Ramoino Paola, Gallus Lorenzo, Beltrame Francesco, Diaspro Alberto, Fato Marco, Rubini Patrizia, Stigliani Sara, Bonanno Giambattista, Usai Cesare
Department for the Study of the Territory and its Resources (DIP.TE.RIS.), University of Genoa, Corso Europa 26, 16132 Genova, Italy.
J Cell Sci. 2006 May 15;119(Pt 10):2056-64. doi: 10.1242/jcs.02931. Epub 2006 Apr 25.
GABAB receptors modulate swimming behavior in Paramecium by inhibiting dihydropyridine-sensitive Ca2+ channels via G-proteins. Prolonged occupancy of GABAB receptors by baclofen results in a decrease in GABAB receptor functions. Since changes in the number of cell-surface GABAA receptors have been postulated to be of importance in modulating inhibitory synaptic transmission in neurons, we have studied the cell-surface expression and maintenance of GABAB receptors in P. primaurelia. In this study, we use immunostaining in electron and confocal microscopy to demonstrate that constitutive internalization of GABAB receptors in P. primaurelia is mediated by clathrin-dependent and -independent endocytosis. Indeed, GABAB receptors colocalize with the adaptin complex AP2, which is implicated in the selective recruitment of integral membrane proteins to clathrin-coated vesicles, and with caveolin 1, which is associated with uncoated membrane invaginations. Furthermore, when endocytosis is blocked with hypertonic medium, cytosol acidification, filipin or with a peptide that disrupts the association between amphiphysin and dynamin, the effect of baclofen on swimming is increased. These results suggest that GABAB receptor endocytosis into clathrin-coated and -uncoated vesicles represents an important mechanism in the modulation of swimming behavior in Paramecium.
GABAB受体通过G蛋白抑制二氢吡啶敏感的Ca2+通道来调节草履虫的游泳行为。巴氯芬对GABAB受体的长期占据会导致GABAB受体功能下降。由于细胞表面GABAA受体数量的变化被认为在调节神经元的抑制性突触传递中具有重要意义，我们研究了绿草履虫中GABAB受体的细胞表面表达和维持情况。在本研究中，我们利用电子显微镜和共聚焦显微镜下的免疫染色来证明，绿草履虫中GABAB受体的组成型内化是由网格蛋白依赖性和非依赖性内吞作用介导的。事实上，GABAB受体与衔接蛋白复合物AP2共定位，AP2参与将整合膜蛋白选择性募集到网格蛋白包被的小泡中，并且与小窝蛋白1共定位，小窝蛋白1与未包被的膜内陷有关。此外，当用高渗培养基、胞质酸化、制霉菌素或用破坏发动蛋白和 amphiphysin 之间关联的肽阻断内吞作用时，巴氯芬对游泳的影响会增强。这些结果表明，GABAB受体向内化到网格蛋白包被和未包被的小泡中是调节草履虫游泳行为的一个重要机制。
