The P2X4 receptor has a widespread distribution in the central nervous system and the periphery, and plays an important role in the function of immune cells and the vascular system. Its upregulation in microglia contributes to neuropathic pain following nerve injury. The mechanisms involved in its regulation are not well understood, although we have previously shown that it is constitutively retrieved from the plasma membrane and resides predominantly within intracellular compartments. Here, we show that the endogenous P2X4 receptors in cultured rat microglia, vascular endothelial cells and freshly isolated peritoneal macrophages are localized predominantly to lysosomes. Lysosomal targeting was mediated through a dileucine-type motif within the N-terminus, together with a previously characterized tyrosine-based endocytic motif within the C-terminus. P2X4 receptors remained stable within the proteolytic environment of the lysosome and resisted degradation by virtue of their N-linked glycans. Stimulation of phagocytosis triggered the accumulation of P2X4 receptors at the phagosome membrane. Stimulating lysosome exocytosis, either by incubating with the Ca2+ ionophore ionomycin, for normal rat kidney (NRK) cells and cultured rat microglia, or the weak base methylamine, for peritoneal macrophages, caused an upregulation of both P2X4 receptors and the lysosomal protein LAMP-1 at the cell surface. Lysosome exocytosis in macrophages potentiated ATP-evoked P2X4 receptor currents across the plasma membrane. Taken together, our data suggest that the P2X4 receptor retains its function within the degradative environment of the lysosome and can subsequently traffic out of lysosomes to upregulate its exposure at the cell surface and phagosome.
The cytolytic ionotropic ATP receptor P2X 7 has several important roles in immune cell regulation, such as cytokine release, apoptosis, and microbial killing. Although P2X 7 receptors are frequently coexpressed with another subtype of P2X receptor, P2X 4 , they are believed not to form heteromeric assemblies but to function only as homomers. Both receptors play a role in neuropathic pain; therefore, understanding how they coordinate the cellular response to ATP is important for the development of effective pain therapies. Here, we provide biochemical and electrophysiological evidence for an association between P2X 4 and P2X 7 that increases the diversity of receptor currents mediated via these two subtypes. The heterologously expressed receptors were coimmunoprecipitated from human embryonic kidney (HEK) 293 cells, and the endogenous P2X 4 and P2X 7 receptors were similarly coimmunoprecipitated from bone marrow-derived macrophages. In HEK293 cells, the fraction of P2X 4 receptors biotinylated at the plasma membrane increased 2-fold in the presence of P2X 7 although there was no change in overall expression. Coexpression of a dominantnegative P2X 4 mutant (C353W) with P2X 7 , inhibited P2X 7 receptor mediated currents by greater than 2-fold, whereas a nonfunctional but non-dominant-negative mutant (S341W) did not. Coexpression of P2X 4 S341W with P2X 7 produced a current that was potentiated by ivermectin and inhibited by 2Ј, 3Ј-O-(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), whereas expression of P2X 7 alone produced a current that was insensitive to both of these compounds at the concentrations used. These results demonstrate a structural and functional interaction between P2X 4 and P2X 7 , which suggests that they associate to form heteromeric receptors.
P2X receptors are cation-selective channels activated by extracellular ATP. The architecture of these receptors is still not completely clear. Here we have addressed this issue by both chemical cross-linking and direct imaging of individual receptors by atomic force microscopy (AFM). Cross-linking of the P2X 2 receptor produced higher order adducts, consistent with the presence of trimers. The mean molecular volume of the receptor determined by AFM (409 nm 3 ) also points to a trimeric structure. P2X 2 receptors bearing His 6 epitope tags were incubated with anti-His 6 antibodies, and the resultant complexes were imaged by AFM. For receptors with two bound antibodies, the mean angle between the antibodies was 123°, again indicating that the receptor is a trimer. In contrast, cross-linking of the P2X 6 receptor did not produce higher order adducts, and the mean molecular volume of the receptor was 145 nm 3 . We conclude that P2X 2 receptors are trimers, whereas the P2X 6 receptor subunits do not form stable oligomers.P2X receptors contain an integral cation-selective ion channel that is opened by the binding of extracellular ATP (1, 2). Seven P2X receptor subunits have been identified, and these subunits associate together to form homo-or hetero-oligomeric receptors. Each subunit has two transmembrane regions (TMRs), 1 which are integrated into the membrane so that both N and C termini are intracellular. The large extracellular domain is glycosylated and contains several cysteine residues that form multiple disulfide bonds.The architecture of P2X receptors is still not clear, although the available evidence favors a trimeric arrangement of subunits, at least for some forms of the receptor. Early indications that the receptors were oligomers arose from electrophysiological studies of both endogenous receptors and exogenously expressed receptors, which revealed that receptor activation had a Hill coefficient greater than 1 (3, 4). Analysis of the mechanism of blockade of the T336C mutant P2X 2 receptor by methanethiosulfonate indicated a trimeric architecture (5). More recently, chemical cross-linking of P2X 1 , P2X 2 , and P2X 3 receptors again pointed to the existence of trimers (6, 7). It is fair to say, however, that this issue has not been completely resolved. For instance, under some circumstances, the P2X 1 , P2X 2 , and P2X 3 receptors have been found to migrate on non-denaturing gels as hexamers and even nonamers (6, 7). Further, the extracellular domain of the P2X 2 receptor behaves as a stable tetramer in solution (8). Finally, the P2X 6 receptor migrates as a tetramer on native gels (7).In the present study, we have combined a chemical crosslinking approach with direct imaging of P2X 2 and P2X 6 receptors by AFM. The ability to visualize single receptors afforded by AFM has permitted the measurement of the molecular volume of the receptors and the determination of the geometry of receptors liganded by antibodies against epitope tags present on the receptor subunits. Our data show convincingly that the P...
Synthetic peptides of the five alternative NH2-terminal sequences of Shaker when applied to the cytoplasmic side of ShB channels that have an NH2-terminal deletion (ShBA6-46) block the channel with potencies correlated with the rate of inactivation in the corresponding variant. These peptides share no sequence similarity and yet three out of the five have apparent dissociation constants between 2 and 15 p.M, suggesting that the specificity requirements for binding are low. To identify the primary structural determinants required for effective block of ShBA6-46, we examined the effects of substitutions made to the 20 residue ShB peptide on association and dissociation rates. Nonpolar residues within the peptide appear to be important in stabilizing the binding through hydrophobic interactions. Substitutions to leucine-7 showed there was a clear correlation between hydrophobicity and the dissociation rate constant (koff) with little effect on the association rate constant (kon). Substituting charged residues for hydrophobic residues within the region 4-8 disrupted binding. Within the COOH-terminal half of the peptide, substitutions that increased the net positive charge increased kon with relatively small changes in koff, suggesting the involvement of long-range electrostatic interactions in increasing the effective concentration of the peptide. Neutralizing charged residues produced small changes in koff. Charges within the region 12-20 act equivalently; alterations which conserved net charge produced little effect on either kon or kof~. The results a~re consistent with this region of the peptide having an extended conformation and suggest that when bound this region makes few contacts with the channel protein and remains relatively unconstrained. Analogous mutations within the NH2-terminal domain of the intact ShB channel produced qualitatively similar effects on blocking and unblocking rates.
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