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Single channel properties of ATP-gated cation channels (P2X receptors) heterologously expressed in Chinese hamster ovary cells
ELSEVIER
Neuroscience Letters 212 (1996) 212-214
HEUROSGIENCE IETT[BS
Single channel properties of ATP-gated cation channels (P2X receptors) heterologously expressed in Chinese hamster ovary cells R.J. E v a n s ~,* Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development S.A., 14 chemin des Aulx, 1228 Plan-Les-Ouates, Geneva, Switzerland
Received 22 March 1996; revised version received 28 May 1996; accepted 12 June 1996
Abstract
The single channel properties of four P2X receptors heterologously expressed in Chinese hamster ovary cells were determined from outside-out patch recordings. P2X! and P2X 4 channels opened in brief flickery bursts with conductances of --18 and 9 pS respectively. P2X 2 receptors had a single channel conductance of --21 pS. Openings of P2X 3 channels were too rapid to be reliably resolved. Application of ATP to macro-patches expressing multiple P2X 2 or P2X 4 receptors evoked sustained currents, in contrast responses of P2X 1 or P2X 3 receptors desensitised during continued agonist application. These data show that four different homomeric P2X receptors form ion channels with distinct single channel properties. Keywords: Ion channels; P2X receptors; Purinergic
P2X receptors for ATP are ligand-gated cation channels present on many neuronal and smooth muscle preparations [ 1]. Functional subtypes of P2X receptors can be discriminated based on pharmacological and kinetic properties. In a limited number of single channel studies unitary conductance estimates for native P2X receptors range from 6 to 60 pS [2,3]. Several P2X receptors have been cloned recently and shown to define a new structural class of ligand-gated ion channels. In the present study, the unitary conductances of heterologously expressed homomeric P2XI_ 4 receptors [4-7] have been determined under identical recording conditions to see if functionally distinct channels are formed and how these correspond to native P2X receptors. The cDNAs encoding human bladder P2X1, and rat P2X 2, P2X 3 and P2X 4 receptors [4-7] were introduced into the Semliki forest virus (SFV) expression vector and used to infect Chinese hamster ovary (CHO) cells as described previously [4]. Responses to ATP were recorded
* Corresponding author, e-mail: [email protected] 1 Present address: Department of Cell Physiology & Pharmacology, Medical Sciences Building, University of Leicester, University Road, Leicester LEt 9HN, UK.
from excised outside-out patches using an Axopatch 200A amplifier (Axon Instruments, USA; patch holding potential - 1 0 0 mV). Initial experiments on P2X 2 receptors demonstrated that, as for native PC12 P2X receptors [8], unitary currents were larger when calcium was reduced from 2 to 0.3 mM and magnesium reduced from 1 mM to nominally magnesium free. Therefore, an extracellular solution of the following composition (in raM) was used, NaCI 150, sucrose 10, HEPES 10, CaCI 2 0.3, and pH adjusted to 7.3-7.4 with NaOH. Using this extracellular solution patches were less stable and had higher levels of background noise. Patch electrodes (10-15 Mff~) were filled with a solution containing (in mM), NaCI 140, EGTA 11, HEPES 10 and pH adjusted to 7.3-7.4 with NaOH (final Na 165). Experiments were carried out at room temperature (25°C). ATP (0.3-10 # M ) was applied rapidly to patches by a double barrelled 'bimorph' system. ATP evoked single channel events were not recorded from uninfected CHO cells (n = 5). SFV infection resulted in a high level of P2X channel expression and all patches expressed multiple P2X channels, therefore channel lifetimes were not analysed. Recordings were filtered at 1 kHz with a low pass Bessel filter and digitised at 5 kHz (Fetchex, PClamp 6, Axon Instruments, USA). Records were analysed using Axo-
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96) 12804-4
213
R.J. Evans/Neuroscience Letters 212 (1996) 212-214
r
B C
20 pA
pA
2.5s
E lpA 200 ms
=
F
200 ms
C
400
_400
$ 200]
l -3
E c
-2 -1 0 amplitude (pA)
i
-3
-2 -1 0 amplitude (pA)
1
Fig. 1. Single channel currents evoked by ATP from heterologously expressed P2X 1 and P2X 2 receptors. (A-C) P2X 1 receptors. (A) Response of an outside-out patch from a CHO cell expressing P2X 1 receptors to 3/~M ATP (application period indicated by bar). (B) ATP evoked single channel events (a 1 s segment of trace from (A)). (C) Amplitude histogram for record shown in (B). (D-F) P2X 2 receptors. (D) Channel activity recorded from an outside-out patch from a CHO cell expressing P2X 2 receptors superfused with ATP (0.3/~M) (recording made within 250 ms of ATP application). (E) Expanded record from the same patch as (D). (F) Amplitude histogram for record shown in (E). Patch potential, -100 mV.
