Effects of drugs of abuse on channelrhodopsin-2 function.

Channelrhodopsins are light activated ion channels used extensively over the past decade to probe the function of genetically defined neuronal populations and distinct neural circuits with high temporal and spatial precision. The widely used Channelrhodopsin-2 variant (ChR2) is an excitatory opsin that undergoes conformational changes in response to blue light, allowing non-selective passage of protons and cations across the plasma membrane thus leading to depolarization. In the addiction neuroscience field, opsins such as ChR2 provide a means to disambiguate the overlapping circuitry involved in mediating the reinforcing and aversive effects of drugs of abuse as well as to determine the plasticity that can occur in these circuits during the development of dependence. Although ChR2 has been widely used in animal models of drug and alcohol self-administration, direct effects of drugs of abuse on ChR2 function may confound its use and lead to misinterpretation of data. As a variety of neuronal ion channels are primary targets of various drugs of abuse, it is critical to determine whether ChR2-mediated currents are modulated by these drugs. In this study, we performed whole-cell electrophysiological recordings in HEK293 cells expressing the commonly used ChR2(H134R) variant and examined the effects of various drugs of abuse and other commonly used agents on light-induced currents. We found no differences in ChR2-mediated currents in the presence of 30 µM nicotine, 30 µM cocaine, 100 µM methamphetamine or 3 mM toluene. Similarly, ChR2 currents were insensitive to 30 mM ethanol but higher concentrations (100-300 mM) produced significant effects on the desensitization and amplitude of light-evoked currents. Tetrahydrocannabinol (1-10 µM) and morphine (30-100 µM) significantly inhibited ChR2 currents while the cannabinoid receptor antagonist AM-251 had no effect. The sodium channel blocker tetrodotoxin (5 µM) and the generic channel blocker/contrast agent gadolinium chloride (10 mM) also reduced ChR2 currents while the divalent ion magnesium (10 mM) had no effect. Together, the results from this study highlight the importance of conducting appropriate control experiments when testing new compounds in combination with optogenetic approaches.

Cysteine substitution of transmembrane domain amino acids alters the ethanol inhibition of GluN1/GluN2A N-methyl-D-aspartate receptors.

N-Methyl-d-aspartate receptors (NMDARs) are inhibited by behaviorally relevant concentrations of ethanol, and residues within transmembrane (TM) domains of NMDARs, including TM3 GluN1 phenylalanine 639 (F639), regulate this sensitivity. In the present study, we used cysteine (C) mutagenesis to determine whether there are additional residues within nearby TM domains that regulate ethanol inhibition on NMDARs. GluN1(F639C)/GluN2A receptors were less inhibited by ethanol than wild-type receptors, and inhibition was restored to wild-type levels following treatment with ethanol-like methanethiosulfonate reagents. Molecular modeling identified six residues in the GluN1 TM1 domain (valine V566; serine S569) and the GluN2A TM4 domain (methionine, M817; V820, F821, and leucine, L824) that were in close vicinity to the TM3 F639 residue, and these were individually mutated to cysteine and tested for ethanol inhibition and receptor function. The F639C-induced decrease in ethanol inhibition was blunted by coexpression of GluN1 TM1 mutants V566C and S569C, and statistically significant interactions were observed for ethanol inhibition among V566C, F639C, and GluN2A TM4 mutants V820C and F821C and S569C, F639C, and GluN2A TM4 mutants F821C and L824C. Ethanol inhibition was also reduced when either GluN1 TM1 mutant V566C or S569C was combined with GluN2A V820C, suggesting a novel TM1:TM4 intrasubunit site of action for ethanol. Cysteines substituted at TM3 and TM4 sites previously suggested to interact with ethanol had less dramatic effects on ethanol inhibition. Overall, the results from these studies suggest that interactions among TM1, TM3, and TM4 amino acids in NMDARs are important determinants of ethanol action at these receptors.

Ethanol inhibition of constitutively open N-methyl-D-aspartate receptors.

N-Methyl-D-aspartate (NMDA) receptors gate a slow and calcium-rich component of the postsynaptic glutamate response. Like all ionotropic glutamate receptors, NMDA subunits contain a highly conserved motif (SYTANLAAF) in the transmembrane (TM) 3 domain that is critically involved in channel gating. Mutation of an alanine in this domain (A7; underlined above) results in constitutively open receptors that show reduced sensitivity to several allosteric modulators. In this study, we examined the effects of ethanol, a substance that inhibits NMDA currents via an unknown mechanism, on tonically active NMDA receptors expressed in human embryonic kidney 293 cells. Ethanol (100 mM) inhibited currents from GluN1(A7R)/GluN2A and GluN1(A7R)/GluN2B receptors by approximately 50%, whereas those from GluN1/GluN2B(A7R) receptors were reduced by less than 10%. In cysteine-substituted GluN1 and GluN2 A7 mutants, estimated ethanol IC50 values for agonist-gated currents were 101, 117, 103, and 69 mM for GluN1(A7C)/GluN2A, GluN1(A7C)/GluN2B, GluN1/GluN2A(A7C), and GluN1/GluN2B(A7C) receptors, respectively. After exposure to the thiol-modifying reagent 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), A7C mutants showed robust agonist-independent currents and reduced sensitivity to ethanol (IC50 values of 371, 256, 715, and 958 mM, respectively, as above). In contrast, cysteine modification of the ligand-binding domain resulted in constitutively open receptors that showed robust ethanol inhibition. Ethanol inhibition of MTSET-treated GluN1(A7C) receptors was further reduced by TM3/TM4 mutations previously shown to reduce ethanol sensitivity of agonist-gated receptors. Overall, these results show that ethanol affects NMDA receptor function at a site distal from agonist binding and appears to exert greater effects via perturbation of GluN2 subunits.

