Elsevier

Neuropharmacology

Volume 39, Issue 11, October 2000, Pages 2054-2066
Neuropharmacology

P2X1 receptor membrane redistribution and down-regulation visualized by using receptor-coupled green fluorescent protein chimeras

https://doi.org/10.1016/S0028-3908(00)00058-7Get rights and content

Abstract

The P2X1 purinergic receptor subtype occurs on smooth muscle cells of the vas deferens and urinary bladder where it is localized in two different size receptor clusters, with the larger beneath autonomic nerve terminal varicosities. 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. P2X1 and a chimera of P2X1 and green fluorescent protein (GFP) were delivered into cells using microinjection, transient transfection or infection with a replication-deficient adenovirus. The P2X1–GFP chimera was used to study the time course of P2X1 receptor clustering in plasma membranes and the internalization of the receptor following prolonged exposure to ATP. Both P2X1 and P2X1–GFP clustered in the plasma membranes of Xenopus oocytes, forming patches 4–6 μm in diameter. Human embryonic kidney 293 (HEK293) cells, infected with the adenovirus, possessed P2X1 antibody-labeled regions in the membrane colocalized with GFP fluorescence. The ED50 for the binding of α,β-methylene adenosine triphosphate (α,β-meATP) to the P2X1–GFP chimera was similar to native P2X1 receptors. ATP-generated whole-cell currents in oocytes or HEK293 cells expressing either P2X1 or P2X1–GFP were similar. Exposure of HEK293 cells to α,β-meATP for 10–20 min in the presence of 5 μM monensin led to the disappearance of P2X1–GFP fluorescence from the surface of the cells. These observations using the P2X1–GFP chimera demonstrate that P2X1 receptors spontaneously form synaptic-size clusters in the plasma membrane that are internalized on exposure to agonists.

Introduction

P2X-type purinergic receptors consist of seven subtypes, classified as P2X1 to P2X7, that form ligand-gated cation channels and that contain cytoplasmic N- and C-termini and a large extracellular domain separating the two transmembrane segments (North and Barnard, 1997). Antibodies against peptides from the C-terminal portion of the P2X1 subtype have been used to determine that P2X1 receptors occur on smooth muscle cells in the vas deferens and urinary bladder (Vulchanova et al., 1996). Recently it has been shown that the P2X1 receptor subtype as well as other subtypes form clusters of two different sizes on the smooth muscle cells of the vas deferens and urinary bladder as well as on blood vessels. There are large numbers of small clusters of about 0.4 μm in diameter distributed over the smooth muscle cells and small numbers of large clusters greater than 1 μm in diameter that are positioned under autonomic nerve terminal varicosities (Hansen et al., 1998, Barden et al., 1999). The large junctional P2X clusters can be observed during normal development to be formed from small clusters of a number of different subtypes, which gather in large numbers in the vicinity of the nerve varicosities (Dutton et al., 1999). Several questions arise from these observations, including whether the formation of large-diameter receptor clusters is conditional on the presence of nerve varicosities and whether the receptors anchored beneath the varicosities are relatively immobile like nicotinic receptors or whether they are more labile as is the case with β2 adrenergic receptors (Barak et al., 1997, Kallal et al., 1998). In order to develop techniques that might answer such questions for tissues both in culture and in vivo, we have created a chimera of the P2X1 subtype with green fluorescent protein (GFP) and a replication-deficient adenovirus, expressing this chimera, for use as a delivery system. The present work describes the use of this chimera and delivery system to trace the formation of large junctional clusters in membranes and to determine the lability of these clusters once they are formed.

Section snippets

Plasmid constructions

pCI–P2X1–GFP was constructed as follows. The plasmid pBKCMV containing a P2X1 cDNA insert from rat vas deferens was obtained (Valera et al., 1994). The P2X1 cDNA was amplified from the plasmid by polymerase chain reaction (PCR) using a forward primer that annealed to the T3 promoter region and a reverse primer that annealed directly upstream of and over the stop codon in P2X1. The reverse primer (5′ CAT GCC ATG GAG GTC CTC ATG TTC TCC 3′) eliminated the stop codon and incorporated an in-frame

Properties of P2X1 and P2X1–GFP in Xenopus oocytes

In order to first confirm that the fusion with GFP did not disturb the functioning of the P2X1 receptor, preliminary microinjection studies were performed on Xenopus oocytes with cRNA made from vectors containing either P2X1 or P2X1–GFP cDNAs and their properties determined. In control oocytes not injected with P2X1 cRNA there was no labeling observed with antibodies against the extracellular domains of the P2X subtypes P2X1 to P2X7 indicating that Xenopus oocytes do not possess endogenous P2X

P2X1–GFP chimera

The present observations show that the P2X1–GFP chimera has properties similar to its wild-type counterpart when either injected as cRNA into Xenopus oocytes or expressed using transient transfection techniques in cultured cells. This indicates that the C-terminal fusion with GFP does not seem to affect the ability of the individual monomers to aggregate into clusters or to function as agonist-activated ion channels. We have also created replication-deficient adenoviruses for the delivery of P2X

Acknowledgments

The authors wish to thank the National Heart Foundation of Australia and the National Health and Medical Research Foundation of Australia for financial support. D.I.C. is a University of Sydney Medical Foundation Fellow and P.P. is a Year 2000 Fellow of the University of Sydney.

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