Ketamine Treatment Translocates Gsalpha from Lipid Rafts in Cultured Glial Cells: Similar Effects to Several Antidepressant Compounds

Abstract

BackgroundPrevious studies have demonstrated that all classes of monoamine‐centric antidepressants increase coupling between the G protein, Gsalpha and adenylyl cyclase, resulting in sustained cAMP production. This effect requires sustained drug treatment and is observed after 3 days in cultured neural or glial cells or 3 weeks in rats. This is apparently due to Gsalpha being released from constraints of a lipid raft environment to cholesterol‐poor regions of the plasma membrane. Consistent with this, both peripheral tissue and postmortem brain from depressed human subjects show a greater proportion of Gsalpha in lipid rafts. In an effort to determine whether the “glutamate‐centric” ketamine showed an antidepressant “biosignature” similar to other antidepressants, studies in cultured cells were initiated. It was hypothesized that ketamine would have an effect similar to antidepressants but along a shorter time course.MethodsC6 glioma cells were treated with 1 μM ketamine for 15 minutes or 24 hours. Cells were harvested and lipid raft fractions prepared and the amount of Gsalpha was assessed in raft and non‐raft membrane fractions by Western blot analysis. A population of C6 cells with stable expression of a fluorescent Gsalpha fusion protein (GFP‐Gsalpha) was treated similarly with ketamine and the mobility of GFP‐Gsa was determined by Fluorescence Recovery After Photobleaching (FRAP). Three‐day treatment of these cells with compounds showing antidepressant activity retard recovery, as the GFP‐Gsalpha is more extensively coupled with adenylyl cyclase, a 12 membrane span protein with slow lateral mobility.ResultsBoth methods produced results consistent with translocation of Gsalpha (or GFP‐Gsalpha) from lipid raft domains to association with adenylyl cyclase in non‐raft domains. Ketamine induced translocation is also dose‐dependent and occurs at the therapeutically relevant concentration of 1uM. FRAP studies also showed that 15 minutes of ketamine treatment was sufficient to achieve substantial translocation of GFP‐Gsalpha. These results suggest that brief treatment of cells with ketamine produces an antidepressant signature similar to that seen after prolonged treatment of well established classes of antidepressants. The NMDA antagonist, memantine, did not show similar effects.ConclusionsBrief ketamine treatment evokes a biochemical hallmark (translocation of Gsalpha) seen after prolonged treatment with several species of drugs that have antidepressant activity. This effect occurs in cells lacking NMDA receptors and is not mimicked by memantine, suggesting a possible additional site for ketamine action. Furthermore, the translocation of GFP‐Gsalpha produced by ketamine and all tested compounds with antidepressant activity (but not mood‐stabilizers, antipsychotics or anxiolytics) might serve as a useful platform for identifying compounds with potential antidepressant activity and for predicting clinical response.Support or Funding InformationSupport: VA BX001149; NIH MH 067631