Regions of the amino terminus of the P2X1 receptor required for modification by phorbol ester and mGluR1α receptors

The potentiation of P2X1 receptor currents by phorbol ester (PMA) treatment and stimulation of mGluR1α receptors was sensitive to inhibition of novel forms of protein kinase C. Potentiation was also reduced by co-expression of an amino terminal P2X1 receptor minigene. Cysteine point mutants of residues Tyr16-Gly30 were expressed in Xenopus oocytes. Peak current amplitudes to ATP for Y16C, T18C and R20C mutants were reduced, however this did not result from a decrease in surface expression of the channels. The majority of the mutants showed changes in the time-course of desensitization of ATP evoked currents indicating the important role of this region in regulation of channel properties. PMA and mGluR1α potentiation was abolished for the mutants Y16C, T18C, R20C, K27C and G30C. Minigenes incorporating either Y16C, K27C, V29C or G30C still inhibited PMA responses. However D17C, T18C or R20C mutant minigenes were no longer effective suggesting that these residues are important for interaction with regulatory factors. These results demonstrate that the conserved YXTXK/R sequence and a region with a conserved glycine residue close to the first transmembrane segment contribute to PMA and GPCR regulation of P2X1 receptors.

Genes encoding seven mammalian P2X receptor subtypes (P2X 1-7 ) have been identified and they assemble to form homo-and hetero-trimeric ATP-gated channels with a range of phenotypes (North 2002). P2X 1 receptors are expressed throughout the body and contribute to a range of physiological processes, e.g. regulation of smooth muscle tone (Valera et al. 1994;Scase et al. 1998;Sage et al. 2000) and thrombosis (Hechler et al. 2003). In the nervous system P2X 1 receptors form heteromeric assemblies and are involved in the pre-synaptic regulation of transmitter release in the auditory brainstem (Watano et al. 2004) and a P2X 1/5 heteromeric receptor has recently been described in astrocytes (Lalo et al. 2008).
P2X receptors constitute a distinct family of ligand gated ion channels with intracellular amino and carboxy termini, two transmembrane segments and a large extracellular loop involved in drug action (Roberts et al. 2006). The intracellular regions of the channels have been shown to be involved in channel regulation (Boue-Grabot et al.2000;Jiang et al.2001;Eickhorst et al.2002;Ennion and Evans 2002;Chaumont et al. 2004;Vial et al. 2004). The amino termini have a relatively standard length of about 30 amino acids (North 2002). This contains a protein kinase C consensus sequence TXK/R preceded by a conserved tyrosine giving rise to a YXTXK/R motif (Tyr 16 -Lys 20 in the P2X 1 receptor) that is conserved in all mammalian and Dictyostelim receptors (Boue-Grabot et al. 2000;Fountain et al. 2007). Mutations of the central threonine led to a speeding of channel desensitization and reduction in peak current amplitude (Boue-Grabot et al.2000;Ennion and Evans 2002) and this residue may be constitutively phosphorylated (Liu et al. 2003). Furthermore, for the P2X 2 receptor when the Cterminal was truncated, the P2X 2 receptor showed faster desensitization, but the normal wild type time-course was recovered by phorbol ester which stimulates PKC (Boue-Grabot et al. 2000). Therefore, the N-terminus may be involved in intracellular regulatory mechanisms.
P2X receptors can be regulated by G-protein coupled receptors (GPCRs) (Ralevic and Burnstock 1998;Paukert et al. 2001;Kunapuli et al. 2003;Vial et al. 2004). For example P2X 1 receptor currents can be potentiated by mGluR1a, P2Y 1 , P2Y 2 and 5-hydroxytryptamine (5-HT) 2A receptors as well as by phorbol ester (phorbol-12-myristate-13-acetate, PMA) treatment and these effects were abolished by the broad spectrum kinase inhibitor staurosporine Ase et al. 2005). This was independent of phosphorylation of the consensus PKC site, as potentiation was still seen when the conserved threonine residue was mutated, and it has been suggested that the modulatory effects may result from action on an interacting protein ). However amino acids in the P2X 1 receptor that contribute to the regulation were not determined.
In this study, we have investigated (i) the role of novel, calcium insensitive, protein kinase C isoforms in the control of the P2X 1 receptor, (ii) the contribution of the N-terminus of the P2X 1 receptor in regulation using over-expression of a minigene and (iii) used cysteine scanning of the 15 residues before the first transmembrane segment to identify for the first time residues involved in regulation of P2X 1 receptors by GPCRs and phorbol ester.

