Bimodal Action of Protons on ATP Currents of Rat PC12 Cells

The mode of action of extracellular protons on ATP-gated P2X2 receptors remains controversial as either enhancement or depression of ATP-mediated currents has been reported. By investigating, at different pH, the electrophysiological effect of ATP on P2X2 receptors and complementing it with receptor modelling, the present study suggests a unified mechanism for both potentiation and inactivation of ATP receptors by protons. Our experiments on patch-clamped PC12 cells showed that, on the same cell, mild acidification potentiated currents induced by low ATP concentrations (<0.1 mM) and attenuated responses to high ATP concentrations (>1 mM) with emergence of current fading and rebound. To clarify the nature of the ATP/H+ interaction, we used the Ding and Sachs's “loop” receptor model which best describes the behavior of such receptors with two open states linked via one inactivated state. No effects by protons could be ascribed to H+-mediated open channel block. However, by assuming that protons facilitated binding of ATP to resting as well as open receptors, the model could closely replicate H+-induced potentiation of currents evoked by low ATP doses plus fading and rebound induced by high ATP doses. The latter phenomenon was due to receptor transition to the inactive state. The present data suggest that the high concentration of protons released with ATP (and catecholamines) from secretory vesicles may allow a dual action of H+ on P2X2 receptors. This condition might also occur on P2X2 receptors of central neurons exposed to low pH during ischemia

Temporal Coding at the Immature Depolarizing GABAergic Synapse

In the developing hippocampus, GABA exerts depolarizing and excitatory actions and contributes to the generation of neuronal network driven giant depolarizing potentials (GDPs). Here, we studied spike time coding at immature GABAergic synapses and its impact on synchronization of the neuronal network during GDPs in the neonatal (postnatal days P2–6) rat hippocampal slices. Using extracellular recordings, we found that the delays of action potentials (APs) evoked by synaptic activation of GABA(A) receptors are long (mean, 65 ms) and variable (within a time window of 10–200 ms). During patch-clamp recordings, depolarizing GABAergic responses were mainly subthreshold and their amplification by persistent sodium conductance was required to trigger APs. AP delays at GABAergic synapses shortened and their variability reduced with an increase in intracellular chloride concentration during whole-cell recordings. Negative shift of the GABA reversal potential (EGABA) with low concentrations of bumetanide, or potentiation of GABA(A) receptors with diazepam reduced GDPs amplitude, desynchronized neuronal firing during GDPs and slowed down GDPs propagation. Partial blockade of GABA(A) receptors with bicuculline increased neuronal synchronization and accelerated GDPs propagation. We propose that spike timing at depolarizing GABA synapses is determined by intracellular chloride concentration. At physiological levels of intracellular chloride GABAergic depolarization does not reach the action potential threshold and amplification of GABAergic responses by non-inactivating sodium conductance is required for postsynaptic AP initiation. Slow and variable excitation at GABAergic synapse determines the level of neuronal synchrony and the rate of GDPs propagation in the developing hippocampus

Agonist-dependence of recovery from desensitization of P2X(3) receptors provides a novel and sensitive approach for their rapid up or downregulation

1. Fast-desensitizing P2X(3) receptors of nociceptive dorsol root ganglion (DRG) neurons are thought to mediate pain sensation. Since P2X(3) receptor efficiency is powerfully modulated by desensitization, its underlying properties were studied with patch-clamp recording. 2. On rat cultured DRG neurons, 2 s application of ATP (EC(50)=1.52 μM), ADP (EC(50)=1.1 μM) or α,β-meATP (EC(50)=1.78 μM) produced similar inward currents that fully desensitized, at the same rate, back to baseline. Recovery from desensitization was much slower after ATP and ADP than after α,β-meATP and, in all cases, it had sigmoidal time course. 3. By alternating the application of ATP and α,β-meATP, we observed complete cross-desensitization indicating that these agonists activated the same receptors. This notion was confirmed by the similar antagonism induced by 2′, 3′-O-(2,4,6,trinitrophenyl)-adenosine triphosphate (TNP-ATP). 4. Recovery from desensitization elicited by ATP was unexpectedly shaped by transient application of α,β-methylene-adenosine triphosphate (α,β-meATP), and vice versa. Thus, short-lasting, full desensitization produced by α,β-meATP protected receptors from long-lasting desensitization induced by subsequent ATP applications. ATP and ADP had similar properties of recovery from desensitization. 5. Low nM concentrations of α,β-meATP (unable to evoke membrane currents) could speed up recovery from ATP-induced desensitization, while low nM concentrations of ATP enhanced it. Ambient ATP levels were found to be in the pM range (52±3 pM). 6. The phenomenon of cross-desensitization and protection was reproduced by rP2X(3) receptors expressed by rat osteoblastic cell 17/2.8 or human embryonic kidney cell 293 cells, indicating P2X(3) receptor specificity. 7. It is suggested that transient application of an agonist that generates rapid recovery from desensitization, is a novel, powerful tool to modulate P2X(3) receptor responsiveness to the natural agonist ATP

Rapid relief of block by mecamylamine of neuronal nicotinic acetylcholine receptors of rat chromaffin cells in vitro: an electrophysiological and modeling study

The mechanism responsible for the blocking action of mecamylamine on neuronal nicotinic acetylcholine receptors (nAChRs) was studied on rat isolated chromaffin cells recorded under whole-cell patch clamp. Mecamylamine strongly depressed (IC50 = 0.34 mu M) inward currents elicited by short pulses of nicotine, an effect slowly reversible on wash. The mecamylamine block was voltage-dependent and promptly relieved by a protocol combining membrane depolarization with a nicotine pulse. Either depolarization or nicotine pulses were insufficient per se to elicit block relief. Block relief was transient; response depression returned in a use-dependent manner. Exposure to mecamylamine failed to block nAChRs if they were not activated by nicotine or if they were activated at positive membrane potentials. These data suggest that mecamylamine could not interact with receptors either at rest or at depolarized level. Other nicotinic antagonists like dihydro-beta-erythroidine or tubocurarine did not share this action of mecamylamine although proadifen partly mimicked it. Mecamylamine is suggested to penetrate and block open nAChRs that would subsequently close and trap this antagonist. Computer modeling indicated that the mechanism of mecamylamine blocking action could be described by assuming that 1) mecamylamine-blocked receptors possessed a much slower, voltage-dependent isomerization rate, 2) the rate constant for mecamylamine unbinding was large and poorly voltage dependent. Hence, channel reopening plus depolarization allowed mecamylamine escape and block relief. In the presence of mecamylamine, therefore, nAChRs acquire the new property of operating as coincidence detectors for concomitant changes in membrane potential and receptor occupancy