PACAP-38 and PACAP(6–38) Degranulate Rat Meningeal Mast Cells via the Orphan MrgB3-Receptor

Infusion of pituitary adenylate cyclase activating peptide-38 (PACAP-38) provokes migraine attacks in migraineurs and headache in non-migraineurs. Adverse events like long-lasting flushing and heat sensation can be terminated with oral antihistamine treatment, indicating the involvement of mast cell activation after PACAP-infusion. Degranulation of rat peritoneal mast cells was provoked by several isoforms of PACAP via previously unknown receptor pharmacology. The effect might thus be mediated either via specific splice variants of the PAC1-receptor or via an unknown receptor for PACAP-38. In the present study, we characterize degranulation of rat meningeal mast cells in response to PACAP-receptor ligands. Furthermore, we investigate if PACAP-38-induced mast cell degranulation is mediated via PAC1-receptor splice variants and/or via the orphan Mas-related G-protein coupled member B3 (MrgB3)-receptor. To address this, the pharmacological effect of different PACAP isoforms on meningeal mast cell degranulation was investigated in the hemisected skull model after toluidine blue staining followed by microscopic quantification. Presence of mRNA encoding PAC1-receptor splice variants and the MrgB3-receptor in rat mast cells was investigated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis. The effect of PACAP isoforms on PAC1- and MrgB3-receptor-expressing Xenopus laevis oocytes were performed by two-electrode voltage-clamp (TEVC) electrophysiology. PACAP-38 is a more potent mast cell degranulating agent than Pituitary Adenylate Cyclase Activating Peptide-27 (PACAP-27) in the meninges. Presence of mRNA encoding the PAC1-receptor and its different splice variants could not be detected in peritoneal mast cells by RT-PCR, whereas the orphan MrgB3-receptor, recently suggested to be a mediator of basic secretagogues-induced mast cell degranulation, was widely present. In PAC1-receptor-expressing Xenopus laevis oocytes both PACAP-38, PACAP-27 and the specific PAC1-receptor agonist maxadilan were equipotent, however, only PACAP-38 showed a significant degranulatory effect on mast cells. We confirmed Pituitary Adenylate Cyclase Activating Peptide(6–38) [PACAP(6–38)] to be a PAC1-receptor antagonist, and we demonstrated that it is a potent mast cell degranulator and have an agonistic effect on MrgB3-receptors expressed in oocytes. The present study provides evidence that PACAP-induced mast cell degranulation in rat is mediated through a putative new PACAP-receptor with the order of potency being: PACAP-38 = PACAP(6–38) > > PACAP-27 = maxadilan. The results suggest that the observed responses are mediated via the orphan MrgB3-receptor

pXOOY: A dual-function vector for expression of membrane proteins in Saccharomyces cerevisiae and Xenopus laevis oocytes

On the quest for solving structures of membrane proteins by X-ray crystallography or cryo-EM, large quantities of ultra-pure protein are a paramount prerequisite. Obtaining enough protein of such high standard is not a trivial task, especially for difficult-to-express membrane proteins. Producing membrane protein for structural studies is often performed in Escherichia coli or Saccharomyces cerevisiae and is frequently complemented with functional studies. Ion channels and electrogenic receptors are traditionally studied in terms of their electrophysiological behavior, which cannot be performed in neither E. coli nor yeast. Therefore, they are frequently characterized in mammalian cells or in Xenopus laevis oocytes. To avoid generating two different plasmids, we here describe the construction of a dual-function plasmid, pXOOY, for membrane protein production in yeast and for electrophysiology in oocytes. pXOOY was constructed such that all elements required for oocyte expression were copied from the dual Xenopus-mammalian vector pXOOM and meticulously introduced into the high-yield yeast expression vector pEMBLyex4. pXOOY is thus designed to preserve the high yield of protein from pEMBLyex4 while simultaneously permitting in vitro transcription for expression in oocytes. We evaluated the performance of pXOOY by comparing expression of two yeast codon optimized human potassium channels, ohERG and ohSlick (Slo2.1) from pXOOY to expression of these channels obtained from the reference vectors pEMBLyex4 and pXOOM. Our proof-of-concept study indicates that accumulation in PAP1500 yeast cells was higher when the channels were expressed from pXOOY, which was verified both qualitatively and quantitatively. Two-electrode voltage clamp measurements in oocytes showed that the pXOOY constructs encoding ohERG and ohSlick gave currents with full preservation of electrophysiological characteristics. Our results show that it is possible to design a dual-function Xenopus-yeast vector without compromising expression in yeast and simultaneously maintaining channel activity in oocytes

pXOOY: A dual-function vector for expression of membrane proteins in Saccharomyces cerevisiae and Xenopus laevis oocytes.

