses exhibited a dose-dependency in all three subunits,
although there were differences in the magnitude of the current
response. The currents were much larger in the cells expressing the
P2X2-1 and P2X2-2 variants than in cells expressing the P2X2-3
variant. In addition, the concentration of ATP required to evoke a
response was much higher in the cells expressing the
P2X2-3 variant compared with the other two
variants (Fig. 2). The EC50 for ATP in cells
expressing the P2X2-1 and
P2X2-2 variants was 10 and 8 µM, respectively,
whereas it was 55 µM for the P2X2-3 variant.
In most cells expressing the P2X2-2 variant, at a holding potential of 60 mV, 100 µM ATP induced a current
amplitude that was larger than the maximum current amplitude that can
be measured with the Axopatch 200A amplifier (i.e., 20 nA). Therefore the dose-response curve for ATP only extend up to 30 µM for the P2X2-2 variants (Fig. 2).
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Rate of desensitization of the ATP-induced response
One of the distinguishing characteristics of different P2X
receptors is the rate of desensitization. The currents evoked by ATP in
the cells expressing the P2X2-1 and
P2X2-2 variants desensitized rapidly in the
presence of the purinoceptor agonists whereas the
P2X2-3 variant desensitized very slowly (Fig.
3A). The desensitization was
more pronounced during prolonged application of a high concentration of
ATP (30 µM for the P2X2-1 and
P2X2-2 variants; 1 mM for the
P2X2-3 variant; Fig. 3A). Because the
P2X2-3 variant exhibited virtually no
desensitization, the current waveforms could not be fit with exponential functions and no time constant was obtained. Because desensitization rate is concentration and activity dependent, the slow
desensitization of the P2X2-3 variant may be
caused by the lack of agonist affinity. The current decay for the
P2X2-1 and P2X2-2
variants was fitted well by a double exponential function. The time
constant () of the current decay was
1 = 3.92 ± 0.6 s and
2 = 15.77 ± 2.99 s (n = 10) for the
P2X2-1 subunit and
1 = 0.99 ± 0.2 s and
2 = 7.56 ± 0.57 s (n = 10) for the
P2X2-2 subunit (Fig. 3B). The
differences in two time constants between the
P2X2-1 and P2X2-2
variants were significant (P < 0.01). In addition, the
difference in the percentage of current remaining at the end of ATP
application to cells expressing the P2X2-1
subunit (4.5 ± 0.9%, n = 10) and the
P2X2-2 subunit (1.7 ± 0.4%,
n = 10) was significant (P < 0.05).
Approximately 95% (P2X2-1) and 98%
(P2X2-2) of the current was desensitized during
the 1-min period of agonist application.
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Effects of purinoceptor agonists
Another distinguishing characteristic of different P2X receptors
is the potency of various agonists such as 2-MeSATP and ,
-MeATP (Burnstock 1990
; Chen and Bobbin 1998
;
Fredholm et al. 1997
; Kennedy and Leff
1995
). Therefore these agonists were used to determine pharmacologic differences among the three P2X2
receptor splice variants. Figure 4 shows
that the three variants exhibited a similar response to 2MeSATP and
,
-MeATP except for differences in current amplitudes. Compared
with ATP, 2-MeSATP exhibited strong agonist activity, whereas
,
-MeATP exhibited weak agonist activity (Fig. 4,
A-C). The order of agonist potency is very similar to that observed in native Deiters' cells (Chen and Bobbin
1998
).
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Effect of purinergic antagonists
During coapplication of ATP with the ATP receptor antagonist
suramin (100 µM), the currents evoked by ATP were reversibly reduced
in HEK293 cells expressing P2X2-1,
P2X2-2, and P2X2-3 splice
variants (Fig. 5, A-D).
Suramin reduced the ATP-induced current to a greater degree in the
cells expressing the P2X2-3 splice variants
(Fig. 5, C and D) and the reduction in cells
expressing the P2X2-1 was not significant (Fig. 5D). During coapplication of ATP with the ATP receptor
antagonist PPADS (100 µM), the currents evoked by ATP were
significantly reduced in HEK293 cells expressing
P2X2-3 splice variants but not in cells
expressing the P2X2-1 or
P2X2-2 splice variants (Fig.
6, A-D). Immediately after
washing out the PPADS the current evoked by ATP was significantly
reduced in cells expressing P2X2-1,
P2X2-2, and P2X2-3 splice
variants (Fig. 6, A-D). A similar delayed block of
ATP-induced currents by PPADS was obtained previously in cells from the
organ of Corti (Chen et al. 1998).
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DISCUSSION |
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Results demonstrate that when P2X2-1, P2X2-2, and P2X2-3 splice variants were individually and transiently transfected in HEK293 cells, each formed homomeric ATP-gated ion channels. The homomeric P2X2-3 receptor differed greatly from P2X2-1 and P2X2-2 receptors in that the former was less sensitive to various agonists and required a higher concentration of agonist to elicit a measurable response. Even at high agonist concentrations the P2X2-3 receptor induced less overall current than the P2X2-1 and P2X2-2 receptors. At the concentrations of agonists tested, the P2X2-3 receptor exhibit little desensitization. However, because desensitization is a function of agonist concentration and induced response, the lack of desensitization may be the result of the low potency of agonists at this receptor.
