Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus.

The cytoplasmic injection of mRNA synthesized in vitro into Xenopus oocytes is widely used for heterologous expression of ion channels and neurotransmitter receptors. We report two new methods for expression of ion channels and receptors in oocytes using vaccinia virus (VV). 1) A recombinant VV carrying the Shaker H4 K+ channel cDNA driven by the VV P7.5 early promoter was injected into oocytes. 2) A recombinant VV containing the bacteriophage T7 RNA polymerase driven by the P7.5 promoter was coinjected along with plasmids containing a T7 promoter and cDNAs for channels and receptors. The functionally expressed proteins include a) voltage-gated ion channels: the Shaker H4 K+ channel and the rat brain IIA Na+ channel, b) a ligand-gated ion channel: the mouse muscle nicotinic acetylcholine receptor (AChR), and c) a G protein-coupled receptor: the rat brain 5HT1C receptor. After virus/cDNA injection into oocytes, these channels and receptors generally showed characteristics and expression levels similar to those observed in mRNA-injected oocytes. However, the AChR expressed at lower levels in virus/cDNA-injected oocytes than in mRNA-injected oocytes. Because our methods bypass mRNA synthesis, they are more rapid and convenient than the mRNA injection method. Potential applications to structure-function studies and expression cloning are discussed.     

An endogenous monocarboxylate transport in Xenopus laevis oocytes

We investigated the existence of an endogenous system for lactate transport in Xenopus laevis oocytes. (36)Cl-uptake studies excluded the involvement of a DIDS-sensitive anion antiporter as a possible pathway for lactate movement. L-[(14)C]lactate uptake was unaffected by superimposed pH gradients, stimulated by the presence of Na(+) in the incubating solution, and severely reduced by the monocarboxylate transporter inhibitor p-chloromercuribenzenesulphonate (pCMBS). Transport exhibited a broad cation specificity and was cis inhibited by other monocarboxylates, mostly by pyruvate. These results suggest that lactate uptake is mediated mainly by a transporter and that the preferred anion is pyruvate. [(14)C]pyruvate uptake exhibited the same pattern of functional properties evidenced for L-lactate. Kinetic parameters were calculated for both monocarboxylates, and a higher affinity for pyruvate was revealed. Various inhibitors of monocarboxylate transporters reduced significantly pyruvate uptake. These studies demonstrate that Xenopus laevis oocytes possess a monocarboxylate transport system that shares some functional features with the members of the mammalian monocarboxylate cotransporters family, but, in the meanwhile, exhibits some particular properties, mainly concerning cation specificity.     

Cyclic AMP phosphodiesterase activities in Xenopus laevis oocytes

Cyclic AMP phosphodiesterase activity has been identified in full-grown Xenopus oocytes in vivo and in vitro. About 50% of the in vitro phosphodiesterase activity was present in the solution fraction and 35% in a partially purified membrane fraction. Both activities exhibited high substrate affinity (Km about 10(-6) M). Sucrose gradient fractionation revealed two forms of phosphodiesterase: a 5 S form (peak I) and a 6.5 S form (peak II). Treatment with trypsin led to the activation of the soluble enzyme with the transformation of peak II into peak I. Ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, calcium dependent regulator, and Fluphenazine did not influence the enzyme activities suggesting that the oocyte phosphodiesterases were not Ca2+-dependent. Intact oocytes were induced to mature by exposure to progesterone; their phosphodiesterase activities and distribution tested in vitro were comparable to those of untreated oocytes.     

Improved technique for studying ion channels expressed in Xenopus oocytes, including fast superfusion.

The study of whole-cell currents from ion channels expressed in Xenopus oocytes with conventional two-electrode voltage clamp has two major limitations. First, the large diameter and spherical geometry of oocytes prevent extremely fast solution changes. Second, the internal medium is not controlled, which limits the experimental versatility of the oocyte expression system. For example, because the internal medium is not controlled, endogenous calcium-activated chloride conductances can contaminate currents measured with channels that are permeable to calcium. We describe a new technique that combines vaseline-gap voltage clamp for oocytes with a fast superfusion system. The vaseline-gap procedure is simplified by having the micropipette that monitors voltage serve a dual role as a perfusion micropipette that controls the internal solution. In addition, the technique provides fast external solution changes that are complete in 30-50 ms. We applied the approach to measure the calcium permeability of a muscle and a neuronal nicotinic acetylcholine receptor. Very fast agonist induced currents were measured without contamination by the secondary activation of calcium-dependent chloride channels.     

Endogenous L-glutamate transport in oocytes of Xenopus laevis.

