Chaoying Li

Ethanol-induced inhibition of a neuronal P2X purinoceptor by an allosteric mechanism

Ethanol inhibits a neuronal P2X purinoceptor by shifting the ATP concentration–response curve to the right in an apparently competitive manner. However, the underlying mechanism has not been determined. We investigated the effects of ethanol on the activation and deactivation time constants for ATP‐activated current in bullfrog dorsal root ganglion neurones. Ethanol decreased the time constant of deactivation of ATP‐gated ion channels without affecting the time constant of activation. The observations are not consistent with a competitive mechanism of inhibition by ethanol, but may be explained by an allosteric action of ethanol to decrease apparent agonist affinity. This represents a novel mechanism of action of ethanol on a neurotransmitter‐gated ion channel.

Distinct ATP-activated currents in different types of neurons dissociated from rat dorsal root ganglion

Rat dorsal root ganglion neurons can be classified into at least three distinct groups based on cell size, afferent fiber diameter, electrophysiological properties, sensitivity to vanilloid agonists such as capsaicin, and function. In the present study, ATP-activated current in these neurons was characterized using whole-cell patch-clamp recording. Small diameter (<30 microm) cells had high capsaicin sensitivity, high affinity for ATP, and rapidly desensitizing ATP-activated current. Medium diameter (30-50 microm) cells had no capsaicin sensitivity, lower affinity for ATP and slowly desensitizing ATP-activated current. Large diameter (>50 microm) cells were insensitive to both capsaicin and ATP. These findings suggest that distinct types of ATP receptor-ion channels are expressed in different types of dorsal root ganglion neurons, and may contribute to the functional differences among these types of neurons.

Inhibition of ATP‐activated current by zinc in dorsal root ganglion neurones of bullfrog

1 The effect of Zn2+ on ATP‐activated current was studied in bullfrog dorsal root ganglion (DRG) neurones using the whole‐cell patch‐clamp technique. 2 Zn2+ (2–800 μm) inhibited current activated by submaximal concentrations of ATP. The Zn2+ concentration that produced 50% inhibition (IC50) of current activated by 2.5 μm ATP was 61 ± 9.8 μm. When ATP concentrations were adjusted to account for chelation of Zn2+, the IC50 of Zn2+ was 86 ± 18 μm. 3 The inhibitory action of Zn2+ on ATP‐gated channels did not appear to be due to a decrease in the concentration of one or more species of ATP. 4 Zn2+ inhibition of ATP‐activated current was independent of membrane potential between −80 and +40 mV, and did not involve a shift in the reversal potential of the current. 5 Zn2+ (100 μm) shifted the ATP concentration‐response curve to the right in a parallel manner, increasing the EC50 for ATP from 2.5 ± 0.5 μm to 5.5 ± 0.4 μm. 6 Zn2+ decreased the time constant of deactivation of ATP‐gated ion channels without affecting the time constant of activation or desensitization. 7 Dithiothreitol (DTT) reversed Zn2+ inhibition of ATP‐activated current. 8 2‐Methylthio ATP, α,β‐methylene ATP and ADP activated current with EC50 values of 2.4 ± 0.3, 50.1 ± 5.8 and 303.1 ± 53.9 μm, respectively. Adenosine, AMP or β,γ‐methylene ATP did not evoke detectable current. 9 Reactive Blue 2 and pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid inhibited ATP‐activated current. 10 The results suggest that Zn2+ can inhibit P2X purinoceptor function by decreasing the affinity of the binding site for ATP. These observations provide the first evidence for this action of Zn2+ on a neurotransmitter‐gated ion channel. Furthermore, the receptor‐channel in these neurones appears to be a novel member of the P2X purinoceptor class.

Inhibition by ethanol of rat P2X4 receptors expressed in Xenopus oocytes

The effect of ethanol on the function of P2X4 receptors expressed in Xenopus oocytes was studied using two‐electrode voltage‐clamp recording. The amplitude of current activated by 1 μM ATP was decreased by ethanol in a concentration‐dependent manner over the concentration range 1–500 mM. The concentration of ethanol that produced 50% inhibition (IC50) of current activated by 1 μM ATP was 58 mM. Ethanol inhibition of ATP‐activated current was not dependent on membrane potential from −60 to +20 mV, and ethanol did not change the reversal potential of ATP‐activated current. Ethanol, 50 mM, shifted the ATP concentration‐response curve to the right, increasing the EC50 for ATP from 9.1 to 16.0 μM, but did not reduce the maximal response to ATP. The results suggest that ethanol may inhibit P2X4 receptors by decreasing the apparent affinity of the binding site for ATP. Since the P2X4 receptor is the most abundant P2X subunit in the brain, these receptors could be important effectors of ethanol action in the central nervous system.