Minigene construction
The amino terminal sequence (Met 1 -Gly 30 ) of the human P2X 1 receptor was amplified from the pcDNA 3.0 vector containing the human P2X 1 receptor cDNA by Polymerase Chain Reaction (PCR) (Techne Genius thermocycler, BioTAQ TM DNA polymerase, Bioline, UK). Start and stop codons at the ends of the minigene as well as restriction sites, EcoRI and HindIII, were introduced using the primers. The minigene sequence was ligated into the plasmid pcDNA3.0 using these two restriction sites at 14°C overnight (T4 DNA ligase, New England Biolabs Ò Inc., Hertfordshire, UK).

Site-directed mutagenesis
Point mutations were introduced into the human P2X 1 plasmid or the minigene construct using the QuikChange TM mutagenesis kit (Stratagene, Amsterdam, Netherlands) according to the manufacturer's instructions as described previously (Ennion et al. 2000) and confirmed by DNA sequencing (Automated ABI Sequencing Service, Leicester University, Leicester, UK).
Xenopus laevis oocytes, stage V, were prepared by enzymatic treatment followed by manual defoliculation as described previously (Ennion et al. 2000). 50 nL of mRNA (1 lg/lL) was injected into isolated Xenopus oocytes using an Inject+Matic microinjector (J.Alejandro Gaby, Geneva, Switzerland). For co-injections with N-termini minigenes the RNA was mixed to give 5 ng wild type (WT) P2X 1 + 10 ng mGluR1a + 35 ng N-termini minigene (or appropriate volume of water was added in the absence of minigene) and injected in a 50 nL volume. Cells were maintained at 18°C in ND96 buffer (concentrations in mM; 96 NaCl, 2 KCl, 1.8 CaCl 2 , 1 MgCl 2 , 5 sodium pyruvate and 5 HEPES, pH 7.5) with 50 lg/mL gentamicin and were used for recording after 2-6 days.
Electrophysiological recordings Two-electrode voltage clamp was used on cRNA-injected oocytes to record currents to applied ATP (Mg salt; Sigma, Poole, UK) as described previously (Ennion et al. 2000). ATP was applied with a fast-flow U-tube perfusion system, applications of ATP were separated by 5 min in order to allow recovery from receptor desensitization.
For oocytes pre-treated with PMA, 100 nM PMA was made in ND96 solution and the oocytes were pre-incubated in the PMA solution for 10 min at 21°C immediately before recording. Comparisons were made between groups of control untreated oocytes and those exposed to PMA. The protein kinase inhibitors Calphostin C(1 lM) (Sigma C6303), K252a (100 nM) (Sigma 05288), Gö6983(200 nM) (Sigma G1918) or Gö6976 (200 nM) (Sigma G1171) were applied to the oocytes for 1 h at 21°C before recording. The inhibitors, when applied alone had no effect on the peak current amplitude or the time-course of P2X 1 receptor currents (data not shown). When looking at the potentiation of the P2X 1 receptor, glutamate (100 lM) with or without the protein kinase inhibitors was bath-perfused for 5 min between the stimulations of the P2X 1 receptor by ATP via the U-tube as described previously. The glutamate was applied to the oocytes when stable responses were observed ).

Western-blotting
The expression levels and molecular weight of the P2X receptor proteins were estimated by western blotting, and both the level of expressed total and cell surface P2X 1 receptors were studied as described previously (Ennion et al. 2000).

Data analysis
All data are shown as mean ± SEM Significant differences between the means of all groups compared to WT were calculated by oneway ANOVA, followed by Dunnett's test for comparisons of individual mutants against control using the GraphPad Prism 5 for Windows (GraphPad Software, San Diego, CA, USA). Student's t tests were also used where appropriate and considered to be significant when p < 0.05. n corresponds to the number of oocytes tested for electrophysiological data.