Effects of ethanol on phosphorylation site mutants of recombinant N-methyl-D-aspartate receptors.

N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by the neurotransmitter glutamate. These channels are highly expressed by brain neurons and are critically involved in excitatory synaptic transmission. Results from previous studies show that both native and recombinant NMDA receptors are inhibited by ethanol at concentrations associated with signs of behavioral impairment and intoxication. Given the important role that NMDA receptors play in synaptic transmission and brain function, it is important to understand the factors that regulate the ethanol inhibition of these receptors. One dynamic mechanism for regulating ethanol action may be via phosphorylation of NMDA subunits by serine-threonine and tyrosine kinases. Both NR1 and NR2 subunits contain multiple sites of phosphorylation; and in the NR1 subunit, most of these are contained within the C1 domain, a carboxy-terminal cassette that is subject to alternative splicing. Although results from our previous studies suggest that single phosphorylation sites do not greatly affect ethanol sensitivity of NMDA receptors, it is likely that in vivo, these subunits are phosphorylated at multiple sites by different kinases. In the present study, we constructed a series of NMDA receptor mutants at serine (S) or threonine (T) residues proposed to be sites of phosphorylation by protein kinase A and various isoforms of protein kinase C. Ethanol (100mM) inhibited currents from wild-type NR1/2A and NR1/2B receptors expressed in human embryonic kidney 293 cells by approximately 25 and 30%, respectively. This inhibition was not different in single-site mutants expressing alanine (A) or aspartate/glutamate (D/E) at positions T879, S896, or T900. The mutant NR1(S890D) showed greater ethanol inhibition than NR1(890A) containing receptors, although this was only observed when it was combined with the NR2A subunit. Ethanol inhibition was not altered by aspartate substitution at four serines (positions 889, 890, 896, and 897) or when T879D was added to the four serine-substituted mutant. Ethanol inhibition was increased when T900E was added to the five serine-/threonine-substituted mutants, but again this was selective for NR2A containing receptors. Together with previously published data, these findings suggest that modification of putative phosphorylation sites could contribute to the overall acute ethanol sensitivity of recombinant NMDA receptors. Supported by R37AA009986.

Ethanol inhibition of recombinant NMDA receptors is not altered by coexpression of CaMKII-alpha or CaMKII-beta.

Previous studies have shown that the N-methyl-d-aspartate (NMDA) receptor is an important target for the actions of ethanol in the brain. N-methyl-d-aspartate receptors are glutamate-activated ion channels that are highly expressed in neurons. They are activated during periods of significant glutamatergic synaptic activity and are an important source of the signaling molecule calcium in the postsynaptic spine. Alterations in the function of NMDA receptors by drugs or disease are associated with deficits in motor, sensory and cognitive processes of the brain. Acutely, ethanol inhibits ion flow through NMDA receptors whereas sustained exposure to ethanol can induce compensatory changes in the density and localization of the receptor. Defining factors that govern the acute ethanol sensitivity of NMDA receptors is an important step in understanding how an individual responds to ethanol. In the present study, we investigated the effect of calcium-calmodulin dependent protein kinase II (CaMKII) on the ethanol sensitivity of recombinant NMDA receptors. Calcium-calmodulin dependent protein kinase II is a major constituent of the postsynaptic density and is critically involved in various forms of learning and memory. NMDA receptor subunits were transiently expressed in human embryonic kidney 293 cells along with CaMKII-alpha or CaMKII-beta tagged with the green fluorescent protein. Whole cell currents were elicited by brief exposures to glutamate and were measured using patch-clamp electrophysiology. Neither CaMKII-alpha or CaMKII-beta had any significant effect on the ethanol inhibition of NR1/2A or NR1/2B receptors. Ethanol inhibition was also unaltered by deletion of CaMKII binding domains in NR1 or NR2 subunits or by phospho-site mutants that mimic or occlude CaMKII phosphorylation. Chronic treatment of cortical neurons with ethanol had no significant effect on the expression of CaMKII-alpha or CaMKII-beta. The results of this study suggest that CaMKII is not involved in regulating the acute ethanol sensitivity of NMDA receptors.

