Conserved extracellular cysteines differentially regulate the potentiation produced by Zn2+ in rat P2X4 receptors.

One feature of the amino acid sequence of P2X receptors identified from mammalian species, Xenopus laevis and zebrafish is the conservation of ten cysteines in the extracellular loop. Little information is available about the role of these conserved ectodomain cysteines in the function of P2X receptors. Here, we investigated the possibility that ten conserved cysteine residues in the extracellular loop of the rat P2X4 receptor may regulate zinc potentiation of the receptor using a series of individual cysteine to alanine point mutations and functional characterization of recombinant receptors expressed in Xenopus oocytes. For the C116A, C132A, C159A, C165A, C217A and C227A mutants, 10 µM zinc did not significantly affect the current activated by an EC40 concentration of ATP. By contrast, 5 µM zinc shifted the ATP concentration-response curve to the right in a parallel manner for both the C261A and C270A mutants and the magnitudes of those shifts were similar to that of the wildtype receptor. Interestingly, for the C126A and C149A mutants, 5µM zinc potentiated ATP-activated current, but increased the maximal response to ATP by 90% and 81% respectively, without significantly changing the EC50 value of ATP. Thus, these results suggest that cysteines and disulfide bonds between cysteines are differentially involved in the potentiation of the rat P2X4 receptor by zinc.

Conserved extracellular cysteines differentially regulate the inhibitory effect of ethanol in rat P2X4 receptors.

Relatively little information is available about the molecular mechanism of ethanol inhibition of P2X receptors. Here, we investigated the possibility that 10 conserved cysteine residues in the extracellular loop of the rat P2X4 receptor may regulate ethanol inhibition of the receptor using a series of individual cysteine to alanine point mutations. Each of the mutated receptors generated robust inward current in response to ATP and the mutations produced less than a sixfold change in the ATP EC50 value. For the C116A, C126A, C149A, and C165A mutants, 100 mM ethanol did not significantly affect the current activated by an EC40 concentration of ATP. By contrast, for the C261A and C270A mutants, ethanol inhibited ATP-activated current in a competitive manner similar to that for the wild-type receptor. Interestingly, for the C132A, C159A, C217A, and C227A mutants, ethanol inhibited ATP-activated current, but decreased the maximal response to ATP by 70-75% without significantly changing the EC50 value of ATP, thus exhibiting a noncompetitive-type inhibition. The results suggest that cysteines and disulfide bonds between cysteines are differentially involved in the inhibition of the rat P2X4 receptor by ethanol.

Activation of nicotinic acetylcholine receptors increases the frequency of spontaneous GABAergic IPSCs in rat basolateral amygdala neurons.

The basolateral amygdala (BLA) is a critical component of the amygdaloid circuit, which is thought to be involved in fear conditioned responses. Using whole cell patch-clamp recording, we found that activation of nicotinic acetylcholine receptors (nAChRs) leads to an action potential-dependent increase in the frequency of spontaneous GABAergic currents in principal neurons in the BLA. These spontaneous GABAergic currents were abolished by a low-Ca2+/high-Mg2+ bathing solution, suggesting that they are spontaneous inhibitory postsynaptic currents (sIPSCs). Blockade of ionotropic glutamate receptors did not prevent this increased frequency of sIPSCs nor did blockade of alpha7 nAChRs. Among the nAChR agonists tested, cystisine was more effective at increasing the frequency of the sIPSCs than nicotine or 1,1-dimethyl-4-phenyl piperazinium iodide, consistent with a major contribution of beta4 nAChR subunits. The nicotinic antagonist, dihydro-beta-erythroidine, was less effective than d-tubocurarine in blocking the increased sIPSC frequency induced by ACh, suggesting that alpha4-containing nAChR subunits do not play a major role in the ACh-induced increased sIPSC frequency. Although alpha2/3/4/7 and beta2/4 nAChR subunits were found in the BLA by RT-PCR, the agonist and antagonist profiles suggest that the ACh-induced increase in sIPSC frequency involves predominantly alpha3beta4-containing nAChR subunits. Consistent with this, alpha-conotoxin-AuIB, a nAChR antagonist selective for the alpha3beta4 subunit combination, inhibited the ACh-induced increase in the frequency of sIPSCs. The observations suggest that nicotinic activation increases the frequency of sIPSCs in the BLA by acting mainly on alpha3beta4-containing nicotinic receptors on GABAergic neurons and may play an important role in the modulation of synaptic transmission in the amygdala.

