Cole S. Nelson

Melatonin activates an outward current and inhibits Ih in rat suprachiasmatic nucleus neurons

Whole-cell voltage-clamp recordings were made from suprachiasmatic nucleus (SCN) neurons maintained in horizontal brain slices. The majority of neurons exhibited spontaneous and evoked excitatory and inhibitory synaptic currents (EPSC and IPSC), mediated by glutamate and GABA respectively. Melatonin had no effect on either the spontaneous or evoked EPSC or IPSC. Application of melatonin (0.1-30 microM) during circadian time (CT) 9-12 activated an outward current at -60 mV and increased the membrane conductance in a concentration-dependent manner. The current was augmented by depolarization, reduced by hyperpolarization and, in some cells, reversed its polarity near the potassium equilibrium potential. Some neurons also responded to melatonin during other times of the circadian day (CT 3-9 or CT 12-15). Hyperpolarizing steps, in a portion of cells, activated an inward cation current which resembled the Ih described in other neurons. Melatonin (10 microM) inhibited activation of the Ih. These data indicate that melatonin may inhibit SCN neurons by activating a potassium current and inhibiting the Ih.

Melatonin receptors activate heteromeric G-protein coupled Kir3 channels.

The effects of melatonin on circadian pacemaker activity in the central nervous system may be the result of melatonin receptor activation of G-protein coupled potassium channels which inhibit the action potential firing of neurons. Xenopus laevis and human1a melatonin receptors stimulated heteromeric G-protein activated inwardly rectifying potassium channels (Kir3.1/Kir3.2) when expressed in vitro in oocytes. Pertussis toxin reduced iodo-melatonin (87.1% reduction) and melatonin (90.3% reduction) stimulated currents in a time-dependent manner for cells expressing X. laevis receptors. A similar pertussis toxin inhibition was observed for human melatonin receptors (melatonin, 78.9% reduction). This suggests a potential role for heteromeric Kir3 channels in the receptor-mediated actions of melatonin in vivo.

Cloning and characterization of Kir3.1 (GIRK1) C-terminal alternative splice variants

Southern blot analysis of RT-PCR products from brain and heart revealed multiple products for a C-terminal region of Kir3.1. Sequencing yielded clones for wild-type Kir3.1 and three Kir3.1 C-terminal alternative splice variants, including a unique alternative exon. Two of these variants encoded truncated Kir3.1 molecules. Tissue distribution and electrophysiological characterization of a single truncated variant, Kir3.1(00) were then examined. Kir3.1 channels are gated by G-protein beta gamma-subunits binding to the C-terminal domain, thus, the truncation of Kir3.1(00) removes a major functional domain. When incorporated into heteromeric channels with other family members (Kir3.1, 3.2 or 3.4) several functional changes were observed: (1) Kir3.1(00) changes G-protein activation of Kir3 channels; (2) Kir3.1(00) is restricted in its ability to assemble with other channel subunits as heteromers; and (3) incorporation of Kir3.1(00) into heteromeric channel complexes alters the kinetics of channel re-activation.

Cyclic AMP Analogs Induce Synthesis, Processing, and Secretion of Prepro Nociceptin/Orphanin FQ-Derived Peptides by NS20Y Neuroblastoma Cells

Recent studies have shown that cAMP analogs can induce expression of prepro (pp) orphanin FA (OFQ)/nociceptin-related gene products in NS20Y mouse neuroblastoma cells (Saito et al. [1996]. J Biol Chem 271, 15615-15622). Additionally, exposure of NS20Y cells to cAMP analogs promoted neurite outgrowth and large dense-core vesicle formation. Even though an OFQ-like precursor (called 27K) was identified in NS20Y cell extracts, no secretion of OFQ-related peptides was detected. We have used reversed-phase high-performance liquid chromatography combined with a specific radioimmunoassay for OFQ(1-17) to determine if NS20Y cells secrete ppOFQ-derived peptides when stimulated by the cAMP analog ctp-cAMP. We found that NS20Y cells secreted abundant amounts of OFQ-derived products when stimulated by cAMP analogs. We also have determined that secretion of OFQ peptides was both time and concentration dependent and reversible on removal of cAMP analogs from the culture medium. In addition, the opioid agonist D-Pen2-D-Pen5-enkephalin inhibited forskolin-stimulated OFQ peptide secretion. Further, the synthetic glucocorticoid dexamethasone virtually abolished ctp-cAMP-stimulated OFQ peptide secretion. These results suggest that the biosynthesis, processing, and secretion of the OFQ neuropeptide transmitter system can be modulated through intracellular cAMP levels and that these functions are regulated by opioids and molecules involved in mediating the stress response. The NS20Y cell system will be extremely valuable for studying the regulation of OFQ-derived peptides by a variety of intra-cellular and extracellular signaling pathways.

Melatonin receptor potentiation of cyclic AMP and the cystic fibrosis transmembrane conductance regulator ion channel

Abstract: We have used the cystic fibrosis transmembrane conductance regulator (CFTR) CI‐ channel as a model system to study the cAMP signal transduction pathways coupled to the Xenopus melatonin receptor. During forskolin (Fsk) stimulation, melatonin reduced the amplitude of the CFTR currents in oocytes injected with in vitro transcribed cRNAs for the Xenopus melatonin receptor and CFTR. Pertussis toxin (Ptx) treatment eliminated melatonin inhibition of Fsk stimulated CFTR currents. In oocytes injected with cRNA for melatonin receptors, serotonin receptors (5‐HT7). and CFTR CI− channels, application of melatonin together with serotonin (5‐HT) activated an additional inward current showing potentiation of adenylyl cyclases by melatonin receptors. Subthreshold activation of 5‐HT7 receptors was sufficient and necessary to permit activation of CFTR channels by melatonin. Preexposure to melatonin desensitized the melatonin receptor mediated response. Therefore, based on this model system, the effects of melatonin in vivo could be either positive or negative modulation of other neuronal inputs, depending on the mode of adenylyl cyclase stimulation.

Synaptic Transmission and Signal Transduction in the Suprachiasmatic Nucleus

Circadian rhythms are daily patterns of physiological processes which persist in the absence of environmental time cues, indicating the existence of an endogenous biological clock. In humans, the circadian clock plays a important role in sleep-wake cycles, jet lag, and the performance ability of shift workers. The circadian clock of mammals is located in the suprachiasmatic nucleus (SCN), which is composed of nuclei located bilaterally of the third ventricle and dorsal to the optic chiasm, each consisting of approximately 8,000 neurons (Van den Pol 1980).