graph 2 software (Axon Instruments, USA). Mean unitary currents were estimated from amplitude histograms (bin size 0.05 pA) generated from segments (0.8-5 s) of traces with openings to a single conductance level and fitted by the sum of two gaussian distributions. Chord conductances were calculated between -100 and 0 mV (reversal potential for P2X currents in above solutions). Application of ATP (3/xM) to outside-out patches of CHO cells expressing P2X 1 receptors evoked transient inward currents. During the continued application of ATP the peak inward current declined (n = 7 patches) and single channel openings could be resolved in three patches (Fig. 1A,B). These were usually brief (<50 ms) or in flickery bursts ( < 3 0 0 ms). The unitary channel amplitude at - 1 0 0 mV was -1.76 _+0.07 pA (n = 3; Fig. 1C).
In patches expressing P2X2 receptors application of 10/xM ATP evoked relatively sustained inward currents comprised of multiple channel openings (n = 12). By reducing the ATP concentration, single channel events (amplitude -2.12 _+0.09 pA (n = 5)) could be resolved (Fig. 1D-F). Channel openings (conductance --21 pS) lasted up to 1 s and were less flickery than those of P2XI, P2X 3 and P2X 4. Inward currents recorded in response to ATP (0.33/tM) in outside-out patches expressing P2X 3 receptors (Fig. 2A) were transient and decayed rapidly during agonist application. During the decline of the response fluctuations in membrane current <3 pA were recorded; however, unitary events could not be reliably resolved (n = 10; Fig. 2B).
A
C 20 pA
B
2.5s
pA
D
100 t
A 200 ms
1O0 ms
==
% -2
-1 0 amplitude (pA)
Fig. 2. The response of heterologously expressed P2X 3 and P2X 4 receptors to ATP. (A,B) P2X 3 receptors. (A) Transient inward current recorded in response to the application of ATP (3/tM, indicated by bar). (B) Expanded record from (A) showing unresolved openings of ATP-gated channels. (C,D) P2X 4 receptors. (C) Unitary currents recorded from an outside-out patch of a CHO cell expressing P2X 4 receptors in response to ATP superfusion (3 ffM) (recording made within 250 ms of ATP application). (D) Amplitude histogram for record shown in (C). Patch potential, -100 mV.
214
R.J. Evans/Neuroscience Letters 212 (1996) 212-214
Application of A T P (1-10/~M) tO outside-out patches expressing P2X 4 receptors evoked sustained inward currents (n = 8 patches). In three patches single channel events were resolved. These were of variable duration, flickery and had an amplitude of - 0 . 8 9 _+0.1 pA (n = 3) (conductance --9 pS; Fig. 2C,D). The present paper provides the first account of the single channel properties of homomeric P2X receptors; it demonstrates that P2X 1, P2X 2, P2X 3 and P2X 4 form functional ion channels with distinct single channel properties. The single channel conductance (--18 pS) and desensitization of P2X1 receptors (time constant of current decay in the continued presence of ATP 100--300 ms) are similar to those reported for native rat vas deferens P2X receptors ( - - 2 0 pS) [1,9] and demonstrate that the properties of native rat vas deferens P2X receptors are likely to be determined by P2X1 subunit expression [1,4]. P2X 2 receptors were originally cloned from rat PC12 cells and P2X 2 RNA is present in a number of autonomic ganglia. The single channel properties of heterologously expressed P2X 2 receptors are essentially the same as those recorded from native PC12 cells (conductance --26 pS) and rat superior cervical ganglion (SCG) neurones recorded under similar conditions (estimated conductance --14 pS at - 1 0 0 mV in 1.2 m M MgCI2, 2.5 mM CaC12 solution) ([8,10] and R.E., unpublished observations). Although multiple P2X receptor RNAs have been demonstrated in PC12 cells (P2X 1 and P2X 2 [1]) and rat SCG neurones (P2X 2 and P2X 4 [11]), the present results suggest that if native rat SCG and PC12 P2X receptors are formed by heteropolymerization of different P2X receptor subunits, their functional properties are dominated by P2X 2 subunit expression. The openings of P2X 3 channels were very rapid and were too fast to be resolved under the present recording conditions. Similar fast transient openings have also been reported for