On the quest for solving structures of membrane proteins by X-ray crystallography or cryo-EM, large quantities of ultra-pure protein are a paramount prerequisite. Obtaining enough protein of such high standard is not a trivial task, especially for difficult-to-express membrane proteins. Producing membrane protein for structural studies is often performed in Escherichia coli or Saccharomyces cerevisiae and is frequently complemented with functional studies. Ion channels and electrogenic receptors are traditionally studied in terms of their electrophysiological behavior, which cannot be performed in neither E. coli nor yeast. Therefore, they are frequently characterized in mammalian cells or in Xenopus laevis oocytes. To avoid generating two different plasmids, we here describe the construction of a dual-function plasmid, pXOOY, for membrane protein production in yeast and for electrophysiology in oocytes. pXOOY was constructed such that all elements required for oocyte expression were copied from the dual Xenopus-mammalian vector pXOOM and meticulously introduced into the high-yield yeast expression vector pEMBLyex4. pXOOY is thus designed to preserve the high yield of protein from pEMBLyex4 while simultaneously permitting in vitro transcription for expression in oocytes. We evaluated the performance of pXOOY by comparing expression of two yeast codon optimized human potassium channels, ohERG and ohSlick (Slo2.1) from pXOOY to expression of these channels obtained from the reference vectors pEMBLyex4 and pXOOM. Our proof-of-concept study indicates that accumulation in PAP1500 yeast cells was higher when the channels were expressed from pXOOY, which was verified both qualitatively and quantitatively. Two-electrode voltage clamp measurements in oocytes showed that the pXOOY constructs encoding ohERG and ohSlick gave currents with full preservation of electrophysiological characteristics. Our results show that it is possible to design a dual-function Xenopus-yeast vector without compromising expression in yeast and simultaneously maintaining channel activity in oocytes

Purification of Functional Human TRP Channels Recombinantly Produced in Yeast

(1) Background: Human transient receptor potential (TRP) channels constitute a large family of ion-conducting membrane proteins that allow the sensation of environmental cues. As the dysfunction of TRP channels contributes to the pathogenesis of many widespread diseases, including cardiac disorders, these proteins also represent important pharmacological targets. TRP channels are typically produced using expensive and laborious mammalian or insect cell-based systems. (2) Methods: We demonstrate an alternative platform exploiting the yeast Saccharomyces cerevisiae capable of delivering high yields of functional human TRP channels. We produce 11 full-length human TRP members originating from four different subfamilies, purify a selected subset of these to a high homogeneity and confirm retained functionality using TRPM8 as a model target. (3) Results: Our findings demonstrate the potential of the described production system for future functional, structural and pharmacological studies of human TRP channels

Reciprocity in the Developmental Regulation of Aquaporins 1, 3 and 5 during Pregnancy and Lactation in the Rat

Milk secretion involves significant flux of water, driven largely by synthesis of lactose within the Golgi apparatus. It has not been determined whether this flux is simply a passive consequence of the osmotic potential between cytosol and Golgi, or whether it involves regulated flow. Aquaporins (AQPs) are membrane water channels that regulate water flux. AQP1, AQP3 and AQP5 have previously been detected in mammary tissue, but evidence of developmental regulation (altered expression according to the developmental and physiological state of the mammary gland) is lacking and their cellular/subcellular location is not well understood. In this paper we present evidence of developmental regulation of all three of these AQPs. Further, there was evidence of reciprocity since expression of the rather abundant AQP3 and less abundant AQP1 increased significantly from pregnancy into lactation, whereas expression of the least abundant AQP5 decreased. It would be tempting to suggest that AQP3 and AQP1 are involved in the secretion of water into milk. Paradoxically, however, it was AQP5 that demonstrated most evidence of expression located at the apical (secretory) membrane. The possibility is discussed that AQP5 is synthesized during pregnancy as a stable protein that functions to regulate water secretion during lactation. AQP3 was identified primarily at the basal and lateral membranes of the secretory cells, suggesting a possible involvement in regulated uptake of water and glycerol. AQP1 was identified primarily at the capillary and secretory cell cytoplasmic level and may again be more concerned with uptake and hence milk synthesis, rather than secretion. The fact that expression was developmentally regulated supports, but does not prove, a regulatory involvement of AQPs in water flux through the milk secretory cell

Expression of the Small Conductance Ca²⁺-Activated Potassium Channel Subtype 3 (SK3) in Rat Uterus after Stimulation with 17β-Estradiol

<div><p>Preterm births accounts for roughly 9% of all births worldwide and can have detrimental or even lethal consequences for the infant. However to develop new treatment that will lower the rate of preterm births, more knowledge is required on the factors contributing to the contraction and relaxation of the myometrium. The small conductance Ca²⁺-activated potassium channel subtype 3 (SK3) has been identified in the myometrium of several species including humans, mice and rats, but with great inter species variation of the expression pattern and regulation. The aim of this study was to investigate the expression of SK3 in the uterus of rats stimulated with 17β-estradiol and progesterone in order to get an in depth understanding of the rat uterine SK3. Using immunohistochemistry SK3 was localized to the glandular and luminal endometrial lamina epitheliali. Furthermore, a weak signal was observed in the myometrium. Using Western blot the protein level of SK3 was found to increase in uteri from animals treated with 17β-estradiol, an effect that was not reflected at the mRNA level. The levels of mRNA for SK3 were significantly lower in the uterus of 17β-estradiol-treated animals than in the uterus of ovariectomized animals. We conclude that the SK channels are present in the endometrial epithelium, and possibly also in the myometrium of the rat uterus. Furthermore, the hormonal effect on SK3 caused by 17β-estradiol includes divergent regulation at mRNA and protein levels.</p></div