Modulation By Aluminum Of ATP-activated Currents Mediated By Rat P2X₄ Receptors Expressed In Xenopus Oocytes

PART 1AIM: To determine the feasibility of establishing the heterologous expression model of P2X4 receptor. METHODS: cRNA of P2X4 was transcribed from cDNA, which was cloned in the pcDNA3 vector. Each oocyte of mature xenopus laevis was injected with transcribed cDNA in vivo and incubated at 18℃for 30 hours. Recording the current induced by P2X4 receptor with voltage clamp technique tested the function of the expressed P2X4 receptor. RESULTS: ATP-activated inward current could be observed in most of the oocytes which were injected with the transcribed P2X4 cRNA, and the ATP induced current were concentration dependent. CONCLUSION:The results demonstrate that the rat P2X4 receptor could expressed in Xenopus Laevis oocytes successfully.PART 2AIM:To explore the characterization of ATP-activated current(IATP)mediated by rat P2X4 receptor expressed in Xenopus Oocytes. METHODS: Two-electrode whole-cell voltage clamp are used to record IATP mediated by rat P2X4 receptor expressed in Xenopus oocytes. RESULTS:(1) The rat P2X4 receptor can be efficiently expressed in Xenopus oocytes,.(2) The activation phase time constant (τon) of IATP are 2.58±0.60 s, 1.80±0.47 s and 1.05±0.24 s respectively, while the desensitizing phase time constant (τdes) of IATP are 9.54±1.56 s,6.52±1.24 s and 4.11±0.78 s respectively,when 1μmol/L,10μmol/L,and 100μmol/L ATP are appied correspondingly.(3) The Concentration-response relationship in ATP is measured as: 16.03±1.81μmol/L of EC50 to 1.02±0.09 of Hill coefficient.(4) PPADS and Suramin at a concentration of 100μmol/L have no effect on ATP-activated current mediated by rat P2X4 receptor.(5)IATP exhibits characteristics of both voltage-dependency and inward-rectification,and (6) IATP doesnot have a significant variation when applying ATP times are 20 s,30 s,45 s,and 60 s.CONCLUSION:The results in this study advance our understanding in the characteristics of ATP-activated current, and prepare for further investigation in the functions of P2X4 receptor and its inner modulating mechanism.PART 3AIM:To investigate the modulation of aluminum on rat P2X4 receptors function and its underlying mechannisms.METHODS: Rat P2X4 receptors were expressed in Xenopus oocytes and studied using the two-electrode voltage clamp.RESULTS(:1)In the presence of 100μmol/L aluminum ,the desensitization time constant(τdes) value of ATP-activated current(IATP) decreased significantly more than without aluminum,while the activation time canstant(τon) increased.(2)In oocytes expressing P2X4 receptors,aluminum cholride,in the concentration range of 3～1000μmol/L,reversibly inhibited current activated by 10μmol/L ATP.Aluminum produced 50% of maximal inhibition of IATP(10μmol/L) was 16.2μmol/L.(3)Aluminum caused the concentration-response curve for IATP a downward displacement by reducing the maximal response,but did not change the EC50 value.(4)The aluminum inhibition of IATP was independent of membrane potential between -80 mV and -20 mV and did not involve a shift in the reversal potential of the current.(5)This block did not involve a gradual decline in the presence of 10μmol/L aluminum at 3-min intervals.Moreever,aluminum modulation of IATP(10μmol/L) was time-dependent.The aluminum-induced inhibition reached its maximum within 60 s pretreatment.CONCLUSION:These results always suggest that aluminum inhibites ATP-activated current mediated by P2X4 receptor in a noncompetitive manner.It may antagonize P2X4 receptors by allosteric modulation,and possibly acts at the binding sites within extracellular regions of the channel.