Sather W A, Detwiler P B
University of Washington, Department of Physiology and Biophysics, Seattle, WA 98195.
Proc Natl Acad Sci U S A. 1987 Dec;84(24):9290-4. doi: 10.1073/pnas.84.24.9290.
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.
在理解光转导方面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，人们开发出了一种功能完备的视网膜视杆细胞外段制剂，该制剂不附着内段，能够在使用特定溶液进行细胞内透析期间对光敏感电流进行电记录。用无核苷酸溶液透析的分离外段未记录到光敏感电流，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）的林格氏溶液中的细胞则产生了光敏感内向暗电流。这表明在黑暗中存在基础水平的cGMP特异性磷酸二酯酶活性。用大于或等于20微摩尔/升的cGMP透析分离的外段会迅速产生光抑制电流。在用浓度高50倍的GTP透析期间，会更缓慢地产生类似大小的电流。显然，cGMP可在外段由鸟苷酸环化酶从GTP合成。仅用cGMP透析的细胞显示出光敏感性降低，加入20微摩尔/升的GTP可将其恢复正常。GTP的这种作用被鸟苷5'-[β - 硫代]二磷酸拮抗。这些发现与生化证据高度一致，表明一种GTP结合蛋白（转导素）在光反应的产生中起关键作用。短暂闪光后光电流的恢复会因用缺乏ATP的溶液或含有鸟苷5'-[γ - 硫代]三磷酸（一种不可水解的GTP类似物）的溶液透析而延迟或消除。这些结果支持这样一种观点，即活化的转导素水解GTP以及视紫红质光产物的ATP依赖性磷酸化对于转导过程的终止都是必要的。
