In this report, we combined bioinformatic, peptidomic, biochemical, and electrophysiological methods to screen novel secretory peptides that have potential biological effects on neural physiology and function. Our in silico screening strategy included more features that specifically targeted secretory peptide processing, such as prohormone cleavage sites, minimal glycosylation, signal peptide motifs, and sequences conserved across different species. The purpose of this screening strategy was to provide higher numbers of potential candidates with minimal false positive selections. Our screening procedure was based on the protein database derived from full length cDNA sequences from human and mouse. As such, it is possible that some genes were not included in these databases. Additionally, we only selected candidate genes with encoded amino acid sequences that were highly conserved across different species. This criterion could rule out potential candidate genes that are species specific. We used whole-cell electrophysiological recordings on single cells as an assay method to provide a simple and quick assessment of the bioactivity of the potential candidate peptides. The sequence of peptide Lv is embedded in the mouse E130203B14Rik gene, which is known as “Mus musculus 0 day neonate eyeball cDNA, a hypothetical Immunoglobulin and major histocompatibility complex domain containing protein” (AK053685.1). In addition to peptide Lv, this gene could encode other bioactive molecules. The mRNA of peptide Lv is expressed in various organs in mice, so it is possible that its actions are not limited to the nervous system. We found that peptide Lv mRNA was heavily distributed in the photoreceptor layer of the retina. Consequently, we found that peptide Lv modulated L-VGCC activities in retinal photoreceptors, which might reveal a potential new mechanism in the regulation of neuronal function and physiology in the retina.
We found that exogenous peptide Lv enhanced calcium currents, and superfusion of L-VGCC inhibitor nitrendipine blocked the peptide Lv-augmented calcium currents, which indicates that peptide Lv indeed enhanced the L-type VGCCs, but not N- or P/Q type. It bears noting that the L-type VGCC is the major calcium channel in retinal photoreceptors 
. Peptide Lv enhanced L-VGCC currents in photoreceptors may in part be through increasing the expression of mRNA and protein levels of the L-VGCCα1 subunits. However, since it took at least 3 hr of peptide Lv treatment to significantly increase mRNA and protein levels of L-VGCCα1 subunits, as well as the L-VGCC currents, we cannot exclude the possibility that our observation of mRNA and protein levels might be a consequence of other posttranscriptional and posttranslational mechanisms, which we have yet to explore. Calcium influx through the L-VGCCs is essential for neurotransmitter release in retinal photoreceptors, bipolar cells, and other non-spiking neurons 
. Treatment with peptide Lv at E12 induced a larger increase of L-VGCC currents than at E18, indicating that peptide Lv might have a developmental stage-dependent effect or a potential trophic factor-like bioactivity. The L-VGCCs are also involved in synaptic plasticity and learning and memory 
. In the hippocampus, long-term potentiation (LTP) is considered to be the major cellular mechanism underlying learning and memory 
. Inactivation of Cav1.2
) in the hippocampus and neocortex impairs L-VGCC dependent LTP 
. We found that peptide Lv increased L-VGCC current density through increasing both mRNA and protein expression of L-VGCCα1 subunits after 4 hr of treatment. Interestingly, we observed high expression of peptide Lv mRNA in the mouse hippocampus. Moreover, peptide Lv elevated intracellular cAMP production and phosphorylation of ERK, both of which are critical for the induction and maintenance of LTP 
. Hence, we postulate a potential role of peptide Lv in the modulation of hippocampal LTP and synaptic plasticity. Since peptide Lv is present in different organs, it will be worthwhile to investigate additional functions of peptide Lv in the future.
The cellular signaling of many peptide hormones or transmitters is mediated through GPCRs. Over 600 GPCRs have been identified in humans and mice 
. There are various intracellular signaling pathways that involve GPCR signaling, depending on which G-protein and downstream effector is coupled 
. Although we have not yet identified the specific receptor(s) for peptide Lv, we found that treatment with peptide Lv increased intracellular cAMP. As a second messenger, cAMP can further activate downstream signaling components, such as Protein kinase A (PKA), RAS, and cAMP response element-binding protein (CREB), which could further regulate gene expression 
. Inhibition with PTX, a Gα-protein inhibitor, dampened the effect of peptide Lv on cAMP production and ERK phosphorylation. Interestingly, treatment with PTX did not have a significant effect on the increase of L-VGCCs by peptide Lv. Treatment with peptide Lv increased cAMP production and pERK within 15 to 30 min, but it took at least 3 hours to increase the mRNA and protein expression of L-VGCCs. In cardiomyocytes, epinephrine enhances L-VGCCs through cAMP-PKA signaling that further phosphorylate L-VGCCs 
, but this pathway might not be how peptide Lv increased L-VGCCs in photoreceptors. Hence, it is possible that peptide Lv may activate different signaling pathways and cellular effects concurrently, and its actions might be cell-type specific, which will require future investigation.
In summary, we developed a new bioinformatics screening strategy, and in combination with various assay methods, we identified a novel bioactive peptide, peptide Lv. The action of peptide Lv in enhancing L-VGCCs, as well as increasing intracellular cAMP and activating ERK, indicates that peptide Lv may be important in modulating neuronal function. Identification of the specific receptor(s), signaling pathways, and other cellular activities of peptide Lv will be important future directions to investigate.