native bullfrog dorsal root ganglia (DRG) P2X receptors, where single channel events could not be resolved even when the corner cut-off frequency of filtering was increased to 6 kHz or patches cooled to 5°C [ 12]. However, in contrast to P2X 3 receptors openings of bullfrog DRG P2X receptors occurred in long flickery bursts. It has been suggested that sensory neuron P2X receptors are formed by heteropolymerization of P2X2 and P2X 3 subunits [6]. The only data available from rat sensory neurons indicates native P2X receptors have a single channel conductance of --36 pS (estimated from [13] with similar ionic conditions to those in this study). This is different from heterologously expressed homomeric P2X 2 or P2X 3 receptors observed in the present study and indicates that if these subunits heteropolymerize to form sensory neuron P2X receptors, a distinct single channel phenotype is produced. P2X 4 receptor RNA is found by in situ hybridization in brain, peripheral ganglia and epithelia. The chord con-
ductance of 8.9 pS for homomeric P2X 4 receptors is similar to that estimated by variance analysis for native P2X channels in rat acinar cells (6 pS) [2]. This confirms previous whole cell pharmacological studies which demonstrated the properties of native P2X receptors in submandibular and acinar cells can be described by homomeric P2X 4 receptors [2,7]. Native P2X receptors with single channel properties different from those of heterologously expressed P2XI_4 receptors, for example the 60 pS rat cardiac ganglion channel [3] indicate the existence of other functionally distinct P2X receptors. These could result from the expression of new P2X receptor clones, heteropolymerization of P2X subunits or different splice variants. I would like to thank Drs. R.A. North and A. Surprenant for their helpful advice and critical reading of the manuscript, K. Lundstrom and Y. Humbert for producing the SFV v i r a l stocks and D. Estoppey and D. Fahmi for cell culture. [1] Surprenant, A., Buell, G. and North, R.A., P2X receptors bring new structure to ligand gated ion channels, Trends Neurosci., 18 (1995) 224-229. [2] Vincent,P., Cationic channels sensitive to extracellularATP in rat lacrimal cells, J. Physiol.,449 (1992) 313-331. [3] Fieber, L. and Adams, D.J., Adenosine triphosphate-evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia, J. Physiol.434 (1991) 239-256. [4] Evans,R.J., Lewis, C., Buell, G., North, R.A. and Surprenant,A., Pharmacological characterisation of heterologously expressed ATP-gated cation channels P2X-purinoceptors,Mol. Pharmacol. 48 (1995) 178-183. [5] Brake, A.J., Wagenbach, M.J. and Julius, D., A new structural motif for ligand-gatedion channels defined by ionotropic ATP receptor, Nature, 371 (1994) 519-523. [6] Lewis, C., Neidhart, S., Holly, C., North, R.A., Buell, G. and Surprenant, A., Co-expression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurones, Nature, 377 (1995) 432-435. [7] Buell, G., Lewis, C., Collo, G., North, R.A. and Surprenant, A., An antagonist-insensitiveP2X receptor in epithelia and brain, EMBO J., 15 (1996) 55~52. [8] Neuhaus, R., Reber, B.F.X. and Reuter, H., Regulation of bradykinin- and ATP-activated Ca2+-permeable channels in rat pheochromocytoma (PC12) cells, J. Neurosci., 11 (1991) 39843990. [9] Nakazawa, K. and Matsuki, N., Adenosine triphosphate-activated inward current in isolated smooth muscle cells from rat vas deferens, Pfltiegers Arch., 409 (1987) 644-646. [10] Cloues, R., Properties of ATP-gated channels recorded from rat sympathetic neurons: voltage dependence and regulation by Zn2+ ions, J. Neurophysiol.,73 (1995) 312-319. [11] Collo, G., North, R.A., Kawashima, E., Merlo-Pich, E., Neidhart, S., Surprenant, A. and Buell, G., Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gatedion channels, J. Neurosci. 16 (1996) 2495-2507. [12] Bean, B.P., Williams, C.A. and Ceelen, P.W., ATP-activated channels in rat and bullfrog sensory neurons: current voltage relation and single-channelbehaviour,J. Neurosci., 10 (1990) 11-19. [13] Krishtal, O.A., Marchenko, S.M. and Obukhov, A.G., Cationic channels activated by extracellular ATP in rat sensory neurons, Neuroscience, 27 (1988) 996-1000.
