Abstract
The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular excitability through Cl- influx and K+ efflux in neurons. Relatively little is known about the link between GABAAR and the K+ channel. The present study was performed to identify the effect of GABAR agonists on K2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABAAR and GABABR agonists. In particular, muscimol, a GABAAR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABAAR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl- influx in GABAergic neurons.
Highlights
Neuronal activity is essential for developing and maintaining neuronal circuits and neurotransmitter switching in developing nervous systems [1]
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Background currents activated by muscimol in B35 cells were blocked in the presence of bicuculline and the TWIK-related K+ (TREK)-2 inhibitor norfluoxetine [14]
Summary
Neuronal activity is essential for developing and maintaining neuronal circuits and neurotransmitter switching in developing nervous systems [1]. B35 cells were blocked in the presence of bicuculline and the TREK-2 inhibitor norfluoxetine [14] These results strongly indicate that the background K+ channel activated by muscimol is TREK-2. TREK-2 channel activity was increased by muscimol in HEK-293 cells transfected with TREK-2, and the activity was blocked in the presence of bicuculline, indicating that the TREK-2 channel is related to the activation of GABAAR. If HEK-293 cells do not express GABAAR, muscimol seems to act directly on the TREK-2 channel. Muscimol may be able to increase TREK-2 currents by binding to the N-terminus of TREK-2 and activating a series of intracellular signals. Specific intracellular signals may be activated to stimulate the N-terminus of TREK-2 after muscimol binds to the receptor in GABAergic neurons. The specific mechanism, needs to be further investigated in brain slices
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