ASSOCIATION OF CYCLIC GMP-GATED CHANNELS AND NA+-CA2+-K+ EXCHANGERS IN BOVINE RETINAL ROD OUTER SEGMENT PLASMA-MEMBRANES

被引:44
作者
BAUER, PJ [1 ]
DRECHSLER, M [1 ]
机构
[1] UNIV FREIBURG, INST BIOPHYS & STRAHLENBIOL, W-7800 FREIBURG, GERMANY
来源
JOURNAL OF PHYSIOLOGY-LONDON | 1992年 / 451卷
关键词
D O I
10.1113/jphysiol.1992.sp019156
中图分类号
Q189 [神经科学];
学科分类号
071006 ;
摘要
1. Cyclic GMP-gated channels and Na+-Ca2+-K+ exchangers from bovine photoreceptors were examined by investigation of the Ca2+ fluxes from vesicles of rod outer segment (ROS) membranes and from proteoliposomes obtained by solubilization of the ROS membrane proteins and reconstitution in soy bean L-alpha-phosphatidylcholine (PC). 2. Whereas vesicles obtained by mild sonication of ROS membranes in a Ca2+-containing buffer yielded a maximal cyclic GMP-induced Ca2+ release of about 2.5% and a maximal Na+-induced Ca2+ release of about 7%, freezing and thawing of ROS membranes prior to sonication elevated these maximal Ca2+ releases to about 17% for cyclic GMP, and to about 34% for Na+. These observations are in agreement with the view that cyclic GMP-gated channels and Na+-Ca2+-K+ exchangers are localized only in the plasma membrane of the photoreceptors (which in bovine ROS makes up about 6% of the total membrane), whereas freezing and thawing results in fusion of disc and plasma membranes, thus leading to a distribution of these proteins over a much larger membrane area. 3. For fused ROS membranes, the cyclic GMP-releasable fraction of Ca2+ of 17% is an upper bound; assuming that the cyclic GMP-gated channels are randomly distributed we estimate that about 37% of the vesicles contain at least one cyclic GMP-gated channel. The mean diameter of the vesicles prepared by sonication was determined to be 0.12+/-0.04-mu-m, and therefore the fused ROS membranes contain about sixteen cyclic GMP-gated channels/mu-m2. If all cyclic GMP-gated channels originated from the plasma membrane, we estimate that the plasma membrane contains about 270 cyclic GMP-gated channels/mu-m2. 4. In vesicles prepared from fused ROS membranes, Na+-Ca2+ exchange after activation of the cyclic GMP-gated channels was about half as great as before activation of the cyclic GMP-gated channels. On the other hand, after an exhaustive Na+-Ca2+ exchange, only little, if any, Ca2+ was released upon addition of cyclic GMP, demonstrating that cyclic GMP-gated channels and Na+-Ca2+-K+ exchangers occur on the same vesicle fraction. This observation suggests that Na+-C2+-K+ exchangers do not distribute independently of the cyclic GMP-gated channels upon membrane fusion but are apparently associated with the cyclic GMP-gated channels. 5. In an attempt to separate cyclic GMP-gated channels and Na+-Ca2+-K+ exchangers on different vesicles, ROS membranes were solubilized with the zwitterionic detergent 3-((3-cholamidopropyl)-dimethylammonio-1-propanesulphonate (CHAPS) in the presence of soy bean PC, and the detergent removed by dialysis; in the resulting proteoliposomes, Na+-Ca2+ exchange markedly reduced the fraction of cyclic GMP-releasable Ca2+. Therefore, even after solubilization and reconstitution of ROS membrane proteins in PC, at least some of the cyclic GMP-gated channels were associated with Na+-Ca2+-K+ exchangers. 6. If saturating cyclic GMP- and Na+-induced Ca2+ releases were subsequently evoked in proteoliposomes, the ratio of the Na+-induced to the cyclic GMP-induced Ca2+ release increased with increasing amounts of PC from values near 1 to above 3. This finding suggests that there are at least as many Na+-Ca2+-K+ exchangers as cyclic GMP-gated channels in the plasma membrane of the photoreceptors. 7. These experiments strongly suggest that in the plasma membranes of the photoreceptors each cyclic GMP-gated channel is associated with at least one Na+-Ca2+-K+ exchanger. Furthermore, there is no indication of aggregation of the cyclic GMP-gated channels since, with the assumption of a random distribution of channel we estimate a channel density in the plasma membrane which is very similar to previously published values.
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页码:109 / 131
页数:23
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