ENHANCEMENT OF ATP-ACTIVATED CURRENT BY PROTONS IN DORSAL ROOT GANGLION NEURONS

Abstract The effect of pH on ATP-activated current in bullfrog dorsal root ganglion neurons was studied using the whole-cell patch-clamp technique. ATP-activated current amplitude was highly dependent upon extracellular pH. An acid pH increased, whereas alkaline pH decreased, ATP-activated current amplitude. The half-maximal pH (EC50) for potentiation of 2.5 μM ATP-activated current was 7.2. Acidification alone did not activate detectable current and, at an acid pH, ATP-activated current was abolished by suramin. Proton-induced enhancement of ATP-activated current was not sensitive to membrane potential between –80 and +40 mV, and did not involve a shift in reversal potential. Lowering pH from 7.2 to 6.5 or elevating pH from 7.2 to 8.0 shifted the ATP concentration/response curve to the left or right, respectively, without changing the maximal response to ATP. Protons increased the time constant of deactivation without affecting the time constant of activation or desensitization of ATP-activated current. Alteration of patch-pipette (intracellular) pH did not affect the enhancement of ATP-activated current by extracellular protons. Diethylpyrocarbonate (DEP), dithiothreitol (DTT), 5,5′-dithio-bis-(2-nitro-benzoic acid) (DTNB), or N-ethylmaleimide (NEM) did not affect enhancement of ATP-activated current by protons. The results suggest that extracellular protons, at physiological concentrations, can regulate the function of P2X purinoceptors by modulating the affinity of the ATP-binding site.

Inhibition of NMDA‐gated ion channels by the P2 purinoceptor antagonists suramin and reactive blue 2 in mouse hippocampal neurones

The action of suramin and reactive blue 2 on N‐methyl‐D‐aspartate (NMDA)‐activated ion current was studied in mouse hippocampal neurones in culture by use of whole‐cell patch‐clamp recording. Suramin and reactive blue 2 inhibited steady‐state current activated by 25 μM NMDA with IC50 values of 68 and 11 μM, respectively. Reactive blue 2 produced a gradual decline of NMDA‐activated current to a steady‐state, but this slow onset was not an indication of use‐dependence, as it could be eliminated by exposure to reactive blue 2 before NMDA application. In addition, NMDA‐activated current recovered completely from inhibition by reactive blue 2 in the absence of agonist. The slow onset of inhibition by reactive blue 2 was not apparently due to an action at an intracellular site, as inclusion of 250 μM reactive blue 2 in the recording pipette did not alter inhibition by 25 μM reactive blue 2 applied externally. Reactive blue 2 and suramin inhibited NMDA‐gated channels in a voltage‐independent manner. Reactive blue 2, 25 μM, decreased the maximal response to NMDA from 1441 to 598 pA without changing its EC50. In contrast, 75 μM suramin increased the EC50 for NMDA from 13 to 35 μM, and decreased the maximal response to NMDA from 1822 to 1498 pA. Schild analysis of suramin inhibition of NMDA‐activated current yielded a nonlinear plot. Both agents decreased the maximal response to glycine without altering its EC50. Suramin and reactive blue 2 appear to inhibit NMDA receptor‐channels in a manner that is noncompetitive with respect to both NMDA and glycine. However, inhibition by suramin differed from that by reactive blue 2, in that suramin significantly increased the EC50 of NMDA.

Cu2+ potently enhances ATP-activated current in rat nodose ganglion neurons.

Several lines of evidence suggest a physiological role for Cu2+ in regulating nervous system function. In the present study using whole-cell patch-clamp recording, Cu2+ greatly enhanced current activated by 10 microM ATP in the majority of rat nodose ganglion neurons. The enhancement was concentration-dependent between 1 and 50 microM Cu2+, and had an EC50 of 6.1 microM. Cu2+ shifted the ATP concentration-response curve to the left in a parallel manner. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally-effective concentration of Zn2+. The observations suggest that Cu2+ increases the affinity of the receptor for ATP by acting at the Zn2+ modulatory site. In addition, a subset of neurons in the nodose ganglion express ATP-gated receptor-channels that are insensitive to modulation by physiological concentrations of Cu2+, Zn2+ and protons.

