Structurally conserved C-RFa revealed prolactin releasing activity in vitro and gene expression changes in pituitary of seasonally acclimatized carp

Here we show the cloning and characterization of a novel homolog of prepro C-RFa cDNA from Cyprinus carpió. The deduced preprohormone precursor of 115 amino acids leads to a mature bioactive peptide of 20 amino acids with identical sequence to other teleost C-RFa. Modeling of the mature C-RFa peptide highlighted significant similarity to homologous human PrRP20, specifically the conserved amphipathic system defined by the C-terminal alpha-helix. Clearly, the synthetic C-RFa peptide stimulated prolactin release from primary cultured fish pituitary cells. For the first time, significant variation was shown in C-RFa mRNA and protein levels in the hypothalamus and pituitary between summer- and winter-acclimatized carp. Furthermore, C-RFa protein distribution in carp central nervous tissue was visualized by immunodetection in fibers and cells in hypothalamus, olfactory tract, cerebellum and pituitary stalk. In conclusion, we demonstrated the structure conservation of C-RFa in teleosts and mammals and immunopositive cells and fibers for C-RFa in brain areas. Finally, the increase of C-RFa expression suggests the participation of this hypothalamic factor in the mechanism of modulation in PRL expression in carp.

Large conductance Ca2+-activated K+ (BK) channel: Activation by Ca2+ and voltage

Large conductance Ca2+-activated K+ (BK) channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 ìM-100 ìM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.