P2X1 receptor mobility and trafficking; regulation by receptor insertion and activation

P>P2X1 receptors for ATP contribute to signalling in a variety of cell types and following stimulation undergo rapid desensitisation (within 1 s), and require similar to 5 min to recover. In HEK293 cells P2X1 receptors C-terminally tagged with enhanced green fluorescent protein (P2X1-eGFP) were predominantly expressed at the cell surface. Following > 90% photo-bleaching of P2X1-eGFP within a 6 mu m2 circle at the cell surface fluorescence recovery after photo-bleaching (FRAP) was fit with a time constant of similar to 60 s and recovered to similar to 75% of pre-bleach levels. Following activation of the P2X1 receptor with alpha,beta-methylene ATP the associated calcium influx doubled the FRAP recovery rate. The protein synthesis inhibitor cycloheximide had only a small effect on repeated FRAP and indicated a limited contribution of new P2X1 receptors to the FRAP. Inhibition of trafficking with brefeldin A reduced recovery and this effect could be reversed following receptor activation. In contrast, the dynamin inhibitor dynasore had no effect on FRAP under unstimulated conditions but reduced the level of recovery following agonist stimulation. In functional studies both brefeldin A and dynasore increased the recovery time from desensitisation. Taken together these studies demonstrate for the first time an important role of receptor recycling on P2X1 receptor responsiveness.