Brain Research Centre, Department of Psychiatry, University of British Columbia, 2211 Wesbrook Mall, Vancouver, British Columbia, Canada V6T 2B5.
Hippocampus. 2011 Sep;21(9):958-67. doi: 10.1002/hipo.20807. Epub 2010 Jun 1.
In cultured hippocampal neurons, transient receptor potential 5 (TRPC5) channels are translocated and inserted into plasma membranes of hippocampal neurons to generate nonselective cation (NSC) currents. We investigated whether TRPC5 channel translocation also contributes to the generation of NSC currents underlying the afterdepolarizations and plateau potentials (PPs) in hippocampal pyramidal cells that are induced by muscarinic receptor activation. Using a biotinylation assay to quantify the change in surface membrane proteins in acute hippocampal slices, we found that muscarinic stimulation significantly enhanced the levels of TRPC5 protein on the membrane surface but not those of TRPC1 or TRPC4 channels. We then investigated the pharmacological sensitivity of the cation current observed during muscarinic stimulation to determine if a component could be due to TRPC5 channels. The TRPC channel antagonists 2-APB and SKF96365 strongly depressed the generation of PPs, the underlying tail currents (I(tail)) and the associated dendritic Ca(2+) influx induced by muscarinic receptor activation in pyramidal neurons. High intracellular concentrations of ATP, which specifically inhibit TRPC5 channels, depressed I(tail). In addition, pretreatment with the calmodulin (CaM) inhibitor W-7, which depresses recombinant TRPC5 currents, inhibited both the cation current (I(tail)) and the surface insertion of TRPC5 channels. Finally, the phosphatidylinositide 3-kinase (PI(3)K) inhibitor wortmannin, which blocks translocation of TRPC5 channels in cell culture, also inhibited both the I(tail) and the surface insertion of TRPC5 channels. Therefore, we conclude that insertion of TRPC5 channels contributes to the generation of the prolonged afterdepolarizations following muscarinic stimulation. This altered plasma membrane expression of TRPC5 channels in pyramidal neurons may play an important role in the generation of prolonged neuronal depolarization and bursting during the epileptiform seizure discharges of epilepsy.
在培养的海马神经元中，瞬时受体电位 5 (TRPC5) 通道易位并插入海马神经元的质膜中，以产生非选择性阳离子 (NSC) 电流。我们研究了 TRPC5 通道易位是否也有助于产生由毒蕈碱受体激活诱导的海马锥体细胞中的后去极化和平台电位 (PP) 所产生的 NSC 电流。我们使用生物素化测定来量化急性海马切片中质膜蛋白的变化，发现毒蕈碱刺激显著增强了膜表面上的 TRPC5 蛋白水平，但没有增强 TRPC1 或 TRPC4 通道的蛋白水平。然后，我们研究了在毒蕈碱刺激过程中观察到的阳离子电流的药理学敏感性，以确定是否可以归因于 TRPC5 通道。TRPC 通道拮抗剂 2-APB 和 SKF96365 强烈抑制了由毒蕈碱受体激活引起的 PP、潜在的尾电流 (I(tail)) 和相关的树突 Ca(2+) 内流的产生。高浓度的细胞内 ATP（特异性抑制 TRPC5 通道）抑制 I(tail)。此外，用钙调蛋白 (CaM) 抑制剂 W-7 预处理，该抑制剂抑制重组 TRPC5 电流，抑制阳离子电流 (I(tail)) 和 TRPC5 通道的表面插入。最后，磷脂酰肌醇 3-激酶 (PI(3)K) 抑制剂wortmannin 阻断细胞培养中 TRPC5 通道的易位，也抑制了 I(tail) 和 TRPC5 通道的表面插入。因此，我们得出结论，TRPC5 通道的插入有助于在毒蕈碱刺激后产生延长的后去极化。这种在锥体神经元中 TRPC5 通道的改变质膜表达可能在癫痫发作期间的延长神经元去极化和爆发中发挥重要作用。
