Membrane Topology of an ATP-gated Ion Channel (P2X Receptor)

Alison Newbolt,Ron Stoop,Caterina Virginio,Annmarie Surprenant,R Alan North,Gary Buell,François Rassendren

doi:10.1074/jbc.273.24.15177

Abstract

Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X2 receptor (about 65 kDa) was reduced by pretreatment with endoglycosidase H. Mutagenesis of one or more of three potential asparagines (N182S, N239S, and N298S) followed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by receptors lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X-S/T) introduced into the N-terminal region (asparagine at position 9, 16, or 26) were not glycosylated. Asparagines were glycosylated when introduced at the C-terminal end of the first hydrophobic domain (positions 62 and 66) and at the N-terminal end of the second hydrophobic domain (position 324). A protein in which the C terminus of one P2X2 subunit was joined to the N terminus of a second P2X2 subunit (from a concatenated cDNA) had twice the molecular mass of the P2X2 receptor subunit, and formed fully functional channels. The experiments provide direct evidence for the topology originally proposed for the P2X receptor, with intracellular N and C termini, two membrane-spanning domains, and a large extracellular loop.

Highlights

Introduction of Artificial NLinked Glycosylation Sites—We introduced three point mutations resulting in consensus sites for N-glycosylation into the ⌬3N-P2X2 receptor between the initiating methionine and the beginning of the first hydrophobic regions (C9N, Y16N, and R28T) (Fig. 3A)
The broad smear of the wild type Western blot may result from the fact that crude membrane preparations were used; these would include plasma membrane and the membrane of intracellular organelles in which the identity and length of sugars added to proteins might be variable
Membrane preparations of oocytes expressing wild type P2X2 receptors and the three double mutants were treated with endoglycosidase H (Endo H); in each case, this led to the appearance of a band that co-migrated with the triply mutant receptor (⌬3N-P2X2) (Fig. 1D)

Summary

Introduction

Introduction of Artificial NLinked Glycosylation Sites—We introduced three point mutations resulting in consensus sites for N-glycosylation into the ⌬3N-P2X2 receptor between the initiating methionine and the beginning of the first hydrophobic regions (C9N, Y16N, and R28T) (Fig. 3A). This experiment failed to provide any evidence for an extracellular location of the N terminus. Five asparagines were individually introduced close to the end of the first hydrophobic domain (Fig. 4A) (Q52N, Q56N, S58N, P62N, and I66N) Two of these (P62N and I66N) had a higher molecular weight than ⌬3N-P2X2; two bands were seen in the case of the P62N mutant, one corresponding to ⌬3N-P2X2 and one at the same position as that seen with I66N. The size of the mobility shift corresponded to that expected from a single glycosylation, and for both P62N and I66N the shift could be prevented by pre-incubation of the membrane preparation with Endo H (Fig. 4C)

Objectives

Results

Conclusion