The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous α and β subunits. Single expression of α subunits generates functional homo-oligomeric GlyRs, whereas the β subunit requires a co-expressed α subunit to assemble into hetero-oligomeric channels of invariant stoichiometry (α 3 β 2 ). Here, we identified eight amino acid residues within the N-terminal region of the α1 subunit that are required for the formation of homo-oligomeric GlyR channels. We show that oligomerization and N-glycosylation of the α1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR α and β subunits.

Many effects of extracellular ATP on cells of the immune system have been attributed to the presence of a so-called P2Z receptor. Recent work has shown that one of the members of the P2X family of ATP-gated receptors Soto et al. 1997;Ralevic & Burnstock, 1998;MacKenzie et al. 1999), designated P2X 7 , shares many phenotypical properties with the P2Z receptor upon heterologous expression, suggesting that the two are identical. The P2X 7 receptor is therefore also referred to as P2Z/P2X 7 receptor (Di Virgilio, 1995;Di Virgilio et al. 1998). A peculiarity of the P2X 7 subunit is its very long intracellular C-terminal tail, which is 196 or 132 amino acids longer than that of the P2X 1 or P2X 2 subunit isoform, respectively.Several studies attempting to characterise the recombinant P2X 7 receptor have provided information about its complex function, which is not yet fully understood. During short applications of ATP lasting a few seconds, the P2X 7 receptor behaves like a typical P2X family member, exhibiting permeability to small cations only. However, upon prolonged or repeated applications of ATP, large non-selective pores are formed in the plasma membrane of some cells expressing P2X 7 (Surprenant et al. 1996;Rassendren et al. 1997;Virginio et al. 1999), which have been attributed to the receptor itself. On the other hand, it has also been suggested that the pores represent distinct entities, which become activated subsequent to the stimulation of P2X 7 receptors (Coutinho-Silva & Persechini, 1997;Schilling et al. 1999).In native cells, ATP elicits different effects over a wide concentration range. For instance, ATP increases the cell 1. The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing either the wild-type human P2X 7 receptor (hP2X 7 ), an N-terminally hexahistidyl-tagged hP2X 7 receptor (His-hP2X 7 ), or a truncated His-hP2X 7 receptor (His-hP2X 7 ∆C) lacking the C-terminal 156 amino acids was investigated using the two-microelectrode voltage clamp technique.2. The activation time course of the wild-type hP2X 7 receptor can be described as the sum of an exponentially growing and an additional almost linearly activating current component.3. The amplitude of the exponentially activating current component of the wild-type hP2X 7 receptor displayed a biphasic dependence on the agonist concentration, which could be best approximated by a model of two equal high-sensitivity and two equal low-sensitivity noncooperative activation sites with apparent dissociation constants of about 4 and 200 µM free ATP 4_ , respectively.4. The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 µM.5. The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X 7 or His-hP2X 7 ∆C.6. Our data indicate that the hP2X 7 receptor possesses at least two types of activation sites, which differ in ATP 4_ sensitivity by a factor of 50. The degree of occupatio...

Human B lymphocytes express an ATP-gated ion channel (P2Z receptor), which shares similarities with the recently identified P2X7 receptor. Using gene specific primers, we have now isolated P2X7 cDNA from the total RNA of human B lymphocytes. This hP2X7 receptor subtype was expressed in Xenopus oocytes and electrophysiologically characterized. The hP2X7 receptor is similar to, but does not completely match, P2Z of human B cells. The hP2X7 receptors resemble the P2Z receptors with regard to the ATP concentration of half maximal activation, reproducibility, permeation characteristics and lack of desensitization of the ATP-evoked currents. However, in contrast to the native lymphocytic P2Z receptor, the time course of activation of hP2X7 displayed an additional linearly increasing current component. Furthermore, a second, small and slowly deactivating current component exists only in hP2X7 expressed in oocytes. The activation and deactivation kinetics as well as permeation characteristics of hP2X7 are different from rat P2X7 recently expressed in oocytes. Unlike in mammalian cells, hP2X7 expressed in Xenopus oocytes is not sufficient to induce large non-selective pores.

The inhibitory glycine receptor (GlyR) in developing spinal neurones is internalized efficiently upon antagonist inhibition. Here we used surface labeling combined with affinity purification to show that homopentameric ␣1 GlyRs generated in Xenopus oocytes are proteolytically nicked into fragments of 35 and 13 kDa upon prolonged incubation. Nicked GlyRs do not exist at the cell surface, indicating that proteolysis occurs exclusively in the endocytotic pathway. Consistent with this interpretation, elevation of the lysosomal pH, but not the proteasome inhibitor lactacystin, prevents GlyR cleavage. Prior to internalization, ␣1 GlyRs are conjugated extensively with ubiquitin in the plasma membrane. Our results are consistent with ubiquitination regulating the endocytosis and subsequent proteolysis of GlyRs residing in the plasma membrane. Ubiquitin-conjugating enzymes thus may have a crucial role in synaptic plasticity by determining postsynaptic receptor numbers.The efficiency of synaptic transmission depends critically on a dense packing of neurotransmitter receptors in the postsynaptic membrane. At fast synapses, ligand-gated ion channels (LGICs) 1 mediate the postsynaptic response. Different lines of evidence indicate that the distribution and density of LGICs in the plasma membrane are regulated tightly. In differentiating muscle fibers, the formation of a densely packed postsynaptic matrix of nicotinic acetylcholine receptors (nAChRs) at the developing motor endplate requires restriction of gene expression to subsynaptic nuclei, efficient internalization and degradation of extrasynaptic receptors, synaptic clustering by rapsyn, and slowing of the turnover of synaptically accumulated nAChRs (1, 2). Inversely, muscle denervation (2) or blockade of neurotransmission by ␣-bungarotoxin (3) causes a loss of nAChRs from the postsynaptic membrane because of an increased rate of protein turnover (4). Because nAChR degradation occurs only after internalization and lysosomal targeting of the receptor protein (4, 5