P2X4 Receptor Is a Glycosylated Cardiac Receptor Mediating a Positive Inotropic Response to ATP

Abstract

Although P2X receptors are suggested to play a role in synaptic neurotransmission, the specific physiological role of each P2X receptor subtype remains largely unknown. We used cultured chick embryo ventricular myocytes as a model to study a potential physiological role of the P2X 4 receptor in mediating the positive inotropic effect of ATP. The chick P2X4 receptor (cP2X 4 R) mRNA was expressed in the heart and the pharmacological features of the ATP-induced positive inotropic response were similar to those of the cP2X 4 R in terms of insensitivity to blockade by known P2 receptor antagonists and the ineffectiveness of adenosine 5-(␣,␤-methylene)triphosphate as an agonist. Treatment of myocytes with antisense oligonucleotides specific to the 5 region of cP2X 4 R abrogated the P2 agonist-stimulated 45 Ca influx. Similarly, antisense oligonucleotide treatment also blocked the 2-methylthio-ATP-stimulated increase in contractile amplitude. The data suggest that the native P2X 4 receptor is involved in mediating the P2 agonist-stimulated response in the heart. In characterizing the biochemical property of the P2X 4 receptor, antibody against cP2X 4 R detected a 44-kDa and a 58-kDa protein in the immunoblot. Inhibition of N-linked glycosylation by tunicamycin converted the 58-kDa protein to the 44-kDa protein, suggesting that the 58-kDa protein was a glycosylated P2X 4 receptor. The nonglycosylated 44-kDa P2X 4 receptor was resistant to various detergent/aqueous extraction, consistent with a role of glycosylation in maintaining its detergent solubility and hydrophilicity. Cross-linking the cell surface proteins with N-hydroxysuccinimide-SS-biotin followed by affinity precipitation with streptavidin-conjugated agarose and subsequent immunoblotting with anti-cP2X 4 R showed that only the glycosylated 58-kDa P2X 4 receptor was expressed on the cell surface, indicating an important role of glycosylation for the receptor's localization on the plasma membrane. These data revealed a novel physiologic function of the P2X 4 receptor and suggested the importance of N-linked glycosylation in its cell surface expression and detergent solubility.P2X receptors are nonselective cation channels gated by ATP (for reviews, see Refs. 1-3). Seven isoforms of P2X receptors identified so far show 35-50% sequence identities based on comparison of the homologous region. Each receptor has two transmembrane domains and a large extracellular loop that contains a number of putative N-glycosylation sites and an ATP binding site. Recent human genome data suggested three additional possible P2X receptors in the human genome, and no P2X counterparts were found in the yeast or Drosophila genome (4). Both N-and C-intracellular termini have been shown to be important in determining the rate of desensitization of the P2X receptor (1-3). Biochemical and elctrophysiological studies suggested homomeric and heteromeric structures, possibly trimer, as the functional form of P2X receptors (1-3, 5-7).In vitro and in vivo studies indicated that P2X receptors ar...

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].…”

“…Deletion of all three glycosylated asparagines of the P2X2 subunits induces an intracellular retention and a complete loss of function of the receptors [34,46]. 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].…”

Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis

“…To investigate whether glycans play a role in protecting the P2X 4 receptor, we first identified how many sites normally were glycosylated. The P2X 4 receptor has seven potential N-linked glycosylation sites (Hu et al, 2002) and shows partial resistance to treatment with endoglycosidase H (Endo H), indicating that it undergoes complex glycosylation in the Golgi (Fig. 3B).…”

A beneficial role of cardiac P2X4 receptors in heart failure: rescue of the calsequestrin overexpression model of cardiomyopathy

“…The P2X 4 R is a ligand-gated ion channel (6,10,12,19,24). It is a member of the extracellular ATP receptor family.…”

“…Recent studies show that activation of the P2X receptor subtype of the extracellular ATP receptor family increases the contractility of both cardiac myocytes and the intact heart (3,4,5,9,17,18,23). P2X receptors are ligand-gated ion channels whose activation results in sodium and calcium entry (6,10,12,19,24). Transgenic overexpression of the P2X 4 subtype of this family of receptor channels exhibits a phenotype of enhanced basal cardiac contractility and output in a working heart model (9).…”