Circadian Regulation of cGMP-Gated Channels of Vertebrate Cone Photoreceptors: Role of cAMP and Ras

Abstract

Circadian oscillators in chicken cone photoreceptors regulate the gating properties of cGMP-gated cationic channels (CNGCs) such that they have a higher apparent affinity for cGMP during the subjective night. Here we show that cAMP, acting through protein kinase A (PKA), Ras, and Erk, is part of the circadian output pathway controlling CNGCs. Endogenous and exogenous cAMP cause activation of Erk and Ras, which are more active at night in cones, and increase the apparent affinity of CNGCs for cGMP. The Ras farnesyl transferase inhibitor manumycin-A, and a dominant-negative form of Ras (RasN17) block the circadian rhythms in CNGC gating, as well as the effects of cAMP. A dominant-negative form of the MEK kinase B-Raf also blocks circadian and cAMP modulation of CNGCs. The circadian output pathway modulating CNGC channels is comprised in part of cAMP 3 PKA 3 Ras 3 B-Raf 3 MEK 3 Erk 3 3 CNGCs. cAMP activation of Ras and Erk occur within minutes, whereas modulation of CNGCs requires Ͼ1 hr. However, cAMP protagonists do not alter rhythms in cPer2 mRNA, and their effects on CNGCs cannot be attributed to clock phase-shifting.

The expression of L‐type voltage‐gated calcium channels in retinal photoreceptors is under circadian control

“…Total RNA from cultured cells or intact retinas was collected using a RNA isolation kit (Qiagen, Valencia, CA, USA), with 500 ng of total RNA from each sample used to quantify expression of cPer2 (a clock gene), chick VGCCa subunit and chick b-actin (loading control) mRNA by quantitative-PCR using the Taqman one-step RT-PCR kit and an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) . The forward and reverse primers for cPer2 and chick b-actin are listed in Ko et al (2003Ko et al ( , 2004b. The forward and reverse primers for the VGCCa1D subunit were 5¢-AAACCTGGAG-GCTTTGATGTCA-3¢ and 5¢-CCGGAGAGGTCGCAATACAC-3¢, respectively (GenBank accession number was AF027607).…”

“…The methods used for quantitative real-time reverse transcription (RT)-PCR were described previously (Ko et al 2003(Ko et al , 2004b. Total RNA from cultured cells or intact retinas was collected using a RNA isolation kit (Qiagen, Valencia, CA, USA), with 500 ng of total RNA from each sample used to quantify expression of cPer2 (a clock gene), chick VGCCa subunit and chick b-actin (loading control) mRNA by quantitative-PCR using the Taqman one-step RT-PCR kit and an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) .…”

“…Samples were collected and prepared as described previously (Ko et al 2001(Ko et al , 2004b. Briefly, cultured cells were washed in ice-cold phosphate-buffered saline and lysed in ristocetin-induced platelet agglutination buffer.…”

“…Zeitgeber time zero was designated as when the lights come on, and Zeitgeber time 12 was the time when the lights turn off. The following experiments were made on the second day of DD, after 6 days of prior entrainment to LD cycles as described previously (Ko et al 2001(Ko et al , 2004b. For in ovo entrainment, retinas were excised from E17 embryos obtained from intact eggs that were exposed to LD 12 h: 12 h for 6 days (from E11 to E16).…”

Circadian proteomics of the mouse retina

“…photoreceptor cells (rods and cones), bipolar cells, ganglion cells, amacrine cells, and horizontal cells, have been extensively characterized [4]. From the view point of circadian biology, a wealth of data exist on cellular, molecular, and system level retinal events that are regulated by local circadian clocks, such as the rhythms in rod disc shedding [5], melatonin release [6], dopamine synthesis [7], electroretinogram b-wave amplitude [8], extracellular pH [9], phototransduction events including iodopsin expression [10] and cGMP-gated channel sensitivity [11,12], and intraocular pressure [13,14]. Many of these rhythms are thought to allow the retina to anticipate day-night variation and to detect light signals even in the dynamic variation of light intensity over 6-log units between day and night.…”