The low potency of agonists suggests the P2X2-3
receptor splice variant, if expressed in vivo as a homomeric receptor, would probably have little sensitivity for endogenous ATP. However, the
homomeric P2X2-3 receptor expressed in HEK293
may not reflect the same sensitivity as the endogenously expressed
receptor. For instance, it may be that in vivo the
P2X2-3 subunit is part of a heteromeric receptor
with one or both of the other variants, in which case the
P2X2-3 receptor would act as a modulator or
modifier. This can be addressed in the future by studying stable
transfected HEK293 cells containing all of the various combinations of
the subunits. In addition, one cannot exclude the possibility that
other variants or proteins not described to date are present which also
have modulatory activity either alone or in combinations with the
identified splice variants. Overall the obstacles for elucidating the
structures that determine the response to ATP in vivo may be elusive as
it has been for other agonists and their receptors. For example,
striking differences have been described between natively and
recombinantly expressed NMDA receptors (for review see Sucher et
al. 1996).
Presumably, the P2X2-3 splice variant found in
the guinea pig cannot be found in the rat. The molecular mechanism
which produces this variant in the guinea pig is the inclusion of an intron which codes for an additional nine amino acids (i.e., intron VIII; Parker et al. 1998). In the rat this intron
contains a stop codon. Therefore if this sequence were included in the
rat mRNA, presumably it would result in a truncated protein lacking the second transmembrane spanning region of the receptor and would therefore be nonfunctional. This raises the question of why the guinea
pig makes this splice variant and the rat does not and what effect this
variant has on the function of the cochlea and other tissue where it
may be expressed (Parker et al.1999
). To date there is
only preliminary data on this question. Chen et al.
(1997)
failed to obtain responses to application of ATP in a
majority of rat OHCs, yet obtained large responses in guinea pig OHCs.
Because the guinea pig OHC response to ATP is a nondesensitizing response (Chen et al. 1997
; Housley et al.
1992
; Nakagawa et al. 1990
), the
P2X2-3 splice variant failed to demonstrate
desensitization and the P2X2-3 splice variant is
absent from the rat; this variant is a candidate for being the receptor
present in guinea pig OHCs and the one being absent from rat OHCs.
It appears that the recombinant P2X2-1 and
P2X2-2 receptors fairly accurately reflect the
activity of the receptors expressed in Deiters' cells and possibly
other supporting cells such as pillar cells in both species
(Chen et al. 1998). The nucleotide sequences of these
guinea pig receptors are very similar to the P2X2-1 and P2X2-2
receptor sequences found in the rat (Parker et al. 1998
)
and so the responses should be similar in both species. The homomeric
P2X2-1 and P2X2-2
receptors both desensitize rapidly and exhibit similar dose response
curves to various agonists including ATP. Therefore they respond
similarly to the receptors on Deiters' cells in both the rat and the
guinea pig (Chen and Bobbin 1998
; Chen et al.
1997
). Brandle et al. (1997)
and Simon et
al. (1997)
found the rat P2X2-2 receptor
had a faster desensitization time constant than the
P2X2-1 receptor when the subunits were expressed
in Xenopus oocytes. A similar relationship was observed
between the guinea pig P2X2-1 and
P2X2-2 receptors in the present study; however,
the guinea pig time constants were approximately twice the rates of those found in the rat. This difference between rat and guinea pig may
be because Brandle et al. (1997)
and Simon et al.
(1997)
made their measurement on receptors expressed in
Xenopus oocytes which might have different
posttranslational modification steps than the HEK293 cells used in this
study. However, similar slower rate constants for rat
P2X2-1 and P2X2-2 using Ca2+ flux
in HEK293 cells and neurons was reported by Koshimizu et al.
(1998)
In summary, P2X2-1, P2X2-2, and P2X2-3 splice variants formed homomeric ATP-gated ion channels when they were individually and transiently transfected into HEK293 cells. These ion channels exhibited differences in the rate of desensitization, magnitude, and pharmacology of the ATP-induced current responses among three splice variants. The distinct properties of these P2X2 receptor splice variants may contribute to the differences in the response to ATP observed in native cochlear cells.
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ACKNOWLEDGMENTS |
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We thank J. Campbell for technical assistance.
This work was supported by the Deafness Research Foundation and National Science Foundation Grant IBN-9817165.
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FOOTNOTES |
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Address for reprint requests: R. P. Bobbin, Kresge Hearing Research Laboratory, LSU Medical Center, 2020 Gravier St., Suite A, New Orleans, LA 70112.
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.
Received 21 September 1999; accepted in final form 1 December 1999.
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REFERENCES |
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