Wen H , Evans RJ .

Wellcome Trust

	Fig. 1. Potentiation of P2X1 receptor currents by PMA and mGluR1α receptor stimulation is sensitive to inhibitors of novel isoforms of protein kinase C. (a) Representative traces of currents evoked by ATP (100 μM) under control conditions (left) and following treatment with PMA (100 nM) or PMA following incubation with the PKC inhibitors (1 h pre-incubation before 10 min of PMA) calphostin C (1 μM), K252a (100 nM), Gö6983 (200 nM) or Gö6976 (200 nM). The lower panel shows a summary of the effects of the inhibitors on PMA potentiation, n=5–19. (b) Sample traces of the effects of PKC inhibitors and mGluR1α mediated potentiation of P2X1 receptor currents. Application of glutamate (100 μM) evoked a transient inward calcium activated chloride current and potentiated the subsequent ATP current. Potentiation was reduced following pre-treatment of the oocytes with the PKC inhibitors. The lower panel shows a summary of the effects, of the inhibitors on glutamate potentiation, n=3–13. ***p< 0.001.
	Fig. 2. The N-termini P2X1 receptor minigene blocks the potentiating effects of PMA and mGluR1α receptor stimulation on P2X1 receptor currents. A minigene encoding the N-terminal sequence of the P2X1 receptor was co-expressed with wild type P2X1 and mGluR1α receptors in the Xenopus oocytes. (a) Upper left panels show representative currents evoked by a maximal concentration of ATP (100 μM, indicated by bar) at control oocytes (WT P2X1) and those following 10 min incubation with PMA (100 nM). Right upper panels show the effects of co-expression of the amino terminal minigene (NH2 minigene) on the effects of PMA. The bar chart shows summary data, n=6–7. (b) Upper panels show sample traces for a given oocyte co-expressing P2X1 and mGluR1α receptors (left) or P2X1 receptors, mGluR1α receptors and the P2X1 receptor amino terminal minigene (right traces). Responses to a maximal concentration of ATP (100 μM, indicated by bar) are shown before and after the application of glutamate (100 μM). Glutamate evoked an inward calcium activated chloride current and potentiated subsequent ATP evoked responses. This potentiation was reduced by co-expression of the P2X1 receptor N-terminal minigene. The bar chart shows a summary of the data, n=6–7. ***p< 0.001.
	Fig. 3. The basic properties of cysteine points mutants of the P2X1 receptor N-terminal. (a) ATP (100 μM) evoked rapidly desensitising responses at WT P2X1 receptors. Desensitising responses were also recorded for the mutants Y16C, T18C and R20C however these were of reduced amplitude. For T18C an insert is provided showing at an increased scale the time-course of the ATP evoked response. (b) Peak current normalised traces showing the more rapid (V22C) and slower (D17C) rates of channel desensitisation of ATP evoked responses compared to WT. (c) Peak current amplitudes of WT and P2X1 receptor mutants to ATP (100 μM). ***p< 0.001. (d) Surface and total expression levels of WT and mutant P2X receptors with reduced peak current amplitudes.
	Fig. 4. PMA potentiation can be abolished by cysteine substitution of amino terminal residues. (a) Sample traces of ATP evoked currents (100 μM application indicated by bar) from oocytes under control conditions and following PMA (100 nM) treatment for WT as well as the mutants D17C, N26C and K27C. (b) Summary of the percentage changes of peak amplitudes by PMA treatment for N-termini cysteine mutants. Cysteine mutants around the conserved PKC consensus site and next to the first transmembrane segment were no longer potentiated by PMA. (c) Effects of mutations of the minigene on PMA potentiation. **p< 0.01, ***p< 0.001.
	Fig. 5. The effects of mGluR1α receptor activation on P2X1 receptor mutants. (a) Sample traces for a given oocyte co-expressing either WT P2X1, D17C or K27C mutant P2X1 receptor with mGluR1α receptors. Responses to a maximal concentration of ATP (100 μM, indicated by bar) are shown before and after the application of glutamate (dotted line). Glutamate (100 μM) evoked an inward calcium activated chloride current and potentiated subsequent ATP evoked responses for WT and D17C mutants but not for the K27C mutant P2X1 receptor. (b) The effects of mGluR1α receptor (100 μM 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 mGluR1α receptor. However, the mGluR1α receptor stimulated potentiation was still robust for the D17C and V29C mutants.
	Fig. 6. The effects of PMA and mGluR1α receptor to R20 substitutions. (a) Example traces of R20C and R20A mutants in response to PMA (100 nM) or mGluR1α receptor stimulation (100 μM glutamate) are shown. Peak amplitudes from control and PMA treated oocytes are shown. In the lower panels ATP evoked currents before and after mGluR1α receptor activation from either R20C (left) or R20A (right) are shown. (b) Summary of effects of amino acid substitution at position R20 at the P2X1 receptor by cysteine, alanine or isoleucine on potentiation by PMA or glutamate. ***p< 0.001.

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