P2X(1) receptor membrane redistribution and down-regulation visualized by using receptor-coupled green fluorescent protein chimeras

The P2X(1) purinergic receptor subtype occurs on smooth muscle cells of the vas deferens and urinary bladder where it is localized in two different si ze receptor clusters, with the larger beneath autonomic nerve terminal vari cosities. We have sought to determine whether these synaptic-size clusters only form in the presence of varicosities and whether they are labile when exposed to agonists. P2X(1) and a chimera of P2X(1) and green fluorescent p rotein (GFP) were delivered into cells using microinjection, transient tran sfection or infection with a replication-deficient adenovirus. The P2X(1)-G FP chimera was used to study the time course of P2X(1) receptor clustering in plasma membranes and the internalization of the receptor following prolo nged exposure to ATP. Both P2X(1) and P2X(1)-GFP clustered in the plasma me mbranes of Xenopus oocytes, forming patches 4-6 mu m in diameter. Human emb ryonic kidney 293 (HEK293) cells, infected with the adenovirus, possessed P 2X(1) antibody-labeled regions in the membrane colocalized with GFP fluores cence. The ED50 for the binding of alpha,beta-methylene adenosine triphosph ate (alpha,beta-meATP) to the P2X(1)-GFP chimera was similar to native P2X( 1) receptors. ATP-generated whole-cell currents in oocytes or HEK293 cells expressing either P2X(1) or P2X(1)-GFP were similar. Exposure of HEK293 cel ls to alpha,beta-meATP for 10-20 min in the presence of 5 mu M monensin led to the disappearance of P2X(1)-GFP fluorescence from the surface of the ce lls. These observations using the P2X(1)-GFP chimera demonstrate that P2X(1 ) receptors spontaneously form synaptic-size clusters in the plasma membran e that are internalized on exposure to agonists. (C) 2000 Elsevier Science Ltd. All rights reserved.