Expression of rat mRNA coding for hormone-stimulated adenylate cyclase in Xenopus oocytes

Beta-Adrenergic agonist-stimulated hyperpolarization, whole-cell cAMP accumulation, and activity of isoproterenol-stimulated membrane-bound adenylate cyclase (EC 4.6.1.1) in Xenopus laevis ovarian oocytes are entirely dependent on the presence of nascent follicle cells. A method was developed to remove rapidly and completely all extra-oocyte cell types to yield defolliculated oocytes that exhibited normal viability and resting membrane potentials yet lacked beta-adrenergic receptor (beta AR)-stimulated responses. Purified follicle membranes contained beta AR-stimulated adenylate cyclase activity, whereas oocyte cell membranes did not. Purified oocyte membrane preparations from X. laevis oocytes previously microinjected with C6-2B rat astrocytoma mRNA, and subsequently defolliculated, exhibited novel beta AR and forskolin-stimulated adenylate cyclase activity. These experiments demonstrate that oocytes expressed rat C6-2B mRNA coding for the beta-adrenergic receptor and the components necessary for forskolin-stimulated adenylate cyclase activity.     

Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.     

Expression of epithelial Na channels in Xenopus oocytes

Epithelial Na channel activity was expressed in oocytes from Xenopus laevis after injection of mRNA from A6 cells, derived from Xenopus kidney. Poly A(+) RNA was extracted from confluent cell monolayers grown on either plastic or permeable supports. 1-50 ng RNA was injected into stage 5-6 oocytes. Na channel activity was assayed as amiloride- sensitive current (INa) under voltage-clamp conditions 1-3 d after injection. INa was not detectable in noninjected or water-injected oocytes. This amiloride-sensitive pathway induced by the mRNA had a number of characteristics in common with that in epithelial cells, including (a) high selectivity for Na over K, (b) high sensitivity to amiloride with an apparent K1 of approximately 100 nM, (c) saturation with respect to external Na with an apparent Km of approximately 10 mM, and (d) a time-dependent activation of current with hyperpolarization of the oocyte membrane. Expression of channel activity was temperature dependent, being slow at 19 degrees C but much more rapid at 25 degrees C. Fractionation of mRNA on a sucrose density gradient revealed that the species of RNA inducing channel activity had a sedimentation coefficient of approximately 17 S. Treatment of filter-grown cells with 300 nM aldosterone for 24 h increased Na transport in the A6 cells by up to fivefold but did not increase the ability of mRNA isolated from those cells to induce channel activity in oocytes. The apparent abundance of mRNA coding for channel activity was 10-fold less in cells grown on plastic than in those grown on filters, but was increased two- to threefold by aldosterone.     

Expression of amino acid transport systems in Xenopus oocytes injected with mRNA of rat small intestine and kidney

Xenopus and Cynops oocytes were injected with exogenous mRNA prepared from rat small intestine and kidney and their electrical responses to amino acids were measured by both the current clamped and the voltage clamped methods. Oocytes injected with mRNA of rat small intestine showed a depolarization response to several neutral and basic amino acids, and almost no response to acidic amino acids. The responses to amino acids increased with incubation time after injection of mRNA, and followed Michaelis-Menten type kinetics. The responses were dependent on both Na+ concentration and membrane potential, and were inactivated by a sulfhydryl reagent, 5,5-dithiobis(2-nitrobenzoate). These results are interpreted as due to the expression of Na+/amino acid cotransporter(s) in oocytes injected with rat small intestine mRNA. On the other hand, the oocyte injected with rat kidney mRNA showed a hyperpolarization response to neutral amino acids, a depolarization response to basic ones, and almost no response to acidic ones in frog Ringer solution. These responses were independent of Na+ concentration and followed Michaelis-Menten type kinetics. These amino acid response characteristics in oocytes injected with rat kidney mRNA are interpreted as due to the expression of facilitated diffusion carrier protein(s) (uniporter) of amino acids in the oocyte.     

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.     

Expression of an inwardly rectifying K+ channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

The vacuolar membrane protein gamma-TIP creates water specific channels in Xenopus oocytes.

