Functional Regulation of P2X₆ Receptors by N-Linked Glycosylation: Identification of a Novel αβ-Methylene ATP-Sensitive Phenotype

Abstract

Investigation of rat recombinant P2X 6 receptors has been limited because of the difficulty in obtaining functional expression in heterologous systems. In this study, we demonstrate glycosylationdependent regulation of recombinant P2X 6 receptor function and associated conferral of a novel phenotype that is sensitive to the P2X 1 and P2X 3 receptor agonist, ␣␤-methylene ATP. In cells functionally expressing P2X 6 receptors, ATP and ␣␤-methylene ATP evoked slowly desensitizing inward currents (EC 50 values, 0.5 and 0.6 M, respectively) with slow kinetics of current decay on agonist washout. 2Ј,3Ј-O-(2,4,6-trinitrophenyl ATP) and iso-pyridoxalphosphate-6-azophenyl-2Ј-5Ј-disulfonate were effective antagonists (IC 50 values, 0.8 and 22 M, respectively); however, suramin was relatively ineffective. Reverse transcription-polymerase chain reaction analysis confirmed the absence of other P2X receptor subunits. Western analysis of membrane fractions from functional and nonfunctional clones confirmed the presence of P2X 6 at the cell membrane but revealed a difference in apparent molecular mass of immunoreactive products (ϳ70 and ϳ60 kDa, respectively). N-glycosidase F treatment of both functional and nonfunctional receptor cell membranes increased the electrophoretic mobilities of immunoreactive products, with both proteins migrating at ϳ55 kDa, demonstrating an increased level of glycosylation of the P2X 6 receptor in functional compared with nonfunctional cells. This study demonstrates that nonfunctional rat recombinant P2X 6 receptors 1) are expressed on the membrane surface of human embryonic kidney cells and 2) are glycosylated. Expression of the novel functional receptor phenotype is associated with further glycosylation, resulting in an apparently larger molecular mass. These results suggest that P2X 6 receptor subunits contribute to ␣␤-methylene ATP sensitivity.

Mouse Leydig cells express multiple P2X receptor subunits

“…These clusters stay in the ER and are not incorporated into the plasma membrane [1]. This point is particularly relevant for the P2X 6 subunits, which requires an extensive glycosylation process in order to form functional homotrimeric channels [23]. The P2X 4 subunit is colocalized with the ER and CN, showing a dense and uniform distribution ( Fig.…”

“…Seven P2X receptor subunits (P2X 1-7 ) were cloned and characterized in vertebrates, and each of them can form two type of assemblies, homomeric, P2X 1 [50], P2X 2 [3], P2X 3 [8], P2X 4 [2], P2X 5 [18], P2X 7 [46], and P2X 6 [23], or heterotrimeric P2X 2/3 [31], P2X 1/5 [48], P2X 4/6 [30], P2X 2/6 [27], P2X 1/2 [5], P2X 1/4 [38], and P2X 4/7 [19]. Each subunit is composed by two transmem-brane regions (TM1 and TM2) forming the channel pore, a large extracellular domain, which contains the ATP binding site and both N and C termini located intracellularly [14,48,22].…”

“…On the other side, P2X 6 receptors form homotrimeric assemblies only after an extensive glycosylation process. When expressed, they show αβmeATP sensitivity and are slowly desensitizing [23].…”

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

“…The N-glycosylation sites within the ectodomain are also essential for trafficking and contribute to the appropriate functionality of P2X1R [37], P2X2R [34,46], P2X4R [23], P2X6R [25], and P2X7R [29]. For P2X1R, any two out of the four naturally occurring N-glycans are sufficient for robust expression of functional receptors at the cell surface [37].…”

“…Glycosylation is also important for P2X4R localization on the plasma membrane [23]. Transiently expressed P2X6 subunits are not properly trafficked to the cell surface, presumably because of a glycosylation defect [25]. Residue N187 of P2X7R, which belongs to an N-linked glycosylation consensus sequence found in six of the seven P2XR family members, is critical for the cell surface expression of this receptor [29].…”