RNA-injected Oocytes Research Articles

Hec1-Dependent Cyclin B2 Stabilization Regulates the G2-M Transition and Early Prometaphase in Mouse Oocytes

SummaryThe functions of the Ndc80/Hec1 subunit of the highly conserved Ndc80 kinetochore complex are normally restricted to M phase when it exerts a pivotal kinetochore-based role. Here, we find that in mouse oocytes, depletion of Hec1 severely compromises the G2-M transition because of impaired activation of cyclin-dependent kinase 1 (Cdk1). Unexpectedly, impaired M phase entry is due to instability of the Cdk1-activating subunit, cyclin B2, which cannot be covered by cyclin B1. Hec1 protects cyclin B2 from destruction by the Cdh1-activated anaphase-promoting complex (APCCdh1) and remains important for cyclin B2 stabilization during early M phase, required for the initial stages of acentrosomal spindle assembly. By late M phase, however, Hec1 and cyclin B2 become uncoupled, and although Hec1 remains stable, APCCdc20 triggers cyclin B2 destruction. These data identify another dimension to Hec1 function centered on M phase entry and early prometaphase progression and challenge the view that cyclin B2 is completely dispensable in mammals.

Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron

Recent studies have shown that overexpression of the transmembrane protein Zrt- and Irt-like protein 14 (Zip14) stimulates the cellular uptake of zinc and nontransferrin-bound iron (NTBI). Here, we directly tested the hypothesis that Zip14 transports free zinc, iron, and other metal ions by using the Xenopus laevis oocyte heterologous expression system, and use of this approach also allowed us to characterize the functional properties of Zip14. Expression of mouse Zip14 in RNA-injected oocytes stimulated the uptake of (55)Fe in the presence of l-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe(2+)) over ferric ion (Fe(3+)). Zip14-mediated (55)Fe(2+) uptake was saturable (K(0.5) ≈ 2 μM), temperature-dependent (apparent activation energy, E(a) = 15 kcal/mol), pH-sensitive, Ca(2+)-dependent, and inhibited by Co(2+), Mn(2+), and Zn(2+). HCO(3)(-) stimulated (55)Fe(2+) transport. These properties are in close agreement with those of NTBI uptake in the perfused rat liver and in isolated hepatocytes reported in the literature. Zip14 also mediated the uptake of (109)Cd(2+), (54)Mn(2+), and (65)Zn(2+) but not (64)Cu (I or II). (65)Zn(2+) uptake also was saturable (K(0.5) ≈ 2 μM) but, notably, the metal-ion inhibition profile and Ca(2+) dependence of Zn(2+) transport differed from those of Fe(2+) transport, and we propose a model to account for these observations. Our data reveal that Zip14 is a complex, broad-scope metal-ion transporter. Whereas zinc appears to be a preferred substrate under normal conditions, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions.

The influence of calcium ions on nickel modulation of NMDA receptor currents

N-Methyl-D-aspartate (NMDA) receptors (NRs) are glutamate-gated channels critical for the functioning of the nervous system. They are assembled from two types of subunits, the essential GluN1 and at least one type of GluN2 (A, B, C, D) subunit. Nickel (Ni) modulates the NR current in a way dependent on the GluN2 subunit present. Besides voltage-dependent and voltage-independent inhibition, in GluN2B-containing channels Ni enhances channel activity. We have recently identified several domains of the channel involved in Ni interaction, but many aspects of this modulation remain elusive. The purpose of the present work is to investigate the role of calcium (Ca) in the effect of Ni on the NR current measured by voltage- and patch-clamp in RNA-injected Xenopus laevis oocytes or in transiently transfected mammalian HEK293 cells expressing GluN1/GluN2B recombinant receptors. In both expression systems, in the presence of a physiological concentration of Ca (1.8 mM), Ni increased the NR current with EC(50) in the μM range, but this potentiation was reduced by decreasing Ca concentration or when Ca was substituted with Ba. In injected oocytes, the effect of Ni in 0.3 mM external Ba was only inhibitory (IC(50) = 65 μM). Increasing the internal calcium buffering by EGTA and BAPTA application, as well as incubation with cytoskeleton perturbing agents, colchicine and cytochalasin-D, did not produce major modifications in the Ni effect. These observations indicate that Ni-mediated potentiation is not dependent on Ca influx and internal Ca concentration, but it is dependent on external Ca, which possibly interacts with the extracellular portion of the protein through a modulatory binding site.

