BOVINE ROF LIGHT SCATTERING & ABSORBANCE STUDY
VOL.
17,
NO.
25, 1978
5347
On the Light-Stimulated Coupling between Rhodopsin and Its Disk Membrane Environment? Rainer Uhl,*.l Klaus Peter Hofmann, and Werner Kreutz
ABSTRACT: Disks from bovine ROS undergo a rapid shrinkage when flash illuminated with green light (Uhl, R., et al. (1977) Biochim. Biophys. Acta 469, 113-122). This can be monitored as a light scattering transient, referred to as the P signal. In this paper the P signal is studied at various temperatures and pH. The temperature dependence of the kinetics reveals that “P” consists of two sequential reaction steps. Both appear to occur within the receptor molecule rhodopsin. The actually observed event, the shrinkage of the disk, is therefore not rate limiting under the tested conditions. Both steps of “P” take place while there is only one spectroscopically detectable reaction of the
rhodopsin molecule, the metarhodopsin I-metarhodopsin I1 transition. This implies that there are intermediates of the rhodopsin photolytic cycle which are not evident as spectroscopically separate species. The amplitude of “P”, i.e., the extent of the disk shrinkage, is independent of the state of the equilibrium between the two photoproducts absorbing at 478 and 380 nm respectively and called MI and MII. A scheme is suggested in which the irreversible decay of MI (478) triggers the disk shrinkage (and maybe transduction), and in which there is an equilibrium between MI1 (380) and a proposed isochromic photoproduct MI’ (478).
T h e visual transduction process can be thought of as a sequence of “communication processes” on various levels of molecular organization in the receptor cell: ( 1 ) the interaction of light with the chromophore and, in turn, with the protein moiety of the pigment molecule rhodopsin; (2) the interaction of rhodopsin with its environment, the disk membrane; (3) the communication between disk and plasma membrane, probably due to a diffusable transmitter. The first “communication step” has been studied extensively, using spectroscopic techniques such as absorption spectroscopy (for a review, see Wald, 1968), flash photolysis (for a review, see Abrahamson & Wiesenfeld, 1972), and resonance Raman (see Lewis, 1976). All of these methods make use of the pigment nature of rhodopsin. The final stages of “communication step” 3 have also been studied very carefully by means of intracellular and extracellular recordings (see Penn & Hagins, 1972; Yau et al., 1977; Kleinschmidt & Dowling, 1975). However, very little is known about the way rhodopsin communicates with its environment. The main reason for this is that the above mentioned techniques only allow the monitoring of events that either occur prior to the coupling between rhodopsin and the disk membrane (rhodopsin photolysis) or much later (rise of the receptor potential). From an analysis of the complex kinetics of the rise of the receptor potential in both rods and cones it has been concluded that a t least 4- 10 first-order kinetic processes are involved in the coupling between rhodopsin and plasma membrane (Penn & Hagins, 1972; Baylor et al., 1974). One or more of these processes may actually reflect the coupling between rhodopsin and its subcellular environment. The complexity of the kinetics of the receptor potential, however, does not permit the resolution of this coupling. Moreover, none of the known reaction steps at the rhodopsin photolytic cycle possesses a rate similar to the ones calculated to fit the kinetic patterns of the light
response; Le., no trace of any of the known rhodopsin reactions can be found in the kinetics of the receptor potential. This leaves a gap between the processes (on the chronological scale and at the morphological level) that can be readily monitored by the above techniques. This paper is an attempt to collect information on processes occurring within this gap. For this we have used a system which, on one hand, is far more physiologically intact than, for instance, rhodopsin in micelles or in reconstituted membranes, and, on the other hand, considerably less complex than the receptor cells in the intact retina. We have used isolated rod outer segments from the retinae of dark adapted cattle eyes. In this system a rapid light induced light scattering transient “P” can be observed which reflects a rapid disk shrinkage, Le., a process where rhodopsin has already communicated with its environment (Uhl et al., 1977). In this paper we shall compare the kinetics of this disk shrinkage with the kinetics of the MI/III transition2 at various temperatures and values of pH. From this comparison a model will be derived that can account for the trigger process of the above structural event. W e shall then speculate that the trigger process of the disk shrinkage can also be the trigger process of transduction and that the two kinetic processes that become obvious from “P” could close the above mentioned gap. This speculation seems legitimate on the basis of the following characteristics of the disk shrinkage phenomenon (Uhl et al., 1977): (1) The extent of the disk shrinkage shows the same action spectrum as rhodopsin bleaching. (2) Its kinetics (half-time of 15-20 ms at room temperature, 6-8 ms at physiological temperature) is fast enough for a process possibly involved in transduction. (3) The fact that “P” reflects an event involving the entire disk implies that “communication processes” beyond the
From the Institute for Biophysics and Radiation Biology of the University of Freiburg, West Germany. Rec eiced October 9, 1977; reuised manuscript received July 14, 1978. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 46). Present address: Department of Chemistry of the University of Guelph, Guelph, Ontario, Canada, N l G 2W1.
I Abbreviations used: ROS, rod outer segments; ~ M l / I l inetarhodopsin , I-metarhodopsin I 1 reaction; “P”, P signal. What we shall call M I / I I transition in this paper is always the spectroscopically observable event, initiated by light and measured as an increase in absorbance at 380 nm or decrease a t 478 nm. The actual underlying molecular events will be dealt with in the discussion.
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0006-2960/78/0417-5347$01 .OO/O
*
0 1978 American Chemical Society
5348
B IOC H EM ISTRY
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