Kainic acid is a potent neurotoxin for certain neurons. Its neurotoxicity is thought to be mediated by an excitatory amino-acid-gated ion channel (ionotropic receptor) possessing nanomolar affinity for kainate. Here we describe a new member of the rat excitatory amino-acid receptor gene family, KA-1, that has a 30% sequence similarity with the previously characterized alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits GluR-A to -D. The pharmacological profile of expressed recombinant KA-1 determined in binding experiments with [3H]kainate is different from that of the cloned AMPA receptors and similar to the mammalian high-affinity kainate receptor (kainate greater than quisqualate greater than glutamate much greater than AMPA) with a dissociation constant of about 5 nM for kainate. The selectively high expression of KA-1 messenger RNA in the CA3 region of the hippocampus closely corresponds to autoradiographically located high-affinity kainate binding sites. This correlation, as well as the particular in vivo pattern of neurodegeneration observed on kainate-induced neurotoxicity, suggests that KA-1 participates in receptors mediating the kainate sensitivity of neurons in the central nervous system.
P2X receptors are a family of ATP-gated ion channels thought to have intracellular N and C termini and two transmembrane segments separating a large extracellular domain. We examined the involvement of the second putative transmembrane domain (TM2) of the P2X2 subunit in ion conduction, using the substituted cysteine accessibility method (SCAM). This method tests the ability of hydrophilic reagents such as Ag+ or the methanethiosulfonates to modify covalently the sulfhydryl side chains exposed to aqueous environments. ATP-gated current was measured in HEK293 cells transiently expressing either wild-type or functional mutant P2X2 receptors containing a cysteine substitution in or around TM2. Application of Ag+ to gating channels had no sustained effect on wild-type P2X2 (WT) but irreversibly altered whole-cell currents in 15 mutants. By contrast, bath application of (2-aminoethyl)methanethiosulfonate (MTSEA) to closed channels inhibited 8 of the 15 residues affected by Ag+ when the channel was gating. Inhibition of the closed channel was prevented in seven of eight mutants when membrane-permeant MTSEA was scavenged by 20 mM intracellular cysteine, indicating that these seven mutants lie on the intracellular side of the channel gate. Further, MTSEA inhibited current through G342C in the absence of intracellular cysteine but augmented the current when cysteine was present, suggesting that this residue may be part of the gate. Taken together, the data help to the identify a functional domain of the channel pore by mapping residues on either side of the channel gate.
P2X receptors are a distinct family of ligand-gated ion channels activated by extracellular ATP. Each of the seven identified subunit proteins (P2X 1 through P2X 7 ) has been reported to form functional homo-oligomeric channels when expressed in heterologous systems. Functional studies of native receptors, together with patterns of subunit gene expression, suggest that hetero-oligomeric assembly among members of this family may also occur. This prediction is supported by reports describing hetero-oligomeric assembly for three different recombinant subunit combinations. In this report, we systematically examined the ability of all members of the P2X receptor family to interact using a co-immunoprecipitation assay. The seven P2X receptor subunits were differentially epitope-tagged and expressed in various combinations in human embryonic kidney 293 cells. It was found that six of the seven subunits formed homooligomeric complexes, the exception being P2X 6 . When co-assembly between pairs of subunits was examined, all were able to form hetero-oligomeric assemblies with the exception of P2X 7 . Whereas P2X 1 , P2X 2 , P2X 5 , and P2X 6 were able to assemble with most subunits, P2X 3 and P2X 4 presented a more restricted pattern of coassociation. These results suggest that hetero-oligomeric assembly might underlie functional discrepancies observed between P2X responses seen in the native and recombinant settings, while providing for an increased diversity of signaling by ATP.Investigation of the native receptors mediating extracellular ATP signaling in tissues has been a difficult task due to the lack of useful pharmacological tools. For this reason, the cloning of ATP receptors (the P2 receptor family) and their recombinant expression has proven extremely useful in elucidating the basic properties of these proteins and for providing a template for further study into native P2 receptors. Two families of proteins mediating the actions of ATP have been identified: the metabotropic G protein-coupled P2Y receptors and the ionotropic P2X receptors (1). The P2X receptors are nonselective ion channels thought to be oligomeric in nature, and they are expressed in many excitable and nonexcitable cells, where they mediate a variety of physiological actions, including smooth muscle contractility, neuroendocrine secretion, and modulation of synaptic transmission (2, 3). In addition, recent reports suggest that they may also play an important role in the transmission of pain perception (4, 5). With the molecular identification of seven P2X receptor subunits, our understanding of the biophysical and pharmacological properties of these channels has been considerably increased. However, relatively little is known about the multimeric organization of this new class of channel receptors.Almost all known ionotropic receptors exist as hetero-oligomers (6). Importantly, their functional and pharmacological properties are directly determined by their subunit composition, with different subunit combinations yielding different phenotypes (e...
P2X receptors are a family of ion channels gated by extracellular ATP. Each member of the family can form functional homomeric channels, but only P2X2 and P2X3 have been shown to combine to form a unique heteromeric channel. Data from in situ hybridization studies suggest that P2X1 and P2X5 may also co-assemble. In this study, we tested this hypothesis by expressing recombinant P2X1 and P2X5 receptor subunits either individually or together in human embryonic kidney 293 cells. In cells expressing the homomeric P2X1 receptor, 30 microM alpha,beta-methylene ATP (alpha,beta-me-ATP) evoked robust currents that completely desensitized in less than 1 sec, whereas alpha,beta-me-ATP failed to evoke current in cells expressing the homomeric P2X5 receptor. By contrast, alpha, beta-me-ATP evoked biphasic currents with a pronounced nondesensitizing plateau phase in cells that co-expressed both subunits. Further, the EC50 for alpha,beta-me-ATP was greater in cells expressing both P2X1 and P2X5 than in cells expressing P2X1 alone (5 and 1.6 microM, respectively). Heteromeric assembly was confirmed using a co-immunoprecipitation assay of epitope-tagged P2X1 and P2X5 subunits. In summary, this study provides biochemical and functional evidence of a novel channel formed by P2X subunit heteropolymerization. This finding suggests that heteromeric subunit assembly constitutes an important mechanism for generating functional diversity of ATP-mediated responses.
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