The vacuolar membrane (tonoplast) of higher plant cells contains an abundant 27 kDa protein called TIP (tonoplast intrinsic protein) that occurs in different isoforms and belongs to a large family of homologous channel-like proteins found in bacteria, plants and animals. In the present study, we identified and characterized the function of gamma-TIP from Arabidopsis thaliana by expression of the protein in Xenopus oocytes. gamma-TIP increased the osmotic water permeability of oocytes 6- to 8-fold, to values in the range 1-1.5 x 10(-2) cm/s. Similar results were obtained with the homologous human erythrocyte protein CHIP28, recently identified as the erythrocyte water channel. The bacterial homolog GlpF did not affect the osmotic water permeability of oocytes, but facilitated glycerol uptake, in accordance with its known function. By contrast, gamma-TIP did not promote glycerol permeability. Voltage clamp experiments provided evidence showing that gamma-TIP induced no electrogenic ion transport in oocytes, especially during osmotic challenge that resulted in massive transport of water. These results allow us to conclude that the various protein members of the MIP family have unique and specific transport functions and that the plant protein gamma-TIP likely functions as a water specific channel in the vacuolar membrane.     

Translation of mRNA for human lymphotoxin in microinjected Xenopus oocytes

Synthesis and secretion of biologically active human lymphotoxin (LT) can be detected in Xenopus laevis oocytes following their inoculation with poly(A+) RNA from human stimulated peripheral blood lymphocytes, but not in oocytes inoculated with RNA from unstimulated lymphocytes or from fibroblastoid cells. In size-fractionating mRNA of stimulated lymphocytes most LT activity is found to be coded for by RNA with an approximate sedimentation value of 19 S.     

Voltage-gated ion channels of inflowing current expressed in Xenopus oocytes after injection of rat brain mRNA

The expression of two types of voltage-gated ion channels of the inflowing current ("fast" sodium channels, sensitive to tetrodotoxin, and high-threshold calcium channels) was detected by electrophysiological methods in the membrane ofXenopus oocytes, after injection of poly(A)⁺-mRNA from the brains of 18- to 20-day-old rats. When Cd²⁺ (200 µmoles/liter) was added to the extracellular solution, the barium current through the expressed calcium channels was completely suppressed, but no sensitivity to D-600 (20 µmoles/liter) and nitrendipine (50 µmoles/liter) was exhibited. A peptide blocker of the high-threshold calcium channels of the neuron membrane, -conotoxin GVIA, in a concentration of 1 µmole/liter led to 20–40 min suppression of the barium current expressed in the oocyte. Steady-state inactivation of this current could be described by the Boltzman formula, using the values of the half-inactivation potential V_1/2=–50 mV and the steepness factor k=14 mV. It is concluded that in potential-dependent and pharmacological properties, the calcium channels expressed in the oocyte, despite the absence of any appreciable time-dependent inactivation, most resemble the high-threshold inactivatable (HTI- or N-type) calcium channels of the neuron membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 344–353, May–June, 1991.     

Rabies mRNA translation in Xenopus laevis oocytes.

Two rabies virus-specific mRNA species were identified by analysis of their encoded proteins after translation of the partially purified species in Xenopus laevis oocytes. One of these coded for the virion surface glycoprotein (G protein), and the other coded for the major structural protein of the virion nucleocapsid (N protein). The G-mRNA sedimented in a sucrose density gradient at about 18S, and the N-mRNA had a sedimentation coefficient of approximately 16S. Their respective translation products were identified in a radioimmunoassay with specific monoclonal antibody probes that recognized only G or N proteins. Immunoprecipitates formed between the radiolabeled viral antigens synthesized in programmed oocytes and their respective monoclonal antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The glycoprotein antigen translated from G-mRNA in oocytes migrated in the gel ahead of the virion G protein with a migration rate that was similar to that of nonglycosylated intracellular glycoproteins from virus-infected cells. The results suggested that the branched-chain carbohydrate of G protein was not required for recognition by the particular monoclonal antibody used. The nucleocapsid antigen translated from N-mRNA in oocytes migrated to the same position in the gel as marker virion N protein. Both the electrophoretic mobility of virus-specific antigens in sodium dodecyl sulfate-polyacrylamide gel and the antibody concentration dependence for immunoprecipitations were criteria for identifying the individual viral proteins encoded by the two rabies mRNA's.     

