Liu Xue, Grove James C R, Hirano Arlene A, Brecha Nicholas C, Barnes Steven
Biomaterials and Live Cell Imaging Institute, Chongqing University of Science and Technology, Chongqing, People's Republic of China; Department of Neurobiology and Jules Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, California;
Department of Neurobiology and Jules Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, California;
J Neurophysiol. 2016 Aug 1;116(2):686-97. doi: 10.1152/jn.00990.2015. Epub 2016 May 18.
Horizontal cells form the first laterally interacting network of inhibitory interneurons in the retina. Dopamine released onto horizontal cells under photic and circadian control modulates horizontal cell function. Using isolated, identified horizontal cells from a connexin-57-iCre × ROSA26-tdTomato transgenic mouse line, we investigated dopaminergic modulation of calcium channel currents (ICa) with whole cell patch-clamp techniques. Dopamine (10 μM) blocked 27% of steady-state ICa, an action blunted to 9% in the presence of the L-type Ca channel blocker verapamil (50 μM). The dopamine type 1 receptor (D1R) agonist SKF38393 (20 μM) inhibited ICa by 24%. The D1R antagonist SCH23390 (20 μM) reduced dopamine and SKF38393 inhibition. Dopamine slowed ICa activation, blocking ICa by 38% early in a voltage step. Enhanced early inhibition of ICa was eliminated by applying voltage prepulses to +120 mV for 100 ms, increasing ICa by 31% and 11% for early and steady-state currents, respectively. Voltage-dependent facilitation of ICa and block of dopamine inhibition after preincubation with a Gβγ-blocking peptide suggested involvement of Gβγ proteins in the D1R-mediated modulation. When the G protein activator guanosine 5'-O-(3-thiotriphosphate) (GTPγS) was added intracellularly, ICa was smaller and showed the same slowed kinetics seen during D1R activation. With GTPγS in the pipette, additional block of ICa by dopamine was only 6%. Strong depolarizing voltage prepulses restored the GTPγS-reduced early ICa amplitude by 36% and steady-state ICa amplitude by 3%. These results suggest that dopaminergic inhibition of ICa via D1Rs is primarily mediated through the action of Gβγ proteins in horizontal cells.
水平细胞在视网膜中形成了抑制性中间神经元的首个横向相互作用网络。在光控和昼夜节律控制下释放到水平细胞上的多巴胺可调节水平细胞的功能。我们使用来自连接蛋白57-iCre×ROSA26-tdTomato转基因小鼠品系的分离、已鉴定的水平细胞，采用全细胞膜片钳技术研究了多巴胺能对钙通道电流（ICa）的调节作用。多巴胺（10μM）可阻断27%的稳态ICa，在存在L型钙通道阻滞剂维拉帕米（50μM）的情况下，该作用减弱至9%。多巴胺1型受体（D1R）激动剂SKF38393（20μM）可使ICa抑制24%。D1R拮抗剂SCH23390（20μM）可减弱多巴胺和SKF38393的抑制作用。多巴胺减缓了ICa的激活，在电压阶跃早期可阻断38%的ICa。通过将电压预脉冲施加到+120mV持续100ms，消除了对ICa的早期增强抑制作用，早期电流和稳态电流的ICa分别增加了31%和11%。在与Gβγ阻断肽预孵育后，ICa的电压依赖性易化和多巴胺抑制的阻断表明Gβγ蛋白参与了D1R介导的调节。当在细胞内加入G蛋白激活剂鸟苷5'-O-（3-硫代三磷酸）（GTPγS）时，ICa较小，并且表现出与D1R激活期间相同的减缓动力学。在移液管中加入GTPγS后，多巴胺对ICa的额外阻断仅为6%。强去极化电压预脉冲使GTPγS降低的早期ICa幅度恢复了36%，稳态ICa幅度恢复了3%。这些结果表明，多巴胺通过D1R对ICa的抑制主要是通过水平细胞中Gβγ蛋白的作用介导的。