Results
Novel protein kinase C isoforms contribute to regulation of the P2X 1 receptor by PMA and GPCRs For oocytes co-expressing P2X 1 and mGluR1a receptors ATP (100 lM, a maximal concentration) evoked transient inward currents that desensitised during the application of ATP. P2X 1 receptor currents were potentiated by 103.1 ± 11.8% (n = 19) following 10 min pre-treatment with PMA (100 nM) (Fig. 1). Similarly, the activation of mGluR1a receptors by 100 lM glutamate for 5 min evoked a transient inward calcium activated chloride current and potentiated the subsequent responses of WT P2X 1 receptors to ATP (100 lM) by 61.0 ± 3.9% (n = 13) (Fig. 1). This is consistent with our previous studies on the P2X 1 receptor . We have previously shown that these effects could be reduced by treatment with the broad spectrum kinase inhibitor staurosporine but were unlikely to involve classical isoforms of protein kinase C as potentiation was not calcium sensitive . To determine whether novel, calcium insensitive, protein kinase C isoforms (PKCd,e,g,h, or l) are involved in the regulation we have used a range of inhibitors ( Fig. 1) [none of these had an effect on peak P2X 1 receptor current amplitude or time course (data not shown)]. Calphostin C(1 lM) acts by competing with the diacyl glycerol and phorbol ester binding site of protein kinase C isoforms and reduced potentiation by PMA and mGluR1a stimulation to 15.5 ± 13.9% (n = 11) and 13.5 ± 2.8% (n = 5). Potentiation was also reduced by the kinase inhibitor K252a (100 nM) (32.6 ± 10.0% and 20.3 ± 5.9%, n = 19, 5 respectively). Gö6983 is a kinase inhibitor at novel PKCs d,e,g,h, but is ineffective at the novel isoform PKCl (also called protein kinase D, PKD)(Martiny- Baron et al. 1993). Potentiation was reduced to )6.9 ± 12.4% (n = 13) and )2.8 ± 3.9% (n = 7) by Gö6983 (200 nM) for PMA and mGlur1a respectively. Gö6976 (200 nM) is an effective inhibitor of the novel calcium insensitive protein kinase C isoform PKCl but is ineffective at the other calcium insensitive novel PKC isoforms (Gschwendt et al. 1996) and reduced PMA and mGluR1a mediated potentiation to )3.2 ± 11.4% (n = 8) and 27.6 ± 0.9% (n = 3). These findings are similar to those reported for 5-HT dependent regulation of the receptor (Ase et al. 2005) and suggest that novel protein kinase C isoforms mediate P2X 1 receptor regulation by both PMA and GPCRs. However our previous studies showed that P2X 1 receptor current potentiation was unaffected by mutation to remove the consensus PKC site, and a change in the phosphorylation status of the receptor was not detected ). These results suggest that the potentiation is mediated by phosphorylation of an interacting regulatory protein .
The amino terminus of the P2X 1 receptor is involved in GPCR and phorbol ester regulation To determine whether the intracellular amino terminus of the P2X 1 receptor contributes to receptor modulation we coexpressed P2X 1 receptors and mGluR1a receptors with a minigene encoding the amino terminus of the P2X 1 receptor. P2X 1 receptor amino terminal minigene expression had no effect on the P2X 1 receptor currents (peak current amplitudes to 100 lM ATP of )6875 ± 298 nA and )6302 ± 304 nA, for control and with the minigene respectively n = 6), or the amplitude of glutamate evoked chloride currents ()4996 ± 807 nA and )4155 ± 1233 nA respectively) demonstrating that the minigene does not regulate P2X 1 or mGluR1a receptor expression or activation (Fig. 2). However co-expression of the amino terminal minigene reduced potentiation by PMA from 128.5 ± 12.6% to 25.8 ± 2.6% (p < 0001, n = 6, 7) and by mGluR1a receptor stimulation from 52.5 ± 3.1% to 28.3 ± 3.3% (p < 0.001, n = 6, 7) (Fig. 2). This suggests that the amino terminus is important in regulation and that the minigene may work by sequestering regulatory factor(s) associated with the receptor.
