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Differential Modulation by Copper and Zinc of P2X2 and P2X4 Receptor Function. The modulation by Cu2+ and Zn2+ of P2X2 and P2X4 receptors expressed in Xenopus oocytes was studied with the two-electrode, voltage-clamp technique. In oocytes expressing P2X2 receptors, both Cu2+ and Zn2+, in the concentration range 1-130 microM, reversibly potentiated current activated by submaximal concentrations of ATP. The Cu2+ and Zn2+ concentrations that produced 50% of maximal potentiation (EC50) of current activated by 50 microM ATP were 16.3 +/- 0.9 (SE) microM and 19.6 +/- 1.5 microM, respectively. Cu2+ and Zn2+ potentiation of ATP-activated current was independent of membrane potential between -80 and +20 mV and did not involve a shift in the reversal potential of the current. Like Zn2+, Cu2+ increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally effective concentration of Zn2+, suggesting a common site or mechanism of action of Cu2+ and Zn2+ on P2X2 receptors. For the P2X4 receptor, Zn2+, from 0.5 to 20 microM enhanced current activated by 5 microM ATP with an EC50 value of 2.4 +/- 0.2 microM. Zn2+ shifted the ATP concentration-response curve to the left in a parallel manner, and potentiation by Zn2+ was voltage independent. By contrast, Cu2+ in a similar concentration range did not affect ATP-activated current in oocytes expressing P2X4 receptors, and Cu2+ did not alter the potentiation of ATP-activated current produced by Zn2+. The results suggest that Cu2+ and Zn2+ differentially modulate the function of P2X2 and P2X4 receptors, perhaps because of differences in a shared site of action on both subunits or the absence of a site for Cu2+ action on the P2X4 receptor.

The effect of dynorphin A(1-13) on N-methyl-D-aspartate (NMDA)-activated currents was investigated in the presence of low extracellular glycine concentrations in Xenopus oocytes expressing recombinant heteromeric NMDA receptors and in cultured hippocampal neurons with the use of voltage-clamp techniques. At an extracellular added glycine concentration of 100 nM, dynorphin A(1-13) (10 microM) greatly increased the amplitude of NMDA-activated currents for all heteromeric subunit combinations tested; on average, the potentiation was: epsilon1/zeta1, 3,377 +/- 1,416% (mean +/- SE); epsilon2/zeta1, 1,897 +/- 893%; epsilon3/zeta1, 4,356 +/- 846%; and epsilon4/zeta1, 1,783 +/- 503%. Potentiation of NMDA-activated current by dynorphin A(1-13) was concentration dependent between 0.1 and 10 microM dynorphin A(1-13), with a half-maximal concentration value of 2.77 microM and an apparent Hill coefficient of 2.53, for epsilon2/zeta1 subunits at 100 nM added extracellular glycine. Percentage potentiation by dynorphin A(1-13) was maximal at the lowest glycine concentrations tested (0.01 and 0.1 microM), and decreased with increasing glycine concentration. No significant potentiation was observed at glycine concentrations > 0.1 microM for epsilon1/zeta1, epsilon2/zeta1, and epsilon4/zeta1 subunits, or at > 1 microM for epsilon3/zeta1 subunits. Potentiation of NMDA-activated currents by dynorphin A(1-13) was not inhibited by 1 microM of the kappa-opioid receptor antagonist nor-binaltorphimine, and potentiation was not observed with 10 microM of the kappa-opioid receptor agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzene-acetamide. Potentiation of NMDA-activated current by dynorphin A(1-13) was inhibited by the glycine antagonist kynurenic acid (50 microM). NMDA-activated current was also potentiated at low glycine concentrations by 10 microM dynorphin A(2-13) or (3-13), both of which have a glycine as the first amino acid, but not by 10 microM dynorphin A(4-13), which does not have glycine as an amino acid. In hippocampal neurons, 10 microM dynorphin A(1-13) or (2-13) potentiated steady-state NMDA-activated current in the absence of added extracellular glycine. The extracellular free glycine concentration, determined by high-performance liquid chromatography, was between 26 and 36 nM for the bathing solution in presence or absence of 10 microM dynorphin A(1-13), (2-13), (3-13), or (4-13), and did not differ significantly among these solutions. The observations are consistent with the potentiation of NMDA-activated current at low extracellular glycine concentrations resulting from an interaction of the glycine amino acids in dynorphin A(1-13) with the glycine coagonist site on the NMDA receptor. Because dynorphin A is an endogenous peptide that can be coreleased with glutamate at glutamatergic synapses, the potentiation of NMDA receptor-mediated responses could be an important physiological regulator of NMDA receptor function at these synapses.

The effect of Mg2+ on ATP-activated current in rat nodose ganglion neurons was investigated with the use of the whole cell patch-clamp technique. Mg2+ decreased the amplitude of ATP-activated current in a concentration-dependent manner over the concentration range of 0.25-8 mM, with a 50% inhibitory concentration value of 1.5 mM for current activated by 10 microM ATP. Mg2+ shifted the ATP concentration-response curve to the right in a parallel manner, increasing the 50% effective concentration value for ATP from 9.2 microM in the absence of added Mg2+ to 25 microM in the presence of 1 mM Mg2+. Mg2+ increased the deactivation rate of ATP-activated current without changing its activation rate. The observations are consistent with an action of Mg2+ to inhibit ATP-gated ion channel function by decreasing the affinity of the agonist binding site on these receptors.

1. The modulation by protons of ATP-gated ion channel responses to ATP and Zn2+ was studied in freshly isolated rat nodose ganglion neurons using the whole cell patch-clamp technique. 2. Reduced external pH enhanced, whereas elevated external pH suppressed, current activated by 10 microM ATP. The pH producing the half-maximal effect (EC50) at this ATP concentration was 7.1. 3. Acidification shifted the ATP concentration-response curve to the left, decreasing the EC50 for ATP, and alkalinization shifted the ATP concentration-response curve to the right, increasing the EC50 for ATP. Fitting the data to a single-site pH model yielded an apparent pKa of the site on the ATP-gated ion channel of 7.6. Between pH 6.8 and 7.8, a change of 0.1 pH unit was calculated to change the ATP EC50 by 4.03 microM. Changing pH did not alter the maximal response to ATP. 4. The potentiating effect of protons appeared to be due to a direct action on the ATP-gated channel, as it could not be explained by an increase in the concentration of one or more species of ATP. 5. Lowering pH also increased the potency of Zn2+ for enhancement of ATP-activated current without altering its maximal response. Changing the pH from 7.3 to 6.8 changed the Zn2+ EC50 from 12 to 1.7 microM. 6. The potentiation of ATP-activated current by protons could not be attributed solely to an increase in the affinity of the receptor for Zn2+, as the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine did not alter the effect of protons. 7. Protons and Zn2+ do not appear to act at the same site on ATP-gated channels, as responses to maximally effective concentrations of Zn2+ were enhanced further by protons and vice versa. 8. These results suggest that protons regulate the function of P2X purinoceptors in rat nodose ganglion neurons by modulating the affinity of the binding sites for ATP and Zn2+ on these receptor channels.