The existence of an endogenous Na(+)-glutamate cotransporter in the oocytes of Xenopus laevis is demonstrated. The transporter does not accept D-glutamate as substrate. The dependence on substrate displays two saturating components with low (K1/2 = 9 mM) and high (K1/2 = 0.35 microM) affinities for L-glutamate. The dependence on external Na+ exhibits a saturating component with a K1/2 value of about 5 mM and a component that has not saturated up to 110 mM Na+. In voltage-clamped oocytes, it is possible to demonstrate that Na(+)-dependent L-glutamate transport is directly coupled to countertransport of Rb+. The analysis of the voltage dependence of the Na+,K(+)-dependent L-glutamate uptake suggests that positive charges are moved inwardly during the transport cycle.     

Kinesin II mediates Vg1 mRNA transport in Xenopus oocytes

The subcellular localization of specific mRNAs is a widespread mechanism for regulating gene expression. In Xenopus oocytes microtubules are required for localization of Vg1 mRNA to the vegetal cortex during the late RNA localization pathway. The factors that mediate microtubule-based RNA transport during the late pathway have been elusive. Here we show that heterotrimeric kinesin II becomes enriched at the vegetal cortex of stage III/IV Xenopus oocytes concomitant with the localization of endogenous Vg1 mRNA. In addition, expression of a dominant negative mutant peptide fragment or injection of a function-blocking antibody, both of which impair the function of heterotrimeric kinesin II, block localization of Vg1 mRNA. We also show that exogenous Vg1 RNA or Xcat-2, another RNA that can use the late pathway, recruits endogenous kinesin II to the vegetal pole and colocalizes with it at the cortex. These data support a model in which kinesin II mediates the transport of specific RNA complexes destined for the vegetal cortex.     

The use of Xenopus oocytes for the study of ion channels

Recently, in addition to the "traditional" research on meiotic reinitiation and fertilization mechanisms, the oocytes of the African frog Xenopus laevis have been exploited for the study of numerous aspects of ion channel function and regulation, such as the properties of several endogenous voltage-dependent channels and the involvement of second messengers in mediation of neurotransmitter-evoked membrane responses. In addition, injection of these cells with exogenous messenger RNA results in production and functional expression of foreign membranal proteins, including various voltage- and neurotransmitter-operated ion channels originating from brain, heart, and other excitable tissues. This method provides unique opportunities for the study of the structure, function, and regulation of these channels. A multidisciplinary approach is required, involving molecular biology, electrophysiology, biochemistry, pharmacology, and cytology.     

Characterization of neutral and cationic amino acid transport in Xenopus oocytes

Amino acid transport was characterized in stage 6 Xenopus laevis oocytes. Most amino acids were taken up by the oocytes by way of both Na+-dependent and saturable Na+-independent processes. Na+-dependent transport of 2-aminoisobutyric acid (AIB) was insensitive to cis- or trans-inhibition by the System A-defining substrate 2-(methylamino)-isobutyric acid (MeAIB), although threonine, leucine, and histidine were found to be effective inhibitors, eliminating greater than 80% of Na+-dependent AIB uptake. Lack of inhibition by arginine eliminates possible mediation by System Bo,+ and suggests uptake by System ASC. The Na+-dependent transport of characteristic System ASC substrates such as alanine, serine, cysteine, and threonine was also insensitive to excess MeAIB. Evidence to support the presence of System Bo,+ was obtained through inhibition analysis of Na+-dependent arginine transport as well arginine inhibition of Na+-dependent threonine uptake. The Na+-independent transport of leucine was subject to trans-stimulation and was inhibited by the presence of excess phenylalanine, histidine, and, to a lesser extent, 2-amino-(2,2,1)-bicycloheptane-2-carboxylic acid (BCH). These observations are consistent with mediation by System L. The characteristics of Na+-independent uptake of threonine are not consistent with assignment to System L, and appear to be reflective of Systems asc and bo,+. In its charged state, histidine appears to be transported by a carrier similar in its specificity to System y+, but is taken up by System L when present as a zwitterion.     

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host''s oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.Download video file.^{(57M, mov)}     

Size selected mRNA induces expression of P-aminohippurate transport in Xenopus oocytes

Xenopus oocytes were injected with size-fractionated mRNA isolated from the renal cortex of rabbit kidney and after 4 days incubation, PAH uptake in oocytes injected with mRNA (0.7-1.3 kb) was 8 to 45 fold that of the water injected controls. The oocyte to medium ratio of accumulated PAH was 1.95. The Km and Vmax for transport were 333 microM and 66.6 nmoles.oocyte-1.min-1, respectively. This Km is similar to that reported for PAH transport in intact kidneys and slices. The uptake of PAH was unaffected by the absence of Na+ or the presence of probenecid. Expression of the transport represents the first step in an effort to clone and identify the gene for PAH transport.     

