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Ligand-gated ion channels underlie and play important roles in synaptic transmission, and it is generally accepted that the ion channel pores have a rigid structure that enables strict regulation of ion permeation. One exception is the P2X ATP-gated channel. After application of ATP, the ion selectivity of the P2X2 channel time-dependently changes, i.e. permeability to large cations gradually increases, and there is significant cell-to-cell variation in the intensity of inward rectification. Here we show P2X2 channel properties are correlated with the expression level: increasing P2X2 expression level in oocytes increases permeability to large cations, decreases inward rectification and increases ligand sensitivity. We also observed that the inward rectification changed in a dose-dependent manner, i.e. when low concentration of ATP was applied to an oocyte with a high expression level, the intensity of inward rectification of the evoked current was weak. Taken together, these results show that the pore properties of P2X2 channel are not static but change dynamically depending on the open channel density. Furthermore, we identified by mutagenesis study that Ile328 located at the outer mouth of the pore is critical for the density-dependent changes of P2X2. Our findings suggest synaptic transmission can be modulated by the local density-dependent changes of channel properties caused, for example, by the presence of clustering molecules.
The primary structure of P2X receptors was determined by the isolation of the first cDNA by the expression cloning method (Brake et al. 1994; Valera et al. 1994), and seven types of P2X cDNAs have been cloned so far (North & Barnard, 1997; North, 2002). All of them have two transmembrane regions with a large extracellular loop, and the functional unit is reported to be composed of three subunits based on the analysis of the weight of the non-denatured proteins (Nicke et al. 1998). This structure, a trimer of two transmembrane type subunits, is in clear contrast with other ligand-gated channels such as nicotinic acetylcholine (nACh) receptors or glutamate receptors (Khakh, 2001), suggesting the possibility of functional differences.
We initially aimed to identify structural determinants of P2X2 receptors involved in the property of inward rectification and prepared various mutants. However, reliable determination of the mutant phenotype was hard, because the intensity of inward rectification varied significantly even in the wild-type (WT). We later noticed that the variation of the inward rectification intensity was due to a difference in expression level. Khakh et al. (2000) also reported the significance of the expression level for the functional interaction between P2X2 and nACH receptors. Triggered by these observations, we decided to focus on the correlation of the expression density and the channel properties of the P2X2 receptor. In the present study, we used Xenopus oocytes as an expression system, and analysed channel properties such as the ion selectivity, inward rectification and ATP sensitivity by recording ATP-evoked current under two-electrode voltage clamp from oocytes with various express levels.