Boaz Gillo

Characterization of the deg-3/des-2 receptor: a nicotinic acetylcholine receptor that mutates to cause neuronal degeneration.

The nicotinic acetylcholine receptor family (nAChR) is a large family of acetylcholine-gated cation channels. Here we characterize the Caenorhabditis elegans DEG-3/DES-2 nAChR, a receptor identified due to its involvement in neuronal degeneration. Pharmacological analysis of a DEG-3/DES-2 receptor expressed in Xenopus oocytes shows that this receptor is preferentially activated by choline. This choline sensitivity of the DEG-3/DES-2 channel can explain its role in neuronal degeneration, as shown by the toxic effects of choline on oocytes expressing the mutant DEG-3/DES-2 channel. We also show that in C. elegans the DEG-3/DES-2 receptor is localized to nonsynaptic regions, including the sensory endings of chemosensory neurons. This localization is in agreement with a role for this receptor in chemosensation of choline, as inferred from a defect in chemotaxis for choline seen in deg-3 mutants. Thus, this work also provides evidence for the diversity of nonsynaptic activities associated with nAChRs.

Functional expression of brain cholecystokinin and bombesin receptors in Xenopus oocytes

Total RNA was extracted from 15-day-old whole rat brains. Microinjection of the RNA into Xenopus laevis oocytes induced electrophysiological responsiveness to cholecystokinin-8 (CCK) and bombesin (BBS) but not to corticotropin-releasing factor (CRF) or somatostatin. The responses to CCK and BBS were similar in shape, time course, and reversal potential to that induced by receptor mediated phospholipid breakdown and that which is induced by intracellular injection of IP3. These responses were not blocked by atropine or by mianserin, did not require extracellular Ca2+ and were completely suppressed by intracellular injection of EGTA.

Activation of ionic currents in Xenopus oocytes by corticotropin-releasing peptides

Oocytes of the African frog Xenopus laevis are shown by electrophysiological methods to possess receptors for corticotropin-releasing factor (CRF), arginine-vasopressin (AVP) and cholecystokinin (CCK). Oocytes surrounded by their follicular cell envelope responded to CRF or AVP with an outward hyperpolarizing current. This current was mediated by an increased conductance of K+ ions. Pretreatment with the adenylate cyclase activator forskolin or with the cAMP phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) potentiated the responses to these peptides indicating that the cAMP second messenger system may mediate the responses. Oocytes stripped of the follicular envelope, which cannot generate cAMP-dependent K+ currents, did not respond to either CRF or AVP. Oocytes exposed to CCK responded with an inward depolarizing current. This current was carried by an increased conductance to Cl-ions. Removal of the follicular cell layer did not affect the response to CCK. The shape, time course, and reversal potential of the Cl-current suggest that CCK acts through the phosphatidylinositol pathway.

Pharmacology of a capacitative Ca2+ entry in Xenopus oocyte

We have characterized pharmacological properties of inositol trisphosphate (InsP3)-mediated calcium entry pathway in Xenopus oocytes via activation of Ca(2+)-dependent Cl- channels (ICl, Ca) as a sensitive indicator for increase in cytosolic [Ca2+]. This type of Ca2+ entry mechanism is known as a capacitative Ca2+ entry (CCE). Voltage-clamped oocytes were maintained in Ca(2+)-free medium and injected with InsP3 which depleted the InsP3-sensitive Ca2+ stores. 10-20 min later, the oocytes were exposed, at 2-3 min intervals, to 5 mM Ca(2+)-containing medium for 5-10 s which evoked repeated inward Cl- current. No effect of external Ca2+ was apparent before InsP3 injection. To determine the pharmacological characteristics of CCE, oocytes were incubated with various chemical agents in Ca(2+)-free solution and exposed to Ca2+ again in presence of the chemical. It was found that organic Ca2+ channel blockers were relatively ineffective in blocking CCE while the inorganic Ca2+ channel blocker La3+ was most efficient in blocking the current. Attempts to measure conductance increase when the Cl- channels were blocked during activation of Ca2+ influx were unsuccessful. Therefore we tested the hypothesis that the Ca2+ influx is mediated via a Ca-H transporter. Lowering the external pH (to pH 6.5) or application of the protonophore carbonylcyanide p-trifluoromethoxyphenyl hydrazone (EC50 = 2 x 10(-8) M) effectively blocked CCE. Since Ca-H countertransport in the plasma membrane is coupled to Ca2+ extrusion by Ca-ATPase in vascular smooth muscle we suggest that the capacitative Ca2+ entry in Xenopus oocytes may possibly arise from slippage of plasma membrane Ca-ATPase coupled to proton countertransport, a mechanism reported in a variety of cells. Ca2+ slippage may arise from the large Ca2+ gradient produced by the Ca2+ depletion protocol.

[6] Hybrid arrest screening in oocytes

Publisher Summary This chapter describes the method of hybrid arrest screening in the oocyte, which is used in cloning of the gonadotropin-releasing hormone (GnRH) receptor. There are two cloning strategies in which the oocyte bioassay has been used, (i) expression cloning and (ii) cloning by hybrid arrest screening. For expression cloning, mRNAs are transcribed in vitro from a cyclic DNA (cDNA) library containing the receptor of interest. A receptor cDNA clone is identified by its expression in the oocyte following injection of the transcribed mRNA. The original pool of cDNA clones is purified by stepwise fractionations of the response-evoking cDNA mixture until a single clone is obtained. To achieve proper expression of a protein in the oocyte, injection of the entire coding sequence of the mRNA is required. In cases where multiple subunits are required for activity, such as in the case of the nicotinic Ach receptor, mRNA for all subunits must be present for the expression of a functional receptor, but this may not always be feasible from a given cDNA library. The second cloning strategy in which the oocyte bioassay is used is the technique of hybrid arrest screening.