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

1. We investigated ethanol inhibition of the rat P2X(4) 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 EC(50) value without affecting E(max). However, 750 or 900 mM ethanol did not produce a further increase in the EC(50) value of the ATP concentration-response curve, suggesting that this inhibition is not competitive. 3. The P2X(4) 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 EC(50) value of the ATP concentration-response curve, instead, ethanol decreased the maximal response to ATP without affecting the EC(50) 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 P2X(4) receptor by an allosteric action to increase the EC(50) value of the ATP concentration-response curve, the P2X(4) receptor mutation H241A alters the mechanism by which ethanol inhibits P2X(4) receptor function, and ethanol and PPADS or suramin appear to inhibit H241A-mutated receptors at independent sites.

Role of extracellular histidines in antagonist sensitivity of the rat P2X4 receptor.

The pharmacological property that most distinguishes rat P2X4 receptors from other P2X receptors is their insensitivity to the purinoceptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). The molecular basis of this insensitivity is not known. Here, we investigated the possibility that histidine residues in the extracellular loop of P2X4 receptors may be involved in the antagonist sensitivity of these receptors. We found that histidine mutation H241A in the rat P2X4 receptor produced receptors that are sensitive to suramin and PPADS. In contrast, mutation H140A or H286A did not significantly alter antagonist sensitivity. In addition, mutation H241A in the human P2X4 receptor significantly increased antagonist sensitivity. The results suggest that histidine 241of P2X4 receptors is involved in regulating the antagonist sensitivity of these receptors.

Role of extracellular histidines in agonist sensitivity of the rat P2X4 receptor.

Relatively little information is available about the relationship between the molecular structure of each of the seven subtypes of P2X receptors and their function. Here, we investigated the possible function of three histidine residues in the extracellular loop of rat P2X(4) receptors. Mutation of histidine 241 to alanine (H241A) in the rat P2X(4) receptor decreased the EC(50) value of the ATP concentration-response curve from 8.4 to 0.7 microM. In contrast, the histidine mutation H140A or H286A slightly increased the EC(50) value. Maximal current responses were significantly larger in oocytes expressing rat H241A-mutated receptors compared to those expressing wildtype, H140A or H286A receptors. In addition, significantly less receptor protein was detected in H241A-expressing oocytes than in oocytes expressing wildtype, H140A or H286A receptors. Moreover, ATP-activated current in H241A-expressing cells activated faster than in wildtype receptor-expressing cells. The increased maximal current amplitude, the decrease in protein expression and the more rapid activation kinetics suggest that the H241A mutation facilitates opening of the receptor-channel (gating).

Arginine 222 in the pre-transmembrane domain 1 of 5-HT3A receptors links agonist binding to channel gating.

Ligand-gated ion channels are integral membrane proteins that mediate fast synaptic transmission. Molecular biological techniques have been extensively used for determining the structure-function relationships of ligand-gated ion channels. However, the transduction mechanisms that link agonist binding to channel gating remain poorly understood. Arginine 222 (Arg-222), located at the distal end of the extracellular N-terminal domain immediately preceding the first transmembrane domain (TM1), is conserved in all 5-HT3A receptors and alpha7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of Arg-222 in the function of 5-HT3A receptors, we mutated the arginine residue to alanine (Ala) and expressed both the wild-type and the mutant receptor in human embryonic kidney 293 cells. Functional studies of expressed wild-type and mutant receptors revealed that the R222A mutation increased the apparent potency of the full agonist, serotonin (5-HT), and the partial agonist, 2-Me-5-HT, 5- and 12-fold, respectively. In addition, the mutation increased the efficacy of 2-Me-5-HT and converted it from a partial agonist to a full agonist. Furthermore, this mutation also converted the 5-HT3 receptor antagonist/very weak partial agonist, apomorphine, to a potent agonist. Kinetic analysis revealed that the R222A mutation increased the rate of receptor activation and desensitization but did not affect rate of deactivation. The results suggest that the pre-TM1 amino acid residue Arg-222 may be involved in the transduction mechanism linking agonist binding to channel gating in 5-HT3A receptors.