Ethanol inhibition of NMDA receptors under conditions of altered protein kinase A activity.

N-methyl-D-aspartate receptors (NMDA) are glutamate-activated ligand-gated ion channels that participate in diverse forms of synaptic plasticity as well as glutamate-dependent excitotoxicity. Inhibition of the NMDA receptor function may underlie some of the behavioral actions associated with acute exposure to ethanol. The sensitivity of NMDA receptors to ethanol is influenced by the subunit composition of the receptor and, by association, with certain cytoskeletal proteins. Previous studies have also suggested that phosphorylation may regulate the sensitivity of NMDA receptors to ethanol. In this study, the ethanol inhibition of recombinant NMDA receptor currents was determined under conditions designed to enhance or inhibit the activity of protein kinase A (PKA). Human embryonic kidney 293 (HEK293) cells were transfected with cDNAs encoding NMDA subunits and channel activity was monitored with whole-cell patch-clamp electrophysiology. Under control recording conditions, ethanol (100 mM) inhibited NR1/2A and NR1/2B receptor currents by approximately 25-30%. The degree of ethanol inhibition was not affected or was slightly enhanced under conditions designed to enhance PKA activity. This included treatment of cells with cAMP analogs, inclusion of phosphatase inhibitors or purified PKA in the pipette filling solution, co-expression of catalytically active PKA, expression of the NR1 PKA-site phosphorylation site mimic (S897D) or by co-expression of the PKA scaffolding protein yotiao or the dopamine D(1) receptor. Ethanol inhibition of NR1/2A and NR1/2B receptors was not altered when PKA effects were suppressed, either by co-expression of a PKI inhibitory peptide or the phosphorylation-deficient NR1 mutants (S897A, S896A, S896A/S897A). In addition, ethanol inhibition of NMDA-induced currents in cultured cortical or hippocampal neurons was not affected by modulators of PKA. These results suggest that PKA does not appear to play a major role in determining the acute ethanol sensitivity of NMDA receptors.

Regulation of MMP-1 expression in vascular endothelial cells by insulin sensitizing thiazolidinediones.

Matrix metalloproteinases (MMPs) have been implicated in the disruption of atherosclerotic plaques that leads to acute coronary events. The present study investigates the effect of thiazolidinediones (TZDs), new antidiabetic drugs, on MMP-1 expression by human vascular endothelial cells. Results show that troglitazone, but not pioglitazone and rosiglitazone, stimulated MMP-1 secretion and mRNA expression in both human umbilical vein and aortic endothelial cells, but had no effect on TIMP-1 and TIMP-2 secretion. Interestingly, troglitazone at high concentrations (> or = 30 micromol/l) inhibited MMP-1 protein synthesis despite a marked stimulation on MMP-1 mRNA. Further studies revealed that troglitazone at higher concentrations inhibits de novo protein synthesis as determined by 35S-methionine/cysteine incorporation, suggesting that the inhibition of MMP-1 synthesis by troglitazone is due to the suppression of total protein synthesis. Finally, our studies showed that high concentrations of troglitazone inhibited the translation initiation factor 4E (eIF4E), but not eIF4G. In summary, the present study demonstrates that insulin sensitizers have different effects on MMP-1 expression, and troglitazone stimulates MMP-1 mRNA expression and protein synthesis at the pharmacological concentrations, but inhibits MMP-1 synthesis at higher doses. This study also suggests that supra-pharmacological concentrations of troglitazone that could be attained in body tissues may inhibit protein synthesis and cause cytotoxicity.

IFN-gamma pretreatment augments immune complex-induced matrix metalloproteinase-1 expression in U937 histiocytes.

We reported recently that immune complexes (ICs) induced matrix metalloproteinase-1 (MMP-1) expression in U937 histiocytes. The present study was undertaken to determine the effect of pretreatment of U937 cells with interferon-gamma (IFN-gamma) on IC-induced MMP-1 expression. Our flow cytometry studies showed that IFN-gamma upregulated the surface expression of FcgammaRI, but not FcgammaRII. Results also showed that pretreatment of the cells with IFN-gamma augmented LDL-containing IC (LDL-IC)-induced MMP-1 secretion in a dose- and time-dependent manner. Furthermore, Northern blot analysis revealed that IFN-gamma pretreatment led to a marked increase in MMP-1 mRNA. Finally, we demonstrated that PD98059 was able to block LDL-IC-induced MMP-1 secretion, regardless of whether the cells were pretreated with IFN-gamma or not, suggesting that IFN-gamma pretreatment did not alter the essential role of the ERK signaling pathway in LDL-IC-induced MMP-1 expression. In conclusion, the present study has demonstrated that IFN-gamma pretreatment augments LDL-IC-induced MMP-1 expression in U937 cells, thus elucidating an immune mechanism potentially involved in plaque destabilization.