Association of cyclic GMP-gated channels and Na(+)-Ca(2+)-K+ exchangers in bovine retinal rod outer segment plasma membranes.
1. Cyclic GMP-gated channels and Na(+)-Ca(2+)-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 Ca(2+)-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(+)-Ca(2+)-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 micron, and therefore the fused ROS membranes contain about sixteen cyclic GMP-gated channels/microns 2. If all cyclic GMP-gated channels originated from the plasma membrane, we estimate that the plasma membrane contains about 270 cyclic GMP-gated channels/microns 2. 4. In vesicles prepared from fused ROS membranes, Na(+)-Ca2+ exchange after 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(+)-Ca(2+)-K+ exchangers occur on the same vesicle fraction. This observation suggests that Na(+)-Ca(2+)-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.(ABSTRACT TRUNCATED AT 400 WORDS)
Bauer
P J
PJ
Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, FRG.
Drechsler
M
M
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism*; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D017136:Ion Transport; D008561:Membrane Fusion; D011188:Potassium / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D012995:Solubility
D002352:Carrier Proteins; D012964:Sodium; D006152:Cyclic GMP; D011188:Potassium; D002118:Calcium