Cysteine 893 is a target of regulatory thiol modifications of GluA1 AMPA receptors

Recent studies indicate that glutamatergic signaling involves, and is regulated by, thiol modifying and redox-active compounds. In this study, we examined the role of a reactive cysteine residue, Cys-893, in the cytosolic C-terminal tail of GluA1 AMPA receptor as a potential regulatory target. Elimination of the thiol function by substitution of serine for Cys-893 led to increased steady-state expression level and strongly reduced interaction with SAP97, a major cytosolic interaction partner of GluA1 C-terminus. Moreover, we found that of the three cysteine residues in GluA1 C-terminal tail, Cys-893 is the predominant target for Snitrosylation induced by exogenous nitric oxide donors in cultured cells and lysates. Co-precipitation experiments provided evidence for native association of SAP97 with neuronal nitric oxide synthase (nNOS) and for the potential coupling of Ca2+- permeable GluA1 receptors with nNOS via SAP97. Our results show that Cys-893 can serve as a molecular target for regulatory thiol modifications of GluA1 receptors, including the effects of nitric oxide.Peer reviewe

Intersubunit Interactions at Putative Sites of Ethanol Action in the M3 and M4 Domains of the NMDA Receptor GluN1 and GluN2B Subunits

Background and Purpose: The N-methyl-D-aspartate (NMDA) receptor is an important target of alcohol action in the brain. Recent studies in this laboratory have demonstrated that alcohol-sensitive positions in the intersubunit interfaces of the M3 and M4 domains of GluN1 and GluN2A subunits interact with respect to ethanol sensitivity and receptor kinetics, and that alcohol-sensitive positions in the M domains of GluN2A and GluN2B subunits differ. In this study we tested for interactions among alcohol-sensitive positions at the M domain intersubunit interfaces in GluN1/GluN2B NMDA receptors. Experimental Approach: We used whole-cell patch-clamp recording in tsA201 cells expressing tryptophan substitution mutants at ethanol-sensitive positions in the GluN1 and GluN2B NMDA receptor subunits to test for interactions among positions. Key Results: Six pairs of positions in GluN1/GluN2B significantly interacted to regulate ethanol inhibition: Gly638/Met824, Gly638/Leu825, Phe639/Leu825, Phe639/Gly826, Met818/Phe637 and Val820/Phe637. Tryptophan substitution at Met824 or Leu825 in GluN2B did not alter ethanol sensitivity but interacted with positions in the GluN1 M3 domain to regulate ethanol action, whereas tryptophan substitution at Gly638, which is the cognate of an ethanol-sensitive position in GluN2A, did not alter ethanol sensitivity or interact with positions in GluN1. Two and three pairs of positions interacted to regulate glutamate steady-state and peak current EC50, respectively, and one pair interacted with respect to macroscopic desensitization. Conclusions: Despite highly-conserved M domain sequences and similar ethanol sensitivity in the GluN2A and GluN2B subunits, the manner in which these subunits interact with the GluN1 subunit to regulate ethanol sensitivity and receptor kinetics differs

Different Sites of Alcohol Action in the NMDA Receptor GluN2A and GluN2B Subunits

The NMDA receptor is a major target of alcohol action in the CNS, and recent behavioral and cellular studies have pointed to the importance of the GluN2B subunit in alcohol action. We and others have previously characterized four amino acid positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN2A subunit that influence both ion channel gating and alcohol sensitivity. In this study, we found that substitution mutations at two of the four corresponding positions in the GluN2B subunit, F637 and G826, influence ethanol sensitivity and ion channel gating. Because position 826 contains a glycine residue in the native protein, we focused our attention on GluN2B(F637). Substitution mutations at GluN2B(F637) significantly altered ethanol IC50 values, glutamate EC50 values for peak (Ip) and steady-state (Iss) current, and steady-state to peak current ratios (Iss:Ip). Changes in apparent glutamate affinity were not due to agonist trapping in desensitized states, as glutamate Iss EC50 values were not correlated with Iss:Ip values. Ethanol sensitivity was correlated with values of both Ip and Iss glutamate EC50, but not with Iss:Ip. Values of ethanol IC50, glutamate EC50, and Iss:Ip for mutants at GluN2B(F637) were highly correlated with the corresponding values for mutants at GluN2A(F636), consistent with similar functional roles of this position in both subunits. These results demonstrate that GluN2B(Phe637) regulates ethanol action and ion channel function of NMDA receptors. However, despite highly conserved M domain sequences, ethanol\u27s actions on GluN2A and GluN2B subunits differ

