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

Modulation by Cadmium Chloride of ATP-actived Currents Mediated by rat P2X4 Receptors Expressed in Xenopus OocytesAIM: To investigate the modulation of Cadmium on rat P2X4 receptors function and its underlying mechannisms.METHODS:cRNA of P2X4 was transcribed from cDNA, which was cloned in the pcDNA3 vector and through rat P2X4 receptor expressed in Xenopus oocytes. Two-electrode whole-cell voltage clamp are used to record I_ATP mediated by rat P2X4 receptor expressed in Xenopus oocytes.RESULTS: (1) The rat P2X4 receptor can be efficiently expressed in Xenopus oocytes, in the concentration range of 3～1000μmol/L, (2) PPADS and Suramin at a concentration of 100μmol/L have no effect on ATP-activated current mediated by rat P2X4 receptor. (3) The maximal cadmium-induced potentiation was obtained with 30 AM, which averaged a 6.2±1.0-fold increase of the receptor-gated current; the magnitude of the potentiation decreased with larger metal concentrations,infer that high cadmium concentrations may even interact at the inhibitory allosteric site.(4)10μmol/L Cadmium potentiated the ATP-evoked currents as evidenced by a significant leftward displacement of the ATP concentration–response curve. Co-application of 10μM cadmium plus ATP almost halved ATP EC50, reducing its value from 17.1±1.7 (n = 4 in the controls) to 10.1±1.7μM (n =4, P<0.01).(5)The effect of cadmium potentiation of ATP-activated current was independent of membrane potential between -140mV and 60mV .The effect was voltage independent.(6)The oocytes were preincubated with 10μM cadmium for 20,60,120,180,or 300 s before a 20-s addition of 1Μm ATP.The cadmium-induced potentiation reached its maxium within 60s.CONCLUSION: These results always suggest that Cadmium potentiated ATP-activated current mediated by P2X4 receptor in a noncompetitive manner, the ATP induced current were concentration dependent under 30μM cadmium. The magnitude of the modulation of Cadmium potentiation decreased with larger metal concentrations, infer that it may antagonize P2X4 receptors by allosteric modulation,and possibly acts at the binding sites within extracellular regions of the channel. The Xenopus Oocytes'Current Response and Mechanism Induced by Temperature change StimulateAIM: To investigate the effect and the modulation of temperature changes on currents in Xenopus oocytes.METHODS: Require the Xenopus oocytes taken out by operation and mechanically isolated into singe cell,base on the two-electrode whole-cell voltage clamp technique.In the operation,Choose the cell that the standing potential > -30mV,the bath was persist in perfusion,the commands voltage was in -50mV. Use the different temperature perfusate to perfusion and record the membrane current of the oocyte。RESULTS: 1. Use 0℃～5℃Ringer's liquid perfusion the oocyte,follow this the oocyte has a inward current swiftly,to accompany the perfusion frequency increased,the inward current of active has changed and the argument in turn to diminution,in the last place,it has become insensitivity to the microtherm perfusion of Ringer's liquid,from sensitive to desensitization is about 2～5hours. 2. With 35℃Ringer's liquid to perfusion,we can record a unitary reaction that the inward current,the amplitude is -56±12nA(n=6),and when perfusion every 8 minutes, along with the the times of the perfusion increased,the amplitude of inward current not display the rejection phenomenon. The currents amplitude activated and has a significant augmentation through application of different temperature of Ringer's liquid(25℃,30℃and 35℃) in the same oocyte..3. The current was voltage dependent.If change the voltage,the current was changed soon.When join 0～5℃Ringer's liquid,the inversion voltage is about -20mV; With chloride choline to substitute with the sodium chloride in the perfusate,the amplitude of current not to have obviously influence.(P>0.05)。when perfusion with 35℃Ringer's liquid,the inversion voltage is -10mV。CONCLUSION: The experimental results showed that the activating current's amplitude and duration change with suddenly changing of the temperature,it can revoke the across membrane ionic current,it proved tha the currents were sensitive to temperature,the across membrane ionic current perhaps originated of different ions activities.