Functional expression of renal organic anion transport in Xenopus laevis oocytes

Secretion of organic anions by the kidney plays a critical role in the elimination of toxic agents from the body. Recent findings in isolated membranes and intact tissue have demonstrated the participation of multiple transport proteins in this process. As a first step toward molecular characterization of these proteins through expression cloning, the studies reported below demonstrate functional expression of both fumarate- and lithium-sensitive glutarate and probenecid-sensitive p-aminohippurate transport in Xenopus oocytes injected with rat kidney poly(A)⁺RNA. Maximal increase in substrate uptake over buffer-injected controls was reached by 5 days after mRNA injection. Expression of size-fractionated mRNA indicated that the active species with respect to both transport activities were in the range of 1.8 to 3.5 kb.     

Molecular mechanisms of organic cation transport in OCT2-expressing Xenopus oocytes

The molecular mechanisms of organic cation transport by rat OCT2 was examined in the Xenopus oocyte expression system. When extracellular Na+ ions were replaced with K+ ions, uptake of tetraethylammonium (TEA) by OCT2-expressing oocytes was decreased, suggesting that TEA uptake by OCT2 is dependent on membrane potential. Kinetic analysis revealed that the decreased TEA uptake by ion substitution was caused at least in part by decreased substrate affinity. Acidification of extracellular buffer resulted in decreased uptake of TEA, whereas TEA efflux from OCT1- and OCT2-expressing oocytes was not stimulated by inward proton gradient, in consistent with basolateral organic cation transport in the kidney. Inhibition of TEA uptake by various organic cations revealed that apparent substrate spectrum of OCT2 was similar with that of OCT1. However, the affinity of procainamide to OCT1 was higher than that to OCT2. Uptake of 1-methyl-4-phenylpyridinium was stimulated by OCT2 as well as OCT1, but uptake of levofloxacin, a zwitterion, was not stimulated by both OCTs. These results suggest that OCT2 is a multispecific organic cation transporter with the characteristics comparable to those of the basolateral organic cation transporter in the kidney.     

Actin in Xenopus oocytes

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.     

Multiple effects of mercuric chloride on hexose transport in Xenopus oocytes.

HgCl(2) had both stimulatory and inhibitory effects on [(3)H]2-deoxyglucose (DG) uptake in Xenopus laevis oocytes. The Hg dose response was complex, with 0.1-10 microM Hg increasing total DG uptake, 30-50 microM Hg inhibiting, and concentrations >100 microM increasing uptake. Analyses of the effects of Hg on DG transport kinetics and cell membrane permeability indicated that low concentrations of Hg stimulated mediated uptake, intermediate concentrations inhibited mediated uptake, but high Hg concentrations increased non-mediated uptake. 10 microM Hg increased the apparent V(max) for DG uptake, but caused little or no change in apparent K(m). Phenylarsine oxide prevented the increase in DG uptake by 10 microM Hg, suggesting that the increase was due to transporter recruitment. Microinjecting low doses of HgCl(2) into the cell increased mediated DG uptake. Higher intracellular doses of Hg increased both mediated and non-mediated DG uptake. Both insulin and Hg cause cell swelling in isotonic media and, for insulin, this swelling has been linked to the mechanism of hormone action. Osmotically swelling Xenopus oocytes stimulated DG transport 2-5-fold and this increase was due to an increased apparent V(max). Exposing cells to 10 microM Hg or 140 nM insulin both increased cellular water content by 18% and increased hexose transport 2-4-fold. These data indicate that low concentrations of Hg and insulin affect hexose transport in a similar manner and that for both an increase cellular water content could be an early event in signaling the increase in hexose transport.     

Ammonium transport by the colonic H+-K+-ATPase expressed in Xenopus oocytes

Functional expression of the rat colonicH⁺-K⁺-ATPasewas obtained by coexpressing its catalytic -subunit and the₁-subunit of theNa⁺-K⁺-ATPasein Xenopus laevis oocytes. We observedthat, in oocytes expressing the rat colonicH⁺-K⁺-ATPasebut not in control oocytes (expressing₁ alone),NH₄Cl induced a decrease in⁸⁶Rb uptake and the initial rateof intracellular acidification induced by extracellularNH₄Cl was enhanced, consistentwith NH⁺₄ influx via the colonicH⁺-K⁺-ATPase.In the absence of extracellularK⁺, only oocytes expressing thecolonicH⁺-K⁺-ATPasewere able to acidify an extracellular medium supplemented withNH₄Cl. In the absence ofextracellular K⁺ and in thepresence of extracellular NH⁺₄, intracellular Na⁺ activity in oocytes expressingthe colonicH⁺-K⁺-ATPasewas lower than that in control oocytes. A kinetic analysis of⁸⁶Rb uptake suggests thatNH⁺₄ acts as a competitive inhibitor of thepump. Taken together, these results are consistent withNH⁺₄ competition forK⁺ on the external site of thecolonicH⁺-K⁺-ATPaseand with NH⁺₄ transport mediated by this pump.