Optical probes of intradiskal processes in rod photoreceptors. II: Light-scattering study of ATP-dependent light reactions.

Rod outer segment (ROS) disks, either stacked or freely floating, respond to flash illumination to yield a specific, ATP-dependent, light-scattering signal AL. In broken ROS AL signals occur only when AD signals have preceded them. The degree to which the preceding AD signal has been completed determines the amplitude of the following AL signal. However, in freshly detached ROS from dark-adapted frogs Al signals with maximal size can be obtained without pre-incubation with exogenous ATP. The energized state, which is restored in broken ROS with the help of ATP, appears to prevail in the living retina and must therefore be considered to be "physiological". AL signals require structurally intact disks. Neither peripheral ROS proteins nor connecting filaments between adjacent disks are necessary. Their structural origin is the same as that of the preceding AD signal, i.e. osmotic disk swelling. AL signals consist of a single slow kinetic component (half-life 10 s at room temperature) and multiphase fast kinetic component (70 ms). The slow phase corresponds to a light-stimulated resumption of ATPase activity (this has been dealt with in a previous paper) whereas the fast component reflects an immediate response of the energized disk to the metarhodopsin I to metarhodopsin II transition. The latter effect is the subject of this paper. A variety of experiments, using different ATPase inhibitors, ionophores and membrane-permeable salts, have been carried out; they are all consistent with notion that AL originates in the disk interior and probes the existence of a proton electrochemical potential difference delta mu (H+) across the disk membrane. A model is presented which can explain all given properties of AL satisfactorily. According to this model the photolysis of rhodopsin causes a proton release in the disk lumen. This, in turn, results in osmotic swelling of the disks, provided that the internal buffer sites have been (at least partially) titrated with protons prior to the flash. Such conditions, i.e. a low internal pH, are provided by the proton transport across the disk membrane, which presumably takes place during the course of the preceding AD signal.