Neuroscience Letters 212 (1996) 212-214
HEUROSGIENCE IETT[BS
Single channel properties of ATP-gated cation channels (P2X receptors) heterologously expressed in Chinese hamster ovary cells R.J. E v a n s ~,* Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development S.A., 14 chemin des Aulx, 1228 Plan-Les-Ouates, Geneva, Switzerland
Received 22 March 1996; revised version received 28 May 1996; accepted 12 June 1996
Abstract
The single channel properties of four P2X receptors heterologously expressed in Chinese hamster ovary cells were determined from outside-out patch recordings. P2X! and P2X 4 channels opened in brief flickery bursts with conductances of --18 and 9 pS respectively. P2X 2 receptors had a single channel conductance of --21 pS. Openings of P2X 3 channels were too rapid to be reliably resolved. Application of ATP to macro-patches expressing multiple P2X 2 or P2X 4 receptors evoked sustained currents, in contrast responses of P2X 1 or P2X 3 receptors desensitised during continued agonist application. These data show that four different homomeric P2X receptors form ion channels with distinct single channel properties. Keywords: Ion channels; P2X receptors; Purinergic
P2X receptors for ATP are ligand-gated cation channels present on many neuronal and smooth muscle preparations [ 1]. Functional subtypes of P2X receptors can be discriminated based on pharmacological and kinetic properties. In a limited number of single channel studies unitary conductance estimates for native P2X receptors range from 6 to 60 pS [2,3]. Several P2X receptors have been cloned recently and shown to define a new structural class of ligand-gated ion channels. In the present study, the unitary conductances of heterologously expressed homomeric P2XI_ 4 receptors [4-7] have been determined under identical recording conditions to see if functionally distinct channels are formed and how these correspond to native P2X receptors. The cDNAs encoding human bladder P2X1, and rat P2X 2, P2X 3 and P2X 4 receptors [4-7] were introduced into the Semliki forest virus (SFV) expression vector and used to infect Chinese hamster ovary (CHO) cells as described previously [4]. Responses to ATP were recorded
* Corresponding author, e-mail: [email protected] 1 Present address: Department of Cell Physiology & Pharmacology, Medical Sciences Building, University of Leicester, University Road, Leicester LEt 9HN, UK.
from excised outside-out patches using an Axopatch 200A amplifier (Axon Instruments, USA; patch holding potential - 1 0 0 mV). Initial experiments on P2X 2 receptors demonstrated that, as for native PC12 P2X receptors [8], unitary currents were larger when calcium was reduced from 2 to 0.3 mM and magnesium reduced from 1 mM to nominally magnesium free. Therefore, an extracellular solution of the following composition (in raM) was used, NaCI 150, sucrose 10, HEPES 10, CaCI 2 0.3, and pH adjusted to 7.3-7.4 with NaOH. Using this extracellular solution patches were less stable and had higher levels of background noise. Patch electrodes (10-15 Mff~) were filled with a solution containing (in mM), NaCI 140, EGTA 11, HEPES 10 and pH adjusted to 7.3-7.4 with NaOH (final Na 165). Experiments were carried out at room temperature (25°C). ATP (0.3-10 # M ) was applied rapidly to patches by a double barrelled 'bimorph' system. ATP evoked single channel events were not recorded from uninfected CHO cells (n = 5). SFV infection resulted in a high level of P2X channel expression and all patches expressed multiple P2X channels, therefore channel lifetimes were not analysed. Recordings were filtered at 1 kHz with a low pass Bessel filter and digitised at 5 kHz (Fetchex, PClamp 6, Axon Instruments, USA). Records were analysed using Axo-
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96) 12804-4
213
R.J. Evans/Neuroscience Letters 212 (1996) 212-214
r
B C
20 pA
pA
2.5s
E lpA 200 ms
=
F
200 ms
C
400
_400
$ 200]
l -3
E c
-2 -1 0 amplitude (pA)
i
-3
-2 -1 0 amplitude (pA)
1
Fig. 1. Single channel currents evoked by ATP from heterologously expressed P2X 1 and P2X 2 receptors. (A-C) P2X 1 receptors. (A) Response of an outside-out patch from a CHO cell expressing P2X 1 receptors to 3/~M ATP (application period indicated by bar). (B) ATP evoked single channel events (a 1 s segment of trace from (A)). (C) Amplitude histogram for record shown in (B). (D-F) P2X 2 receptors. (D) Channel activity recorded from an outside-out patch from a CHO cell expressing P2X 2 receptors superfused with ATP (0.3/~M) (recording made within 250 ms of ATP application). (E) Expanded record from the same patch as (D). (F) Amplitude histogram for record shown in (E). Patch potential, -100 mV.
