MT2-like melatonin receptor modulates amplitude receptor potential in visual cells of crayfish during a 24-hour cycle

Retinular photoreceptors are structures involved in the expression and synchronization of the circadian rhythm of sensitivity to light in crayfish. To determine whether melatonin possesses a differential effect upon the receptor potential (RP) amplitude of retinular photoreceptors circadian time (CT)-dependent, we conducted experiments by means of applying melatonin every 2 h during a 24-hour cycle. Melatonin with 100 nM increased RP amplitude during subjective day to a greater degree than during subjective night. To determine whether MT2 melatonin receptors regulate the melatonin-produced effect, we carried out two experiments, circadian times (CTs) 6 and 18, which included the following: (1) application of the MT2 receptor selective agonist 8-M-PDOT and antagonist DH97, and (2) the specific binding of [125I]-melatonin in eyestalk membranes. The amount of 10 nM of 8-M-PDOT increased RP amplitude in a similar manner to melatonin, and 1 nM DH97 abolished the increase produced by melatonin and 8-M-PDOT. Binding of [125I]-melatonin was saturable and specific. Scatchard analysis revealed an affinity constant (Kd) of 1.1 nM and a total number of binding sites (Bmax) of 6 fmol/mg protein at CT 6, and a Kd of 1.46 nM and Bmax of 7 fmol/mg protein at CT 18. Our results indicate that melatonin increased RP amplitude of crayfish retinular photoreceptors through MT2-like melatonin receptors. These data support the idea that melatonin is a signal of darkness for the circadian system in crayfish retinular cells.