Sequence- or Position-Specific Mutations in the Carboxyl-Terminal FL Motif of the Kidney Sodium Bicarbonate Cotransporter (NBC1) Disrupt Its Basolateral Targeting and α-Helical Structure

The sodium-bicarbonate cotransporter NBC1 is targeted exclusively at the basolateral membrane. Mutagenesis of a dihydrophobic FL motif (residues 1013-1014) in the C-terminal domain disrupts the targeting of NBC1. In the present study, we determined the precise constraints of the FL motif required for basolateral targeting of NBC1 by expressing epitope-tagged wild-type and mutant NBC1 in MDCK cells and RNA-injected Xenopus oocytes and examining their subcellular localization. We assayed the functional activity of the mutants by measuring bicarbonate-induced currents in oocytes. Wild-type NBC1 (containing PFLS) was expressed exclusively on the basolateral membrane in MDCK cells. Reversal of the FL motif (PLFS) had no effect on basolateral targeting or activity. Shifting the FL motif one residue upstream (FLPS) resulted in mistargeting of the apical membrane but the FLPS mutant retained its functional activity in oocytes. Shifting the FL motif one residue downstream resulted in a mutant (PSFL) that did not efficiently translocate to the plasma membrane and was instead colocalized with the ER marker, protein disulfide isomerase (PDI). Analysis of circular dichroism (CD) revealed that a short peptide, 20 amino acid residues, of wild-type NBC1 contained a significant alpha-helical structure, whereas peptides in which the FL motif was reversed or C-terminally shifted were disordered. We therefore propose that the specific orientation and the precise location of the FL motif in the primary sequence of NBC1 are strict requirements for the alpha-helical structure of the C-terminal cytoplasmic domain and for targeting of NBC1 to the basolateral membrane.

An Innexin-Dependent Cell Network Establishes Left-Right Neuronal Asymmetry in C. elegans

Gap junctions are widespread in immature neuronal circuits, but their functional significance is poorly understood. We show here that a transient network formed by the innexin gap-junction protein NSY-5 coordinates left-right asymmetry in the developing nervous system of Caenorhabditis elegans. nsy-5 is required for the left and right AWC olfactory neurons to establish stochastic, asymmetric patterns of gene expression during embryogenesis. nsy-5-dependent gap junctions in the embryo transiently connect the AWC cell bodies with those of numerous other neurons. Both AWCs and several other classes of nsy-5-expressing neurons participate in signaling that coordinates left-right AWC asymmetry. The right AWC can respond to nsy-5 directly, but the left AWC requires nsy-5 function in multiple cells of the network. NSY-5 forms hemichannels and intercellular gap-junction channels in Xenopus oocytes, consistent with a combination of cell-intrinsic and network functions. These results provide insight into gap-junction activity in developing circuits.

Functional Nicotinic Acetylcholine Receptors That Mediate Ganglionic Transmission in Cardiac Parasympathetic Neurons

Nicotinic acetylcholine receptors (nAChRs) mediate ganglionic transmission in the peripheral autonomic nervous system in mammals. Functional neuronal nAChRs have been shown to assemble from a combination of alpha and beta subunits, including alpha3, alpha5, alpha7, beta2, and beta4 in RNA-injected oocytes, but the subunit composition of functional neuronal nAChRs in vivo in mammals remains unknown. We examined the subunit composition of functional nAChRs in the intracardiac parasympathetic ganglion in a physiologically intact system in vivo. We report here that localized perfusion of the canine intracardiac ganglion in situ with an antagonist specific for nAChRs containing an alpha3/beta2 subunit interface (alpha-conotoxin MII 100-200 nm) resulted in reversible attenuation of the sinus cycle length (SCL) response by approximately 70% to electrical stimulation of the preganglionic vagus nerve. Perfusion with antagonist specific for receptors containing an alpha3/beta4 subunit interface (alpha-conotoxin AuIB 1 micrometer) resulted in attenuation in SCL responses (approximately 20%) compared with baseline when applied by itself, but not in animals pretreated with alpha-conotoxin MII. Perfusion of the ganglion with alpha-bungarotoxin (1 micrometer, which blocks alpha7 receptors) caused a reduction in SCL response by approximately 30% compared with baseline when perfused on its own and when added after blockade with MII and AuIB. Perfusion with hexamethonium bromide resulted in complete blockade of ganglionic transmission, confirming total perfusion of the ganglion and the nicotinic nature of ganglionic transmission at this synapse. Immunohistochemistry using monoclonal antibodies against specific nicotinic subunits confirmed the presence of alpha3, alpha7, beta2, and beta4 subunits. We conclude that functional ganglionic transmission in the canine intracardiac ganglion is mediated primarily by receptors containing an alpha3/beta2 subunit interface, with a smaller contribution by receptors containing alpha7 nAChRs. Despite the presence of beta4 subunits in functional channels, a contribution of a distinct alpha3/beta4 receptor population that does not include an alpha3/beta2 subunit interface was less clear.

