Sensitive light scattering probe of i rod photoreceptor membranes (vision/guanosine triphosphate-binding protein/phosphodiesterase/pr

Light excitation of as little as 0.05% of the rhodopsin in a retinal rod membrane suspension reduces the near-IR optical transmission by 25%. This transmission de- crease requires the presence of guanosine triphosphate, is op- posite in sign and 25 times larger in amplitude than a GTP- dependent light-scattering signal previously reported in rod outer segment suspensions (Kuhn, H., Bennett, N., Michel- Vallez, M. & Chabre, M. (1981) Proc. Natl. Acad. Sci. USA, 78, 6873-6877), and is kinetically complex. The initial phase of the optical transmission decrease begins after about a 50-ms lag (at 0.05% bleach) and has a first-order time constant of 300-500 ms. The scattering signal returns to the preactinic baseline in a time dependent on the amount of GTP added. A nonhydrolyzable GTP analogue, guanylyl imidodiphosphate, produces a scattering signal that does not return to the preac- tinic baseline. Adenosine triphosphate strongly inhibits the re- turn of the GTP-dependent transmission decrease to the preac- tinic baseline. This effect of ATP on the GTP signal apparently requires ATP hydrolysis because it is inhibited by the simulta- neous presence of adenylyl imidodiphosphate, a nonhydrolyza- ble analogue of ATP. The light-scattering signal and the veloci- ty of the activation of a rod outer segment phosphodiesterase saturate when >0.05% of the rhodopsin is bleached and both show nearly identical dependence on light stimulus. It is sug- gested that these nucleotide-dependent light-scattering signals arise from changes in the state of membrane aggregation that are controlled by enzymatic processes. This hypothesis is sup- ported by the large amplitude of the signals, sedimentation ex- periments, and a strong membrane concentration dependence. The ATP effects can be rationalized within the above hypothe- sis as being due to ATP-dependent rhodopsin phosphorylation that adds negative charges to the membrane surface and tends to keep the membranes disaggregated. An additional signal, which increases light transmission, is produced by a second, much more intense flash. The latter signal is interpreted as the result of proton binding by bleached rhodopsin molecules that decreases the negative charge repulsion between the mem- branes and allows increased aggregation.