Intracellular biochemical manipulation of phototransduction in detached rod outer segments.
Intracellular biochemical manipulation of phototransduction in detached rod outer segments.
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分离杆外节光转导的细胞内生化操作。
DOI：
10.1073/pnas.84.24.9290
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发表时间：
1987
影响因子：
11.1
通讯作者：
Detwiler,PB
中科院分区：
文献类型：
--
作者：
Sather,WA;Detwiler,PB
理解光转导的最新进展主要来自对无细胞系统的研究。为了研究生理条件下的转导过程，开发了一种功能齐全的视网膜杆外段制备方法，该方法允许在细胞内透析过程中用确定的溶液电记录光敏电流。用无核苷酸溶液透析的分离外节没有记录到光敏电流，而从视网膜分离到含有3-异丁基-1-甲基黄嘌呤（一种磷酸二酯酶抑制剂）的林格溶液中的细胞会产生光敏的向内暗电流。这表明在黑暗中存在cGMP特异性磷酸二酯酶活性的基础水平。用大于或等于20 μ M cGMP透析的分离的外节段迅速产生光抑制电流。在GTP浓度高50倍的透析过程中，产生类似大小的电流的速度更慢。显然，cGMP可以通过鸟苷酸环化酶由GTP合成。单独用cGMP透析的细胞显示出降低的光敏感性，其通过添加20 μ M GTP恢复至正常。鸟苷5 '-[β-硫代]二磷酸可拮抗GTP的这种作用。这些发现与生物化学证据非常一致，表明GTP结合蛋白（transducin）在产生对光的反应中起着关键作用。用缺乏ATP或含有鸟苷5 '-[γ-硫代]三磷酸（一种不可水解的GTP类似物）的溶液透析，可延迟或消除短暂闪光后的光电流恢复。这些结果支持这样的观点，即GTP水解激活transducin和ATP依赖的磷酸化的视紫红质光产物是必要的终止的转导过程。
Recent progress in understanding phototransduction has come primarily from studies on cell-free systems. To investigate the transduction process under physiological conditions, a fully functional preparation of retinal rod outer segments without attached inner segments was developed that allows electrical recording of light-sensitive current during intracellular dialysis with defined solutions. No light-sensitive current is recorded from detached outer segments dialyzed with nucleotide-free solutions, whereas cells detached from the retina into Ringer's solution containing 3-isobutyl-1-methyl-xanthine (a phosphodiesterase inhibitor) develop a light-sensitive inward dark current. This indicates that there is a basal level of cGMP-specific phosphodiesterase activity in the dark. Detached outer segments dialyzed with greater than or equal to 20 microM cGMP rapidly develop a light-suppressible current. A current of similar magnitude is generated more slowly during dialysis with a 50-fold greater concentration of GTP. Apparently, cGMP can be synthesized from GTP by guanylate cyclase in the outer segment. Cells dialyzed with cGMP alone show a reduced light sensitivity that is restored to normal by addition of 20 microM GTP. This action of GTP is antagonized by guanosine 5'-[beta-thio]diphosphate. These findings are in good agreement with biochemical evidence indicating that a GTP-binding protein (transducin) plays a pivotal role in the generation of responses to light. The recovery of photocurrent following a brief flash is delayed or abolished by dialysis with solutions that lack ATP or contain guanosine 5'-[gamma-thio]triphosphate, a nonhydrolyzable GTP analog. These results support the view that both GTP hydrolysis by activated transducin and ATP-dependent phosphorylation of a rhodopsin photoproduct are necessary for termination of the transduction process.