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S.E. Dryer, G.Y. Ko, M. Ko; An Essential Role for Ras in a Circadian Output Pathway in Vertebrate Cones . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2872.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Cyclic GMP-gated cationic channels (CNGCs) are the last step in phototransduction in the vertebrate retina. We have previously shown that an intrinsic circadian oscillator in cones regulates the gating properties CNGCs such that they have a significantly higher affinity for activating ligand during the subjective night. This effect persists for several days in constant darkness, and is dependent on rhythms in Erk MAP kinase, which is more active during the subjective night. A common mechanism for activation of Erk entails small GTPases such as Ras, Rac, and Rap. Ras, in particular, causes activation of protein kinases of the Raf family, typically leading to activation of MEK enzymes and stimulation of Erk. Here we have examined whether Ras is part of the pathway that leads to circadian modulation of CNGCs in chick cones Methods: Inside-out patch clamp recordings from cultured chick retinal photoreceptors after LD 12:12 entrainment in vivo or in vitro; immunochemical assays of Erk and Ras activation. Biolistic transfection of chick cones with DNA constructs encoding dominant-negative forms of Ras (e.g. RasN17) and related proteins; and GFP (to allow visualization of transfected cells during electrophysiology). Results: Ras activity in cone photoreceptors is much greater during the subjective night (CT16-22) than the subjective day (CT4-10) in chick cones free-running on the second day of DD, and peaks at the same circadian time as Erk activation. To test whether this rhythm is part of the circadian output pathway leading to modulation of CNGCs, we co-transfected cultured cones with expression vectors encoding Ras N17, a dominant-negative form of Ras, and GFP. Transfections were done after 5 days entrainment to LD 12:12 cycles, and patch clamp recordings were done on the next day. This ensured that Ras inhibition was not disrupting entrainment of the cells. Cells expressing RasN17 did not exhibit the normal nocturnal increase in the affinity of CNGCs for cGMP, whereas control cells expressing GFP alone exhibited normal circadian behavior. Moreover, RasN17 also blocked the ability of cAMP treatment to evoke increases in CNGC affinity during the subjective day. A similar pattern was observed in cells transfected with B-RafKN, a dominant-negative form of B-Raf, a MAPKKK that leads to Erk activation in some cell types. This was also seen in cells treated with manumycin-A, a farnesyl transferase inhibitor that blocks Ras activation. Conclusions: Ras activation is rhythmic, and is essential for circadian control of CNGCs. The pathway leading to Erk activation is unusual, albeit not without precedent, in that it may also include B-Raf, instead of Raf-1.
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