Expression of sperm Ca 2+-activated K + channels in Xenopus oocytes and their modulation by extracellular ATP

Ionic fluxes across the sperm membrane have been shown to be important in the initiating process of sperm activation and gamete interaction; however, electrophysiological investigation of the ion channels involved has been precluded by the small size of the sperm, especially in mammalian species. In the present study sperm ion channels were expressed in Xenopus oocytes by injection of RNAs of spermatogenic cells isolated from the rat testes. The RNA-injected oocytes responded to ATP, a factor known to regulate sperm activation, with the activation of an outwardly rectifying whole-cell current which was dependent on K + concentrations and inhibitable by K + channel blockers, charybdotoxin (CTX) and tetraethylammonium (TEA). The ATP-induced current could be mimicked by a Ca 2+ ionophore but suppressed by a Ca 2+ chelator applied intracellularly, indicating a Ca 2+ dependence of the current. Single-channel measurements on RNA-injected oocytes revealed channels of large conductance which could be blocked by CTX and TEA. Co-injection of germ cell RNAs with the antisense RNA for a mouse gene encoding slowpoke `Maxi' Ca 2+-activated K + channels resulted in significant reduction of the ATP- and ionomycin-induced current. The expression of the `Maxi' Ca 2+-activated K + channels in sperm collected from the rat epididymis was also confirmed by Western blot analysis. These results suggest that sperm possess Ca 2+-activated K + channels which may be involved in the process of sperm activation.

Functional expression of a Ca 2+-activated K + channel in Xenopus oocytes injected with RNAs from the rat testis

The present study investigated the feasibility of using Xenopus oocytes to express sperm ion channel by injection of RNAs extracted from the rat testis. The RNA-injected oocytes expressed an outwardly rectifying current which was dependent on K + concentration and inhibitable by K + channel blockers, charybdotoxin (CTX) and tetraethylammonium (TEA). The Ca 2+ ionophore, ionomycin, could also stimulate current activation with similar current characteristics in the RNA-injected oocytes, suggesting the expression of a Ca 2+-activated K + channel. Immunolocalization indicated predominant Ca 2+-activated K + channel immunoreactivity associated with spermatogenic cells. Reverse transcriptase-polymerase chain reaction studies confirmed the expression of the Ca 2+-activated K + channel mRNA in isolated spermatogenic cells. Our results suggest that ion channels and/or receptors of spermatogenic cells could be investigated using the Xenopus oocyte as an expression system. The present study also suggests that sperm may possess a Ca 2+-activated K + channel which has been implicated in the process of sperm activation and gamete interaction.

The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor.

1. The gamma-aminobutyric acid (GABA)-modulatory and GABA-mimetic actions of etomidate at mammalian GABA(A) receptors are favoured by beta2- or beta3- versus beta1-subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (beta2 N290, beta3 N289, beta1 S290). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two-point voltage clamp technique to determine the influence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate-insensitive invertebrate GABA receptor (Rdl) of Drosophila melanogaster. 2. Complementary RNA-injected oocytes expressing the wild type Rdl GABA receptor and voltage-clamped at -60 mV responded to bath applied GABA with a concentration-dependent inward current response and a calculated EC50 for GABA of 20+/-0.4 microM. Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine (RdlM314N) or a serine (RdlM314S) also exhibited a concentration-dependent inward current response to GABA, but in both cases with a reduced EC50 of 4.8+/-0.2 microM. 3. Utilizing the appropriate GABA EC10, etomidate (300 microM) had little effect on the agonist-evoked current of the wild type Rdl receptor. By contrast, at RdlM314N receptors, etomidate produced a clear concentration-dependent enhancement of GABA-evoked currents with a calculated EC50 of 64+/-3 microM and an Emax of 68+/-2% (of the maximum response to GABA). 4. The actions of etomidate at RdlM314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 microM R-(+)-etomidate and S-(-)-etomidate enhancing the current induced by GABA (EC10) to 52+/-6% and 12+/-1% of the GABA maximum respectively. 5. The effects of this mutation were selective for etomidate as the GABA-modulatory actions of 1 mM pentobarbitone at wild type Rdl (49+/-4% of the GABA maximum) and RdlM314N receptors (53+/-2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5alpha-pregnan-3alpha-ol-20-one (Rdl = 25+/-4% of the GABA maximum) was not altered by this mutation (RdlM314N = 18+/-3% of the GABA maximum). 6. Etomidate acting at beta1 (S290)-containing mammalian GABA(A) receptors is known to produce only a modest GABA-modulatory effect. Similarly, etomidate acting at RdlM314S receptors produced an enhancement of GABA but the magnitude of the effect was reduced compared to RdlM314N receptors. 7. Etomidate acting at human alpha6beta3gamma2L receptors is known to produce a large enhancement of GABA-evoked currents and at higher concentrations this anaesthetic directly activates the GABA(A) receptor complex. Mutation of the human beta3 subunit asparagine to methionine (beta3 N289M found in the equivalent position in Rdl completely inhibited both the GABA-modulatory and GABA-mimetic action of etomidate (10-300 microM) acting at alpha6beta3 N289Mgamma2L receptors. 8. It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modification of their function by etomidate can be influenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction. Furthermore, this study provides a clear example of the specificity of anaesthetic action.