graph 2 software (Axon Instruments, USA). Mean unitary currents were estimated from amplitude histograms (bin size 0.05 pA) generated from segments (0.8-5 s) of traces with openings to a single conductance level and fitted by the sum of two gaussian distributions. Chord conductances were calculated between -100 and 0 mV (reversal potential for P2X currents in above solutions). Application of ATP (3/xM) to outside-out patches of CHO cells expressing P2X 1 receptors evoked transient inward currents. During the continued application of ATP the peak inward current declined (n = 7 patches) and single channel openings could be resolved in three patches (Fig. 1A,B). These were usually brief (<50 ms) or in flickery bursts ( < 3 0 0 ms). The unitary channel amplitude at - 1 0 0 mV was -1.76 _+0.07 pA (n = 3; Fig. 1C).
In patches expressing P2X2 receptors application of 10/xM ATP evoked relatively sustained inward currents comprised of multiple channel openings (n = 12). By reducing the ATP concentration, single channel events (amplitude -2.12 _+0.09 pA (n = 5)) could be resolved (Fig. 1D-F). Channel openings (conductance --21 pS) lasted up to 1 s and were less flickery than those of P2XI, P2X 3 and P2X 4. Inward currents recorded in response to ATP (0.33/tM) in outside-out patches expressing P2X 3 receptors (Fig. 2A) were transient and decayed rapidly during agonist application. During the decline of the response fluctuations in membrane current <3 pA were recorded; however, unitary events could not be reliably resolved (n = 10; Fig. 2B).
A
C 20 pA
B
2.5s
pA
D
100 t
A 200 ms
1O0 ms
==
% -2
-1 0 amplitude (pA)
Fig. 2. The response of heterologously expressed P2X 3 and P2X 4 receptors to ATP. (A,B) P2X 3 receptors. (A) Transient inward current recorded in response to the application of ATP (3/tM, indicated by bar). (B) Expanded record from (A) showing unresolved openings of ATP-gated channels. (C,D) P2X 4 receptors. (C) Unitary currents recorded from an outside-out patch of a CHO cell expressing P2X 4 receptors in response to ATP superfusion (3 ffM) (recording made within 250 ms of ATP application). (D) Amplitude histogram for record shown in (C). Patch potential, -100 mV.
214
R.J. Evans/Neuroscience Letters 212 (1996) 212-214
Application of A T P (1-10/~M) tO outside-out patches expressing P2X 4 receptors evoked sustained inward currents (n = 8 patches). In three patches single channel events were resolved. These were of variable duration, flickery and had an amplitude of - 0 . 8 9 _+0.1 pA (n = 3) (conductance --9 pS; Fig. 2C,D). The present paper provides the first account of the single channel properties of homomeric P2X receptors; it demonstrates that P2X 1, P2X 2, P2X 3 and P2X 4 form functional ion channels with distinct single channel properties. The single channel conductance (--18 pS) and desensitization of P2X1 receptors (time constant of current decay in the continued presence of ATP 100--300 ms) are similar to those reported for native rat vas deferens P2X receptors ( - - 2 0 pS) [1,9] and demonstrate that the properties of native rat vas deferens P2X receptors are likely to be determined by P2X1 subunit expression [1,4]. P2X 2 receptors were originally cloned from rat PC12 cells and P2X 2 RNA is present in a number of autonomic ganglia. The single channel properties of heterologously expressed P2X 2 receptors are essentially the same as those recorded from native PC12 cells (conductance --26 pS) and rat superior cervical ganglion (SCG) neurones recorded under similar conditions (estimated conductance --14 pS at - 1 0 0 mV in 1.2 m M MgCI2, 2.5 mM CaC12 solution) ([8,10] and R.E., unpublished observations). Although multiple P2X receptor RNAs have been demonstrated in PC12 cells (P2X 1 and P2X 2 [1]) and rat SCG neurones (P2X 2 and P2X 4 [11]), the present results suggest that if native rat SCG and PC12 P2X receptors are formed by heteropolymerization of different P2X receptor subunits, their functional properties are dominated by P2X 2 subunit expression. The openings of P2X 3 channels were very rapid and were too fast to be resolved under the present recording conditions. Similar fast transient openings have also been reported for