Harayama Nobuya, Kayano Tomohiko, Moriya Taiki, Kitamura Naoki, Shibuya Izumi, Tanaka-Yamamoto Keiko, Uezono Yasuhito, Ueta Yoichi, Sata Takeyoshi
Critical Care Medicine, University Hospital, University of Occupational and Environmental Health, Kitakyushu 807-8556, Japan.
Laboratory of Veterinary Physiology, Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan.
Brain Res. 2014 Dec 3;1591:1-13. doi: 10.1016/j.brainres.2014.10.022. Epub 2014 Oct 23.
While magnocellular neurons in the supraoptic nucleus (SON) possess rich Gi/o-mediated mechanisms, molecular and cellular properties of G-protein-activated inwardly rectifying K(+) (GIRK) channels have been controversial. Here, properties of GIRK channels are examined by RT-PCR and whole-cell patch-clamp techniques in rat SON neurons. Patch clamp experiments showed that the selective GABAB agonist, baclofen, enhanced currents in a high K(+) condition. The baclofen-enhanced currents exhibited evident inward rectification and were blocked by the selective GABAB antagonist, CGP55845A, the IRK channel blocker, Ba(2+), and the selective GIRK channel blocker, tertiapin, indicating that baclofen activates GIRK channels via GABAB receptors. The GIRK currents were abolished by N-ethylmaleimide pretreatment, and prolonged by GTPγS inclusion in the patch pipette, suggesting that Gi/o proteins are involved. RT-PCR analysis revealed mRNAs for all four GIRK 1-4 channels and for both GABABR1 and GABABR2 receptors in rat SON. However, the concentration-dependency of the baclofen-induced activation of GIRK currents had an EC50 of 110 µM, which is about 100 times higher than that of baclofen-induced inhibition of voltage-dependent Ca(2+) channels. Moreover, baclofen caused no significant changes in the membrane potential and the firing rate. These results suggest that although GIRK channels can be activated by GABAB receptors via the Gi/o pathway, this occurs at high agonist concentrations, and thus may not be a physiological mechanism regulating the function of SON neurons. This property that the membrane potential receives little influence from GIRK currents seems to be uncommon for CNS neurons possessing rich Gi/o-coupled receptors, and could be a special feature of rat SON neurons.
虽然视上核（SON）中的大细胞神经元拥有丰富的Gi/o介导机制，但G蛋白激活的内向整流钾通道（GIRK）的分子和细胞特性一直存在争议。在此，通过逆转录聚合酶链反应（RT-PCR）和全细胞膜片钳技术研究大鼠SON神经元中GIRK通道的特性。膜片钳实验表明，选择性GABAB激动剂巴氯芬在高钾条件下增强电流。巴氯芬增强的电流表现出明显的内向整流，并被选择性GABAB拮抗剂CGP55845A、内向整流钾通道阻滞剂Ba(2+)和选择性GIRK通道阻滞剂特律平阻断，表明巴氯芬通过GABAB受体激活GIRK通道。N-乙基马来酰亚胺预处理可消除GIRK电流，而膜片钳微管中加入GTPγS可延长该电流，提示Gi/o蛋白参与其中。RT-PCR分析显示大鼠SON中存在所有四种GIRK 1 - 4通道以及GABABR1和GABABR2受体的mRNA。然而，巴氯芬诱导GIRK电流激活的浓度依赖性的半数有效浓度（EC50）为110 μM，这比巴氯芬诱导电压依赖性Ca(2+)通道抑制的浓度高约100倍。此外，巴氯芬对膜电位和放电频率无显著影响。这些结果表明，尽管GIRK通道可通过Gi/o途径被GABAB受体激活，但这发生在高激动剂浓度下，因此可能不是调节SON神经元功能的生理机制。这种膜电位受GIRK电流影响较小的特性在拥有丰富Gi/o偶联受体的中枢神经系统神经元中似乎并不常见，可能是大鼠SON神经元的一个特殊特征。
