Modulation Of Neuronal Activity By TWIK-1 Membrane Expression In Hippocampal Astrocytes

Background:Tandem of two pore domains K⁺channel TWIK-1 is an abundantly expressed potassium channel in mature brain astrocyte.But it is mainly located in the cytoplasm and function is unknown.Our recent experiments showed that the activation of the G_i/G_o conjugated metabotropic glutamate receptor 3?mGluR3?in hippocampal astrocyte stimulates the TWIK-1 to the membrane and enhances the extracellular NH₄⁺uptake.mGluR3 is the specifically and highly expressed mGluR type in mature mouse hippocampal astrocyte.Based on that NH₄⁺is a substrate for the glutamate-glutamine cycle?GGC?which involves in the replenishment of neurotransmitters,such as glutamate,we hypothesize that TWIK-1 activity-dependent membrane expression may regulate GGC to precisely match the requirements of neuronal transmitter synthesis.Objective:Investigate the effects of membrane expression of astrocyte TWIK-1 on the functional activities of hippocampal neurons and the involvement of GGC in this process.Methods:Wild-type and TWIK-1 knockout?TWIK-1 KO?C57BL/6J mice were used.We selected the hippocampal Schaffer collateral-CA1 pathway,a classic neural pathway to study synaptic plasticity as well as learning and memory.Brain slices were used for electrophysiology and immunofluorescence.In addition,single cell isolation,western blot were also used.Results:?1?Field potential recordings showed that activation of astrocyte mGluR3enhanced the amplitude of long-term potentiation?LTP?in hippocampus.Combined with single cell staining and previous results,it suggests that the activation of mGluR3 may increase LTP through TWIK-1 membrane translocation.?2?The LTP amplitude decreased in TWIK-1 KO mice.Meanwhile,the whole cell patch clamp revealed that the paired pulse facilitation ratio of pyramidal neurons increased in hippocampal CA1 region,indicating that the presynaptic transmitter release was reduced.It suggests that TWIK-1 channel's translocation promote synaptic transmission of hippocampal neurons and enhances synaptic plasticity.?3?Then we analyzed expression of synaptic marker proteins by western blot,synaptophysin and PSD95.The results showed no significant difference between Wild-type and TWIK-1KO mice,which likely precluded the lack of TWIK-1 effect on synaptic structure at the molecular level.?4?TWIK-1 deficiency also showed no obvious effect on dendritic morphology of pyramidal neurons by biocytin filling.?5?Inhibit the key catalytic enzymes of GGC,glutamine synthetase and pyruvate carboxylase inhibitors were used.The potentiation of LTP induced by mGluR3's activation was disappeared after the application of inhibitors.Conclusions:The above results indicate that the activation of astrocyte TWIK-1membrane translocation may affect neuronal activity by regulating GGC.This study shows a light on the physiological function of TWIK-1,a potassium channel highly expressed in astrocytes.It further elucidates astrocyte roles in central nervous system homeostasis and cognitive function in mammalian cells.