Effects of point cysteine substitutions on basic P2X 1 receptor properties We used cysteine substitution mutagenesis to investigate the contribution of the 15 amino acids before the first transmembrane segment to channel properties. This region includes the conserved YXTXK/R motif and SCAM (substituted cysteine accessibility method) analysis of P2X 2 recep-tors suggested that this part of the amino terminal may play a role in channel function (Jiang et al. 2001). ATP (100 lM) evoked fast desensitizing inward currents from all of the mutants. There was no effect on the peak current amplitude for 12 of the mutants (Fig. 3, Table 1) however responses were reduced for mutants Y16C, T18C, and R20C (Fig. 3, Table 1). Western blotting showed there was no obvious difference in either total or surface expression levels for these mutants compared to WT (Fig. 3) demonstrating that the Upper panels show sample traces for a given oocyte co-expressing P2X 1 and mGluR1a receptors (left) or P2X 1 receptors, mGluR1a receptors and the P2X 1 receptor amino terminal minigene (right traces). Responses to a maximal concentration of ATP (100 lM, indicated by bar) are shown before and after the application of glutamate (100 lM). Glutamate evoked an inward calcium activated chloride current and potentiated subsequent ATP evoked responses. This potentiation was reduced by co-expression of the P2X 1 receptor N-terminal minigene. The bar chart shows a summary of the data, n = 6-7. ***p < 0.001. reduction in current amplitude does not result from deficiencies in receptor trafficking. There were no major changes in ATP potency at the mutant receptors; 1 lM ATP, an EC 50 concentration at the WT receptor, evoked between 30% and 70% of the maximum current amplitudes for each of the mutants. The time-course of desensitisation (time for the peak currents to 100 lM ATP to decay to 50%) was unaffected for the mutants Y16C, R20C, V24C, N26C and G30C. Gly 30 is conserved throughout the mammalian and Dictyostelium families (Fountain et al. 2007). However, it is interesting that mutation to cysteine had no effect on the current amplitude of timecourse of P2X 1 (this study) or P2X 2 (Jiang et al. 2001) receptor currents. This shows that the flexibility associated with the glycine residue is not essential for normal channel function. For the remainder of the mutants, there were significant changes in time-course. Slowed desensitisation was recorded for D17C, P19C, L23C and K28C; with D17C responses slowed almost 6 fold (Fig. 3, Table 1). Faster desensitisation was seen for the remainder of the mutants with the greatest change seen for V22C with an 3 fold speeding (greater than for T18C) in the decay of current evoked during the continued presence of ATP but no effect on peak current amplitude (see Fig. 3b, Table 1). Studies on the P2X 2 receptor showed for the majority of analogous mutants (13/15) there was no effect on the time-course of the response (Jiang et al. 2001). This may reflect that P2X 2 receptor currents are relatively non-desensitising whereas for the P2X 1 receptor there is a rapid transition once the channel is open to the desensitised state, and this is more sensitive to disruption of the amino terminus.
Cysteine substitution can block PMA potentiation WT P2X 1 receptors were potentiated by 116.5 ± 14.5% (n = 11) following 10 min application of PMA. A similar level of potentiation was seen for the mutants P19C, V22C, L23C, V24C, V25C, N26C and K28C. For M21C, potentiation was reduced to about 50%, no potentiation was seen for the mutants Y16C, D17C, T18C, R20C, V29C and G30C (Fig. 4), and the responses were inhibited for mutant K27C. These results suggest a cluster of residues comprising the conserved YXTXK/R motif and those close to the first transmembrane segment of the P2X 1 receptor are involved in PMA regulation.

Mutant minigenes identify residues important in interaction with regulatory factors
Cysteine mutants that reduced PMA potentiation could be important for mediating the interaction with a regulatory factor and/or important in conformational changes induced by the regulatory factor. To address these roles we introduced the individual cysteint mutants that reduced PMA potentiation into the N terminal minigene. It was predicted that mutation of residues in the minigene important for interaction with regulatory factors/proteins would remove the inhibitory/ sequestering effect of the minigene. Minigenes expressing the mutants D17C, T18C and R20C no longer inhibited the PMA induced potentiation (Fig. 4) consistent with a role of these residues in mediating interactions with regulatory factors. It is unlikely that these effects result from the disruption of the protein kinase C consensus in the minigene as PMA is still effective at potentiating P2X 1 receptors where the consensus for phosphorylation has been mutated , and this study). In contrast Y16C, K27C, V29C and G30C mutant minigenes still inhibited PMA potentiation and suggested that these residues may be important in conformational changes on the P2X 1 receptor associated with PMA potentiation.
Differential sensitivity of cysteine mutants to mGluR1a receptor and PMA For the mutants where PMA no longer had an effect or reduced the response, we also tested to see whether mGluR1a receptor dependent regulation was also attenuated. ATP (100 lM) was applied repeatedly at 5 min intervals to obtain reproducible responses. Glutamate potentiated WT P2X 1 receptor currents by 67.8 ± 4.8% (n = 8). For most (4/ 6) of the mutants that showed no potentiation with PMA treatment the effects of mGluR1a receptor stimulation were also reduced significantly (Fig. 5). However, the mGluR1a receptor regulation was not affected by the mutations D17C and V29C (that abolished PMA potentiation) where the application of glutamate gave potent potentiations of 114.5 ± 4.7% (n = 4) and 66.2 ± 6.9% (n = 5) respectively. These results demonstrate that PMA and mGluR1a stimulation have some differences in their regulatory mechanisms.
