P2X2 characteristics of the ATP receptor coupled to [Ca2+]i increases in cultured Purkinje neurons from neonatal rat cerebellum
Introduction
Besides being the exchange currency in intracellular energy metabolism, ATP is also an extracellular messenger. In the central nervous system (CNS), fast synaptic transmission mediated by ATP has been demonstrated in the medial habenula and locus coeruleus (Edwards et al., 1992, Nieber et al., 1997). This action is mediated by ionotropic P2X receptors present in postsynaptic neurons. P2X receptors are a new family of ligand-gated ionic channels with two transmembrane segments. They are unrelated, both in terms of primary sequence and membrane topology, to other receptor channels such as the nicotinic acetylcholine receptor or ionotropic glutamate receptors (see reviews by Buell et al., 1996a, Soto et al., 1997). P2X receptors are supposed to be oligomeric integral membrane proteins formed by the assembly of several subunits. At least seven P2X subunits have been cloned so far. Unlike the rule for other families, homo-oligomeric assemblies formed by only one subunit type result in functional channels in heterologous expression systems. It is thought that P2X receptors at some locations are formed by such homomeric channels. The macroscopic properties of homo-oligomeric receptors are widely different. P2X1 and P2X3 subunits form rapidly-inactivating channels activated by the non-hydrolizable ATP analog α,β-meATP (Valera et al., 1994, Chen et al., 1995, Lewis et al., 1995). P2X2-composed receptors are not activated by α,β-meATP and elicit almost non-inactivating inward currents (Brake et al., 1994). The P2 antagonists suramin and PPADS block these three receptors. On the contrary, P2X4 and P2X6 are not sensitive to these antagonists and show intermediate inactivation (Bo et al., 1995, Buell et al., 1996b, Collo et al., 1996, Séguéla et al., 1996, Soto et al., 1996). In addition to homomeric channels P2X receptors may be formed by hetero-oligomerization of different subunits. P2X receptors present in sensory ganglia seem to be formed by P2X2 and P2X3 subunit co-assembly. The properties of these channels are a mix of the properties of contributing subunits: sensory neurons display slowly inactivating, H+-enhanced (P2X2) ATP receptors sensitive to α,β-meATP (P2X1) as an agonist (Chen et al., 1995, Lewis et al., 1995, Stoop et al., 1997). This suggests that the pharmacological and kinetic properties of each subunit are somewhat intrinsic features of the protein that can be used to identify the subunit composition of endogenous ATP receptors.
The expression of P2X3 and P2X5 subunits is highly localized in nervous tissue. They are found only in ganglionic sensory neurons and equivalent locations, such as the mesencephalic trigeminal nucleus (Collo et al., 1996, Vulchanova et al., 1997). However, other P2X subunits are widely distributed throughout the CNS. The more abundantly expressed mRNAs are those of P2X4 and P2X6 subunits (Collo et al., 1996). They are found from the cortex to the midbrain, brain stem nuclei and the spinal cord. Particularly rich levels of expression are found in the pyramidal cell layer of the hippocampus and in the Purkinje cell layer of the cerebellar cortex. The distribution of these two subunits overlaps widely. Using antibodies directed against the C-terminal region of the P2X4 protein, a predominantly postsynaptic location has been observed, except for the olfactory bulb and spinal cord, where P2X4 may also reside in presynaptic structures (Lê et al., 1998). P2X1 and P2X2 subunits are also widely expressed in the CNS, although in much lower levels in adults. Higher expression of the mRNA of these subunits is found in neonatal animals (Kidd et al., 1995). Immunolocalization of the P2X2 protein in adult rats suggests that the expression of P2X2 subunits may be more important than what has been assumed from mRNA data (Kanjhan et al., 1996). Furthermore, the heterogeneity of P2X proteins may be increased by alternative splicing. At least three additional isoforms of the P2X2 subunit (P2X2(b)-P2X2(d)) and one of the human P2X4 subunit have been described (Simon et al., 1997, Dhulipala et al., 1998). The overlapping distribution of several P2X subunits raises questions about the nature of the endogenously assembled P2X receptors. As an example, the expression of P2X1, P2X2, P2X4 and P2X6 subunits has been found in cerebellar cortex (Kidd et al., 1995, Collo et al., 1996, Kanjhan et al., 1996). Do all these subunits contribute to an homogeneous population of hetero-oligomeric receptors? or do several separate P2X receptor populations coexist?. We have recently shown the presence of functional ATP-gated channels coupled to [Ca2+]i signaling in cerebellar Purkinje neurons (Mateo et al., 1998). Here we study the pharmacological properties of endogenous ATP receptors in these cells to identify the subunits contributing to them.
Section snippets
Cultures of rat cerebellar neurons
Cerebellar cells were obtained from 7–8 day-old Wistar rat pups as described previously. Briefly 8–10 cerebella were pooled in isolation buffer (composition in mM: NaCl, 130; KCl, 4; Na2HPO4, 10; MgSO4, 1.5; HEPES, 10; glucose, 15; bovine serum albumin, 0.05; pH 7.4, plus penicillin, 50 U/ml, and streptomycin, 50 μg/ml), cut into small pieces and incubated for 20 min at 37°C with 0.25 mg/ml trypsin. Digestion was stopped by addition of isolation buffer containing 0.25 mg/ml soybean trypsin
Agonistic effect of ATP analogs
Individual cells were selected for [Ca2+]i recording based on a set of morphological features described by Mateo et al. (1998) and were positively identified as Purkinje neurons by specific immunolabeling after the [Ca2+]i experiments. Previously we had shown that anti-calbindin antibodies labeled cells with the same morphological appearance as those cells used for [Ca2+]i recording but we did not identify individual recorded cells. Here we unequivocally identify, in a cell-by-cell basis, each
Discussion
Cerebellar cultures are very heterogeneous, they contain mostly granule cells but also other types of interneurons, in addition to Purkinje cells. Furthermore, postnatally-obtained Purkinje cells do not develop easily in culture, hindering their identification. Despite this heterogeneity, immunolabeling of identified cells has allowed us to unequivocally ascribe the observed properties to Purkinje neurons. These cells show the highest levels of expression of mRNA for antagonist-insensitive P2X4
Acknowledgements
This work was funded by grants from DGICYT, Spain (PM 95-0072), from the European Union BIOMED 2 programme (UE 96-0012) and from Ramón Areces Foundation (Spain). M.G-L. holds a predoctoral fellowship from Ramón Areces Foundation.
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