Expression of Na/Pi cotransport from opossum kidney cells in Xenopus laevis oocytes

Xenopus laevis oocytes have been used for the expression of Na/P_i-cotransport activity by injections of poly(A)⁺ RNA (mRNA) isolated from an established renal cell line (OK cells). 3–5 days after mRNA injection, Na-dependent phosphate (P_i) uptake by oocytes was increased in a dose-dependent manner; there was no increase in Na-independent P_i uptake. Sucrose density-gradient fractionation indicated that the mRNA species encoding this activity is 2.4–2.8 kb in length. In Northern blots, using a cDNA probe related to human kidney-cortex Na/P_i-cotransport activity (NaPi-3), hybridization with a mRNA-species of 2.4–2.6 kb was obtained. Kinetic characterization ([P_i], [Na]) showed that expressed transport activity has properties similar to apical Na/P_i cotransport in OK cells.     

Inactivation of cloned Na channels expressed in Xenopus oocytes

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein.     

Photocrosslinking of proteins to maternal mRNA in Xenopus oocytes

Ultraviolet irradiation was used to covalently crosslink poly(A) RNA and associated proteins in Xenopus oocytes and reticulocytes. Each cell type contained similar as well as unique crosslinked proteins. The somatic cells contained a single 78-kDa 3' poly(A) tract binding protein while oocyte poly(A), however, was bound by this protein and at least three additional proteins. Based on the mass of poly(A) RNA, oocytes in their earliest stages of growth contained crosslinked proteins that were generally more prevalent than in fully grown oocytes. An investigation of possible messenger RNA-specific proteins was undertaken by a series of RNA injection experiments. Two radiolabeled SP6-derived mRNAs were injected into oocytes; the first, globin mRNA, assembled into polysomes, while the second, a maternal mRNA termed G10, entered a nontranslating ribonucleoprotein compartment. Following the induction of oocyte maturation, additional globin mRNA was recruited onto polysomes while G10 mRNA remained a nontranslating mRNP. The proteins that can be crosslinked to these injected mRNAs were detected by 32P nucleotide transfer. Each mRNA associated with shared as well as unique proteins, some of which were detected only in mature oocytes. The possible function of these proteins is discussed.     

Expression of an outward-rectifying potassium channel from maize mRNA and complementary RNA in Xenopus oocytes.

Injection of Xenopus oocytes with poly(A)+ mRNA isolated from different plants (maize, cucumber, and squash) results in the appearance of a voltage- and time-dependent, potassium-selective, outward current that is similar to the outward-rectifying potassium current recorded in many higher plant cells. Maize shoots were found to be especially enriched in mRNA encoding such activity. A cDNA library of maize shoot mRNA was constructed in the vector lambda ZAPII and was used to synthesize RNA complementary to the cDNA (cRNA). Injection of the cRNA gave rise to an outward-rectifying potassium current with properties similar to the currents obtained by poly(A)+ mRNA injection. These results demonstrate that higher plant mRNA can be properly translated into a product that produces a voltage-regulated potassium channel in the plasma membrane of Xenopus oocytes. Thus, Xenopus oocytes can be used as a heterologous expression system for the functional identification and isolation of plant ion channel genes as well as for the study of structure-function relationship of plant ion channels.     

Separate fractions of mRNA from Torpedo electric organ induce chloride channels and acetylcholine receptors in Xenopus oocytes.

Poly(A)+ mRNA extracted from the electric organ of Torpedo was fractionated by sucrose density gradient centrifugation. After injection into Xenopus oocytes one mRNA fraction induced the appearance of chloride channels in the oocyte membrane. Many of these channels were normally open, and the ensuing chloride current kept the resting potential of injected oocytes close to the chloride equilibrium potential. When the membrane was hyperpolarized, the chloride current was reduced. A separate fraction of mRNA induced the incorporation of acetylcholine receptors into the oocyte membrane. When translated in a cell-free system this fraction directed the synthesis of the alpha, beta, gamma, and delta subunits of the acetylcholine receptor. In contrast, the mRNA fraction that induced the chloride channels caused the synthesis of the delta subunit, a very small amount of alpha, and no detectable beta or gamma subunits. This suggests that the size of the mRNA coding for the chloride channel is similar to the preponderant species of mRNA coding for the delta subunit of the acetylcholine receptor.     

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA

Summary— Using the whole cell voltage-clamp technique and a Cl free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 ± 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of I_Ba could be detected (exogenous I_Ba). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 ± 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous I_Ba could be divided into two components: a “fast component” and a “slow component” probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous I_Ba was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.     

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.     

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA

Using the whole cell voltage-clamp technique and a Cl free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 ± 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of I_Ba could be detected (exogenous I_Ba). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 ± 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous I_Ba could be divided into two components: a “fast component” and a “slow component” probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous I_Ba was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.