(b) (a) Fig. 5 The effects of mGluR1a receptor activation on P2X 1 receptor mutants. (a) Sample traces for a given oocyte co-expressing either WT P2X 1 , D17C or K27C mutant P2X 1 receptor with mGluR1a receptors. Responses to a maximal concentration of ATP (100 lM, indicated by bar) are shown before and after the application of glutamate (dotted line). Glutamate (100 lM) evoked an inward calcium activated chloride current and potentiated subsequent ATP evoked responses for WT and D17C mutants but not for the K27C mutant P2X 1 receptor. (b) The effects of mGluR1a receptor (100 lM glutamate) and PMA (100 nM) on WT and the cysteine mutants are shown (+++p < 0.001 comparing mGluR1 receptor regulation of mutants to WT, ***p < 0.001 for mutants treated with PMA compared to the WT effect). For most of the mutants unable to exhibit the PMA potentiation, no potentiation was seen following the activation of mGluR1a receptor. However, the mGluR1a receptor stimulated potentiation was still robust for the D17C and V29C mutants.
(a) (b) Fig. 6 The effects of PMA and mGluR1a receptor to R20 substitutions. (a) Example traces of R20C and R20A mutants in response to PMA (100 nM) or mGluR1a receptor stimulation (100 lM glutamate) are shown. Peak amplitudes from control and PMA treated oocytes are shown. In the lower panels ATP evoked currents before and after mGluR1a receptor activation from either R20C (left) or R20A (right) are shown. (b) Summary of effects of amino acid substitution at position R20 at the P2X 1 receptor by cysteine, alanine or isoleucine on potentiation by PMA or glutamate. ***p < 0.001.
PMA treatment had no effect on channel permeability The permeability of some P2X receptors changes with time (Khakh et al. 1999;Virginio et al. 1999) and can lead to an increase in currents for the P2X 4 receptor (Khakh et al. 1999). TRPV1 receptors show a similar change in permeability on prolonged agonist application and this is potentiated by activation of PKC (Chung et al. 2008). We tested whether the potentiation in current at the P2X 1 receptor following PMA treatment resulted from an increase in permeability. Under control conditions when dimethylamine was the only external cation P2X 1 receptor currents reversed at )10.2 ± 0.9 mV (n = 5) (consistent with previous studies on the receptor, Evans et al. 1996). The reversal potential for dimethylamine was unaffected ()11.1 ± 1.9 mV, n = 8) following PMA treatment demonstrating that the potentiation of P2X 1 receptor currents does not result from an increase in channel permeability.

Discussion
P2X 1 receptor mediated responses can be potentiated by phorbol ester and Ga q coupled GPCR stimulation and this may allow for regulation of P2X receptor signalling . The > 80% reduction by calphostin C of P2X 1 receptor potentiation by PMA or mGluR1a stimulation supports the role of protein kinase C in receptor regulation.
Our previous study showed that potentiation was not dependent on a rise in intracellular calcium ) and suggests that novel, calcium insensitive, and not classical calcium sensitive PKC isoforms are involved. Amongst the novel PKC isoforms Gö6976 is selective for PKCl (PKD) and abolished potentiation by PMA and reduced by 60% mGluR1a modulation. This indicates a role of PKCl (PKD) in the regulation of P2X 1 receptors, as reported previously for 5-HT regulation (Ase et al. 2005).
