Calcineurin serves in the circadian output pathway to regulate the daily rhythm of l-type voltage-gated calcium channels in the retina

The L-type voltage-gated calcium channels (L-VGCCs) in avian retinal cone photoreceptors are under circadian control, in which the protein expression of the a1 subunits and the current density are greater at night than during the day. Both Ras-mitogen-activated protein kinase (MAPK) and Ras- phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT) signaling pathways are part of the circadian output that regulate the L-VGCC rhythm, while cAMP-dependent signaling is further upstream of Ras to regulate the circadian outputs in photoreceptors. However, there are missing links between cAMP-dependent signaling and Ras in the circadian output regulation of L-VGCCs. In this study, we report that calcineurin, a Ca ²⁺ /calmodulin-dependent serine (ser)/threonine (thr) phosphatase, participates in the circadian output pathway to regulate L-VGCCs through modulating both Ras-MAPK and Ras-PI3K-AKT signaling. The activity of calcineurin, but not its protein expression, was under circadian regulation. Application of a calcineurin inhibitor, FK-506 or cyclosporine A, reduced the L-VGCC current density at night with a corresponding decrease in L-VGCCa1D protein expression, but the circadian rhythm of L-VGCCa1D mRNA levels were not affected. Inhibition of calcineurin further reduced the phosphorylation of ERK and AKT (at thr 308) and inhibited the activation of Ras, but inhibitors of MAPK or PI3K signaling did not affect the circadian rhythm of calcineurin activity. However, inhibition of adenylate cyclase significantly dampened the circadian rhythm of calcineurin activity. These results suggest that calcineurin is upstream of MAPK and PI3KAKT but downstream of cAMP in the circadian regulation of L-VGCCs.