native bullfrog dorsal root ganglia (DRG) P2X receptors, where single channel events could not be resolved even when the corner cut-off frequency of filtering was increased to 6 kHz or patches cooled to 5°C [ 12]. However, in contrast to P2X 3 receptors openings of bullfrog DRG P2X receptors occurred in long flickery bursts. It has been suggested that sensory neuron P2X receptors are formed by heteropolymerization of P2X2 and P2X 3 subunits [6]. The only data available from rat sensory neurons indicates native P2X receptors have a single channel conductance of --36 pS (estimated from [13] with similar ionic conditions to those in this study). This is different from heterologously expressed homomeric P2X 2 or P2X 3 receptors observed in the present study and indicates that if these subunits heteropolymerize to form sensory neuron P2X receptors, a distinct single channel phenotype is produced. P2X 4 receptor RNA is found by in situ hybridization in brain, peripheral ganglia and epithelia. The chord con-
ductance of 8.9 pS for homomeric P2X 4 receptors is similar to that estimated by variance analysis for native P2X channels in rat acinar cells (6 pS) [2]. This confirms previous whole cell pharmacological studies which demonstrated the properties of native P2X receptors in submandibular and acinar cells can be described by homomeric P2X 4 receptors [2,7]. Native P2X receptors with single channel properties different from those of heterologously expressed P2XI_4 receptors, for example the 60 pS rat cardiac ganglion channel [3] indicate the existence of other functionally distinct P2X receptors. These could result from the expression of new P2X receptor clones, heteropolymerization of P2X subunits or different splice variants. I would like to thank Drs. R.A. North and A. Surprenant for their helpful advice and critical reading of the manuscript, K. Lundstrom and Y. Humbert for producing the SFV v i r a l stocks and D. Estoppey and D. Fahmi for cell culture. [1] Surprenant, A., Buell, G. and North, R.A., P2X receptors bring new structure to ligand gated ion channels, Trends Neurosci., 18 (1995) 224-229. [2] Vincent,P., Cationic channels sensitive to extracellularATP in rat lacrimal cells, J. Physiol.,449 (1992) 313-331. [3] Fieber, L. and Adams, D.J., Adenosine triphosphate-evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia, J. Physiol.434 (1991) 239-256. [4] Evans,R.J., Lewis, C., Buell, G., North, R.A. and Surprenant,A., Pharmacological characterisation of heterologously expressed ATP-gated cation channels P2X-purinoceptors,Mol. Pharmacol. 48 (1995) 178-183. [5] Brake, A.J., Wagenbach, M.J. and Julius, D., A new structural motif for ligand-gatedion channels defined by ionotropic ATP receptor, Nature, 371 (1994) 519-523. [6] Lewis, C., Neidhart, S., Holly, C., North, R.A., Buell, G. and Surprenant, A., Co-expression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurones, Nature, 377 (1995) 432-435. [7] Buell, G., Lewis, C., Collo, G., North, R.A. and Surprenant, A., An antagonist-insensitiveP2X receptor in epithelia and brain, EMBO J., 15 (1996) 55~52. [8] Neuhaus, R., Reber, B.F.X. and Reuter, H., Regulation of bradykinin- and ATP-activated Ca2+-permeable channels in rat pheochromocytoma (PC12) cells, J. Neurosci., 11 (1991) 39843990. [9] Nakazawa, K. and Matsuki, N., Adenosine triphosphate-activated inward current in isolated smooth muscle cells from rat vas deferens, Pfltiegers Arch., 409 (1987) 644-646. [10] Cloues, R., Properties of ATP-gated channels recorded from rat sympathetic neurons: voltage dependence and regulation by Zn2+ ions, J. Neurophysiol.,73 (1995) 312-319. [11] Collo, G., North, R.A., Kawashima, E., Merlo-Pich, E., Neidhart, S., Surprenant, A. and Buell, G., Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gatedion channels, J. Neurosci. 16 (1996) 2495-2507. [12] Bean, B.P., Williams, C.A. and Ceelen, P.W., ATP-activated channels in rat and bullfrog sensory neurons: current voltage relation and single-channelbehaviour,J. Neurosci., 10 (1990) 11-19. [13] Krishtal, O.A., Marchenko, S.M. and Obukhov, A.G., Cationic channels activated by extracellular ATP in rat sensory neurons, Neuroscience, 27 (1988) 996-1000.