The mechanism by which ethanol inhibits rat P2X4 receptors is altered by mutation of histidine 241

1 We investigated ethanol inhibition of the rat P2X4 receptor and the contribution of the three histidine residues in the extracellular loop of this receptor to ethanol inhibition of receptor function, using site‐directed mutagenesis and electrophysiological characterization of recombinant receptors. 2 In the wild‐type receptor, 50, 200 and 500 mM ethanol increasingly shifted the ATP concentration–response curve to the right in a parallel manner, increasing the EC50 value without affecting Emax. However, 750 or 900 mM ethanol did not produce a further increase in the EC50 value of the ATP concentration–response curve, suggesting that this inhibition is not competitive. 3 The P2X4 receptor mutations H140A and H286A did not significantly alter ethanol inhibition of ATP‐activated current. By contrast, the mutation H241A changed the mechanism by which ethanol inhibits receptor function; viz., ethanol inhibition was not associated with an increased EC50 value of the ATP concentration–response curve, instead, ethanol decreased the maximal response to ATP without affecting the EC50 value of the ATP concentration–response curve. 4 Ethanol inhibition of the H241A mutant was voltage independent between −60 and +20 mV and ethanol did not alter the reversal potential of ATP‐activated current. In addition, ethanol decreased the desensitization rate of the H241A‐mediated current. 5 The purinoceptor antagonists, suramin and pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), did not alter the magnitude of ethanol inhibition of ATP‐activated current in the H241A mutant. 6 The results suggest that ethanol inhibits the wild‐type rat P2X4 receptor by an allosteric action to increase the EC50 value of the ATP concentration–response curve, the P2X4 receptor mutation H241A alters the mechanism by which ethanol inhibits P2X4 receptor function, and ethanol and PPADS or suramin appear to inhibit H241A‐mutated receptors at independent sites.

Phosphorylation of the GABAA receptor γ2L subunit in rat sensory neurons may not be necessary for ethanol sensitivity

The effect of ethanol on the current activated by 2.5 to 40 microM gamma-aminobutyric acid (GABA) was studied in freshly isolated rat dorsal root ganglion (DRG) neurons under voltage clamp in the whole-cell and perforated-patch recording configurations. Our results confirmed that GABAA-activated current in these neurons was insensitive to ethanol at concentrations from 2.5 to 100 mM [G. White, D.M. Lovinger, F.F. Weight, Ethanol inhibits NMDA-activated current but does not alter GABA-activated current in an isolated adult mammalian neuron, Brain Res. 507 (1990) 332-336.]. In addition, the ethanol sensitivity of GABA receptors was studied under conditions that promote phosphorylation of the PKC site on the gamma2L subunit. The presence of the gamma2L and other subunit mRNAs was detected by reverse transcription (RT) of total RNA purified from adult DRG followed by polymerase chain reaction (PCR) using subunit specific primer sets. We found that the GABA response remained insensitive to 2.5-100 mM ethanol despite: (i) the extracellular preapplication of 5, 20 or 500 nM phorbol 12-myristate 13-acetate (PMA); (ii) raising free intracellular Ca2+ ([Ca2+]i) from 7 to 100 or 600 nM by altering the intracellular Ca2+/EGTA ratio; (iii) intracellular application of PKC (0.247 U ml-1 ); and (iv) combining the intracellular application of 1 microM okadaic acid and 30 microM peptide 3 with the extracellular application of 20 nM PMA. These results suggest that phosphorylation of the gamma2L subunit is not the only requirement for ethanol sensitivity of GABAA receptors.

1,9-Dideoxyforskolin does not mimick all cAMP and protein kinase a independent effects of forskolin on GABA activated ion currents in adult rat sensory neurons

The effect of forskolin on GABAA receptor activated events has been the subject of recent investigations, the conclusions of which are conflicting. Forskolin can reduce current amplitude and increase the rate of decay of current activated by 100 microM GABA and these effects are not mimicked by 1,9-dideoxyforskolin (Tehrani et al., Synapse, 4 (1989) 126-131). On the other hand, both forskolin and 1,9-dideoxyforskolin inhibit 36Cl- flux induced by lower concentrations of muscimol (Heuschneider and Schwartz, Proc. Natl. Acad. Sci. USA, 86 (1989) 2938-2942). Using the whole-cell patch clamp technique to measure GABA activated current in dorsal root ganglion neurons that were freshly isolated from adult rats, we have confirmed the finding of Tehrani et al. (Synapse, 4 (1989) 126-131) using 100 microM GABA; however, the effects of forskolin that were not mimicked by 1,9-dideoxyforskolin were not blocked by the kinase inhibitor H-7 (50 microM). In contrast, at lower concentrations of GABA (10-20 microM), both forskolin and 1,9-dideoxyforskolin increased the decay rate of GABA activated current. In addition, all effects of forskolin occurred within 200 ms of application of forskolin and the effects were not blocked or occluded by H-7, 10 microM cAMP, or the active subunit of protein kinase A. We conclude that: (1) 1,9-dideoxyforskolin is not a reliable indicator of forskolin specificity in this system because its effects are dependent upon GABA concentration; and (2) the most prominent effects of forskolin on amplitude and decay time course of GABA activated ion current are not mediated by cAMP or protein kinase A (PKA).