Efferent Innervation to Limulus Eyes In Vivo Phosphorylates a 122 kD Protein.

Efferent fibers innervate all of the eyes of the horseshoe crab, Limulus polyphemus. Driven by a circadian clock located in the central nervous system, the activity of the fibers at night is responsible for anatomical, biochemical, and physiological changes in the eyes, which increase their ability to detect and respond to light. We showed previously that octopamine, a putative efferent neurotransmitter, stimulates the phosphorylation of a 122 kD protein in in vitro preparations of both ventral and lateral eyes by means of a cAMP-dependent mechanism. We now report that phosphorylation of the 122 kD protein in the lateral eye is enhanced in vivo: (1) at night, in correlation with efferent nerve input activated by the circadian clock; and (2) during the day, in response to electrical stimulation of efferent axons. We show further that the 122 kD protein is enriched in, and may be restricted to, tissues that contain photoreceptors. We postulate that this protein is involved in the efferent-stimulated increase in retinal sensitivity.

Light-regulated proteins in Limulus ventral photoreceptor cells.

The protein intermediates of the photoresponse and the modulation of this response in invertebrate photoreceptors are largely unknown. As a first step toward identifying these proteins, we have examined light-stimulated changes in protein phosphorylation in preparations of Limulus photoreceptors. Here we show that light modulates the level of phosphorylation of three proteins associated with Limulus ventral photoreceptors: the upper band of a 46-kD protein doublet (46A) and a 122-kD protein, which become more heavily phosphorylated in response to light, and the lower component of the 46-kD doublet (46B), which is phosphorylated in dark-adapted cells, but not in cells maintained in the light. In dark-adapted preparations, 46A is phosphorylated within 30 s after a flash of light and dephosphorylates over a period of many minutes. It is also a major substrate for calcium/calmodulin-dependent protein kinase (Wiebe et al., 1989); therefore, we speculate that 46A is involved in some aspect of dark adaptation. Interestingly, the level of phosphorylation of 46A is the same when measured from preparations maintained in complete darkness or ambient light for at least 1.5 h. The 122-kD phosphoprotein is the same protein which becomes phosphorylated in response to efferent innervation to Limulus eyes (Edwards et al., 1988) and the efferent neurotransmitter, octopamine (Edwards and Battelle, 1987). It may be involved in the increase in retinal sensitivity and the enhanced response of photoreceptors to light that is initiated by efferent innervation. Its role in light-stimulated processes is not clear. The level of phosphorylation of 46B may be most relevant to the long-term state of adaptation of the photoreceptor cell to light and dark.

Calcium/calmodulin-stimulated phosphorylation of photoreceptor proteins in Limulus.

Calcium (Ca2+) is thought to play a major role in the photoresponse of both vertebrates and invertebrates, but the mechanisms through which Ca2+ exerts its effects are unclear. In many systems, some effects of Ca2+ on cellular processes are thought to be mediated via activation of calcium/calmodulin protein kinase (CaCAM-PK) and the phosphorylation of specific proteins. Thus, protein substrates for CaCAM-PK in photoreceptor cells may be important in mediating the effects of Ca2+ on the photoresponse. In this study, we identify eight substrates for CaCAM-PK found in both the ventral and lateral eyes of Limulus. We focus on a characterization of one of these, a 46-kD substrate. We show that its subcellular distribution in ventral photoreceptors and its isoelectric forms are identical to the 46-kD light-stimulated phosphoprotein (46A) described by Edwards et al. (1989). Furthermore, we present evidence that 46A is unique to photoreceptor cells, and that it is present throughout the cell. Based on the results of this study, and the previous study by Edwards et al. (1989), we propose that 46A is involved in mediating the effects of Ca2+ on Limulus photoreceptor cell function, and that it may be involved in dark adaptation.