The difference in effectiveness of the PKCl inhibitor between PMA and mGluR1a stimulation suggests that there are differences in the regulatory pathways used. This is consistent with mutagenesis studies that showed D17C and V29C mutants abolished PMA potentiation but had no effect on the mGluR1a stimulation. In addition the minigene had a greater inhibitory effect on PMA stimulation than on that evoked by mGluR1a stimulation even though the extent of PMA potentiation at 120% was significantly higher that the 55% potentiation evoked by mGluR1a stimulation. This suggests that mGluR1a stimulation recruits additional regulatory pathways compared to PMA. The reduction in potentiation by Gö6983 (effective at the concentration used at novel PKC isoforms with the exception of PKCl; IC 50 100 times higher than that used in the current study) suggests that one or more of the isoforms PKC d,e,g, h are also involved. mGluR1a potentiation was still observed when the proposed 'phosphorylatable' threonine was mutated or the consensus PKC sequence was disrupted by Arg 20 mutation (this study and Vial et al. 2004). In addition we could not detect any changes in P2X 1 receptor phosphorylation on PMA stimulation and studies on the P2X 3 receptor also failed to detect receptor phosphorylation associated with PKC regulation (Franklin et al. 2007). These studies rule out a role of phosphorylation at the consensus PKC site as a mechanism of action and raise the question where the novel PKC isoforms act? Interacting proteins that regulate the function of P2X 7 and P2X 2 receptors have been described (Adinolfi et al. 2003;Masin et al. 2006) and we suggest that modulation of the P2X 1 receptor occurs through the phosphorylation by a novel PKC of a regulatory protein that interacts with the P2X 1 receptor.
Minigenes have been used widely to study regulation (Del Gatto et al. 1996;Wu et al. 2004) including work on P2X receptors (Boue-Grabot et al. 2000). The over-expression of a minigene encoding the amino terminus of the P2X 1 receptor reduced PMA and mGluR1a receptor potentiation of P2X 1 receptor currents and demonstrated that the amino terminus plays an important role in receptor regulation possibly through providing a docking site for a regulatory protein and therefore over-expression of the minigene led to sequestering of this regulatory protein. The minigene, like the range of PKC inhibitors used, had no effect on the P2X 1 receptor currents under resting conditions suggesting that the receptor is not basally regulated, and that PMA and GPCR stimulation provide an auxiliary mechanism to modify channel properties. The greater reduction on the PMA effect compared to GPCR stimulation by the amino terminal minigene suggests that GPCR stimulation could have additional regulatory effects (and is consistent with mutations that abolish PMA effects with little action on mGluR1a stimulation). These results also suggest that the C terminus may also play a significant role in the regulation of the P2X 1 receptor (this is supported by studies with a C terminal minigene H. W. and R. J. E., unpublished observations). In addition the abolition of PMA potentiation by the mutations D17C, R20A and V29C but no effect on mGlur1a stimulation indicates further that there are subtle differences in the regulation following PMA and mGlur1a stimulation, however the molecular basis of this remains to be elucidated.
The cysteine mutagenesis of the P2X 1 receptor amino terminal identified residues that were important for PMA regulation. The introduction of these mutants into the minigene in a second round of mutagenesis identified substitutions that abolished the inhibitory effect of the minigene (D17C, T18C and R20C) and those that had no effect on the inhibitory actions of the minigene (Y16C, K27C, V29C and G30C). The abolition of the inhibitory effect of the minigene when Asp 17 , Thr 18 or Arg 20 were mutated demonstrates that these residues are likely to play an important role in the sequestering action of the minigene in competing with the P2X 1 receptor for the regulatory factor/ protein and identifies for the first time residues that involved in direct association with the regulatory factor/protein. The results with the minigene also show that resides Tyr 16 , Lys 27 , Val 29 and Gly 30 are not important in the interaction directly with the regulatory factor but mediate the changes in the P2X 1 receptor that lead to potentiation of the response. One possibility is that these residues could contribute to the gating of the P2X 1 receptor or regulation of channel conductance. Responses at the Y16C, P19C and G30C mutants were reduced by cysteine reactive MTS reagents for the P2X 1 (see Fig. S1; Appendix S1) and this is consistent with the findings for the P19C and G30C mutants at the P2X 2 receptor (Y16C mutant was non-functional see above) (Jiang et al. 2001). In previous studies we have shown that MTS reagents that result in a change in ATP potency at P2X 1 receptors modify the time-course of the currents (Roberts and Evans 2007). The reduction in amplitude by MTSEA of Y16C, P19C and G30C mutant P2X 1 receptors with no effect on the time-course of the response suggests that these residues are involved in ionic permeation through the channel. Taken together these results give rise to a mechanism whereby following PMA stimulation Asp 17 , Thr 18 and Arg 20 are involved in interaction with a regulatory factor that is phosphorylated, and this subsequently results in a change in channel properties involving residues Tyr 16 , Lys 27 , Val 29 and Gly 30 .
In summary this study has identified two amino terminal regions, YXTXK/R and Lys 27 -Gly 30 that are involved in regulation by PMA and ionic permeation. This raises the possibility that there may be some interaction between these two regions to regulate channel properties. Structural models of the intracellular regions however will be required for fuller interpretation of these results.