In the nervous system purinergic excitatory synapses use ATP to mediate fast synaptic transmission via activation of P2X receptor cation channels. In sensory neurons the ATP-induced current is mediated primarily by homo- and heteromeric P2X2 and P2X3 receptors (Dunn et al. 2001). We have recently shown that currents mediated by homomeric P2X2 receptors are attenuated under hypoxic conditions (Mason et al. 2003). This study examined the effect of hypoxia on homomeric P2X3 and heteromeric P2X2/3 receptors.
Whole-cell currents were recorded from P2X2/3 or P2X3 receptors stably expressed in HEK293 cells (Kawashima et al. 1998). Pipettes were filled with (mM): 10 NaCl, 117 KCl, 2 MgSO4, 1 CaCl2, 11 EGTA, 2 Na-ATP, 11 Hepes (pH 7.2), and cells were continuously perfused with 135 NaCl, 5 KCl, 1 EGTA, 5 Hepes and 10 glucose (pH 7.4). Cells were held at a potential of -70 mV and the cells perfused with either ATP or the P2X3-selective agonist α,β-MeATP. Data are given as means ± S.E.M. and statistical analysis was performed using Student’s paired or unpaired t test with P < 0.05 regarded as significant.
Perfusion of 0.5 µM ATP induced an inward current in cells expressing the P2X2/3 receptor that showed little desensitization during repeated exposures under normoxic conditions (-70.0 ± 7.0 pA pF-1 to -66.4 ± 9.2 pA pF-1, mean of first three and last three exposures respectively, n = 5). Exposure to a hypoxic ATP (0.5 µM) solution (PJ{special} 25 mm Hg, EC solution bubbled with 100 % N2) reduced the mean whole-cell current density from -100.8 ± 24.4 pA pF-1 to -62.8 ± 13.8 pA pF-1 (P < 0.05, n = 5). Similar results were obtained with hypoxic 10 µM α,β-MeATP (-32.7 ± 11.5 pA pF-1 to -19.5 ± 6.6 pA pF-1, P < 0.05, n = 6). In contrast, in cells expressing the P2X3 receptor, perfusion of ATP induced a rapidly desensitizing inward current. The magnitudes of the ATP-induced current densities were similar under normoxic (-18.7 ± 6.2 pA pF-1, n = 8) and hypoxic (-24.4 ± 8.9 pA pF-1, n = 8) conditions. Similar results were obtained with 10 µM α,β-MeATP (normoxic -18.7 ± 5.9 pA pF-1, n = 8; hypoxic -19.7 ± 11.2 pA pF-1, n = 8).
In summary, this is the first report indicating that hypoxia selectively modulates the response of P2X receptors to ATP and suggests that the presence of the P2X2 receptor subunit in either homo- or heteromeric receptor channels is critical in mediating the response to hypoxia in the nervous system.
The authors would like to thank R.A. North (Institute of Molecular Physiology, University of Sheffield) for kindly providing us with the P2X stable cell lines. This work was funded by the Welcome Trust 065887/Z/01/Z.