Dhingra Anuradha, Sulaiman Pyroja, Xu Ying, Fina Marie E, Veh Rüdiger W, Vardi Noga
Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6058, USA.
J Comp Neurol. 2008 Oct 10;510(5):484-96. doi: 10.1002/cne.21807.
Retinal ON bipolar cells make up about 70% of all bipolar cells. Glutamate hyperpolarizes these cells by binding to the metabotropic glutamate receptor mGluR6, activating the G-protein G(o1), and closing an unidentified cation channel. To facilitate investigation of ON bipolar cells, we here report on the production of a transgenic mouse (Grm6-GFP) in which enhanced green fluorescent protein (EGFP), under control of mGluR6 promoter, was expressed in all and only ON bipolar cells. We used the mouse to determine density of ON bipolar cells, which in central retina was 29,600 cells/mm(2). We further sorted the fluorescent cells and created a pure ON bipolar cDNA library that was negative for photoreceptor unique genes. With this library, we determined expression of 27 genes of interest. We obtained positive transcripts for G(o) interactors: regulators of G-protein signaling (RGS), Ret-RGS1 (a variant of RGS20), RGS16, RGS7, purkinje cell protein 2 (PCP2, also called L7 or GPSM4), synembryn (RIC-8), LGN (GPSM2), RAP1GAP, and Gbeta5; cGMP modulators: guanylyl cyclase (GC) 1alpha1, GC1beta1, phosphodiesterase (PDE) 1C, and PDE9A; and channels: inwardly rectifying potassium channel Kir2.4, transient receptor potential TRPC2, and sperm-specific cation channels CatSper 2-4. The following transcripts were not found in our library: AGS3 (GPSM1), RGS10, RGS19 (GAIP), calbindin, GC1alpha2, GC1beta2, PDE5, PDE2A, amiloride-sensitive sodium channel ACCN4, and CatSper1. We then localized Kir2.4 to several cell types and showed that, in ON bipolar cells, the channel concentrates in their dendritic tips. The channels and modulators found in ON bipolar cells likely shape their light response. Additional uses of the Grm6-GFP mouse are also discussed.
视网膜ON双极细胞约占所有双极细胞的70%。谷氨酸通过与代谢型谷氨酸受体mGluR6结合，激活G蛋白G(o1)，并关闭一个未确定的阳离子通道，使这些细胞发生超极化。为便于对ON双极细胞进行研究，我们在此报告一种转基因小鼠（Grm6-GFP）的产生，其中增强型绿色荧光蛋白（EGFP）在mGluR6启动子的控制下，仅在所有ON双极细胞中表达。我们使用该小鼠来确定ON双极细胞的密度，在视网膜中央其密度为29,600个细胞/mm²。我们进一步对荧光细胞进行分选，并创建了一个对光感受器特异基因呈阴性的纯ON双极cDNA文库。利用这个文库，我们确定了27个感兴趣基因的表达情况。我们获得了G(o)相互作用蛋白的阳性转录本：G蛋白信号调节因子（RGS）、Ret-RGS1（RGS20的一个变体）、RGS16、RGS7、浦肯野细胞蛋白2（PCP2，也称为L7或GPSM4）、synembryn（RIC-8）、LGN（GPSM2）、RAP1GAP和Gbeta5；cGMP调节剂：鸟苷酸环化酶（GC）1α1、GC1β1、磷酸二酯酶（PDE）1C和PDE9A；以及通道：内向整流钾通道Kir2.4、瞬时受体电位TRPC2和精子特异性阳离子通道CatSper 2 - 4。在我们的文库中未发现以下转录本：AGS3（GPSM1）、RGS10、RGS19（GAIP）、钙结合蛋白、GC1α2、GC1β2、PDE5、PDE2A、阿米洛利敏感钠通道ACCN4和CatSper1。然后我们将Kir2.4定位到几种细胞类型，并表明在ON双极细胞中，该通道集中在其树突尖端。在ON双极细胞中发现的通道和调节剂可能塑造了它们的光反应。还讨论了Grm6-GFP小鼠的其他用途。