Mutations at F637 in the NMDA receptor NR2A subunit M3 domain influence agonist potency, ion channel gating and alcohol action

Background and purpose: NMDA receptors are important molecular targets of ethanol action in the CNS. Previous studies have identified a site in membrane-associated domain 3 (M3) of the NR1 subunit and two sites in M4 of the NR2A subunit that influence alcohol action; the sites in NR2A M4 also regulate ion channel gating. The purpose of this study was to determine whether mutations at the site in the NR2A subunit corresponding to the NR1 M3 site influence alcohol action and ion channel gating. Experimental approach: We investigated the effects of mutations at phenylalanine (F) 637 of the NR2A subunit using whole-cell and single-channel patch-clamp electrophysiological recording in transiently-transfected HEK 293 cells. Key results: Mutations at F637 in the NR2A subunit altered peak and steady-state glutamate EC50 values, maximal steady-state to peak current ratios (Iss:Ip), mean open time, and ethanol IC50 values. Differences in glutamate potency among the mutants were not due to changes in desensitization. Ethanol IC50 values were significantly correlated with glutamate EC50 values, but not with maximal Iss:Ip or mean open time. Ethanol IC50 values were linearly and inversely related to molecular volume of the substituent. Conclusions and implications: These results demonstrate that NR2A(F637) influences NMDA receptor affinity, ion channel gating, and ethanol sensitivity. The changes in NMDA receptor affinity are likely to be the result of altered ion channel gating. In contrast to the cognate site in the NR1 subunit, the action of ethanol does not appear to involve occupation of a critical volume at NR2A(F637)

Agonist- and antagonist-induced up-regulation of surface 5-HT₃A receptors

BACKGROUND AND PURPOSE: The 5-HT(3) receptor is a member of the pentameric ligand-gated ion channel family and is pharmacologically targeted to treat irritable bowel syndrome and nausea/emesis. Furthermore, many antidepressants elevate extracellular concentrations of 5-HT. This study investigates the functional consequences of exposure of recombinant 5-HT(3)A receptors to agonists and antagonists. EXPERIMENTAL APPROACH: We used HEK cells stably expressing recombinant 5-HT(3)A receptors and the ND7/23 (mouse neuroblastoma/dorsal root ganglion hybrid) cell line, which expresses endogenous 5-HT(3) receptors. Surface expression of recombinant 5-HT(3)A receptors, modified to contain the bungarotoxin (BTX) binding sequence, was quantified using fluorescence microscopy to image BTX-conjugated fluorophores. Whole cell voltage-clamp electrophysiology was used to measure the density of current mediated by 5-HT(3)A receptors. KEY RESULTS: 5-HT(3)A receptors were up-regulated by the prolonged presence of agonists (5-HT and m-chlorophenylbiguanide) and antagonists (MDL-72222 and morphine). The up-regulation of 5-HT(3)A receptors by 5-HT and MDL-72222 was time- and concentration-dependent but was independent of newly translated receptors. The phenomenon was observed for recombinant rodent and human 5-HT(3)A receptors and for endogenous 5-HT(3) receptors in neuronal ND7/23 cells. CONCLUSIONS AND IMPLICATIONS: Up-regulation of 5-HT(3)A receptors, following exposure to either agonists or antagonists suggests that this phenomenon may occur in response to different therapeutic agents. Medications that elevate 5-HT levels, such as the antidepressant inhibitors of 5-HT reuptake and antiemetic inhibitors of 5-HT(3) receptor function, may both raise receptor expression. However, this will require further investigation in vivo