Characterization of ion currents elicited by a stream of fluid during spontaneous and ligand-induced chloride current oscillation in Xenopus laevis oocytes.

During Ca2+-activated C- current oscillations a mechanical deformation of the Xenopus laevis oocyte by a fluid stream evokes transient inward currents of high amplitude (stream evoked inward current, Ii,st). This current can be observed either in native or RNA-injected oocytes expressing ligand-controlled ion channels from rat brain. Ii,st reversed at the equilibrium potential of chloride and was blocked by 9-anthracene carboxylic acid (2 mM). Power spectral analysis of the oscillations did not reveal a correlation between the features of the oscillations and the amplitude of Ii,st. Antagonists of stretch-activated cation channels [gadolinium (100 microM) and lanthanum (1mM)] did not block Ii,st. Calcium channel blockers [cobalt and manganese (10 mM)] did not inhibited Ii,st and Ii,st could also be elicited in calcium-free medium. Preloading oocytes with pertussis toxin (PTX) for 17 h prevented current oscillations and Ii,st caffeine (10 mM), an antagonist of the liberation of calcium from intracellular stores, inhibited Ii,st. Our results proride evidence for modulation of the mechanosensitivity of chloride currents by activation of intracellular second messenger cascades.

DrosophilaSerotonin Transporters Have Voltage-Dependent Uptake Coupled to a Serotonin-Gated Ion Channel

Serotonin (5HT) transporters (SERTs) couple to existing ion gradients to transport 5HT into presynaptic terminals. In mammalian SERTs, the transport cycle is reported as electroneutral, with a translocation of zero net charge, and 5HT uptake is independent of membrane voltage. Yet mammalian SERTs exhibit 5HT-induced currents, and Drosophila SERTs (dSERTs) show voltage-dependent uptake. Thus, the relationship between uptake and current remains controversial; furthermore, the number of 5HT molecules translocated per ion channel event is unknown. To investigate this, we have used heterologous expression of cloned dSERTs to measure 5HT flux and dSERT currents concurrently under voltage clamp, and we have used fluctuation analysis to measure the size of the elementary ionic events in the same cells. RNA-injected Xenopus oocytes accumulate 5HT, and paroxetine or desipramine inhibit this uptake. RNA-injected oocytes also display paroxetine-sensitive 5HT-induced currents and 5HT-independent leak currents. Na replacement decreases the uptake and the induced currents. 5HT-induced current and 5HT uptake both increase at negative potentials, where 5HT carries approximately 5% of the induced current. Recently, several groups have reported similar phenomena for other transporters, in which transmitter-induced currents exceed the predictions of coupled transport. We now provide evidence that in dSERT, approximately 500 5HT molecules are translocated per channel opening, which, at -20 mV, carries approximately 10,000 electronic charges. These data support a model in which 500 SERT cycles occur for each 5HT-induced channel opening or a model in which 500 5HT molecules and 10,000 electronic charges pass through a common pore.

Functional Expression of a P 2T ADP Receptor in Xenopus Oocytes Injected With Megakaryocyte (CMK 11-5) RNA

Since the P2T purinergic (ADP) receptor is unique to the megakaryocytic/platelet lineage, cells of this lineage were screened for the relative effects of ADP and ATP in intracellular Ca2+ levels. Like platelets, CMK 11-5 cells responded with an increase in intracellular Ca2+ mobilization in response to ADP but not to ATP or adenosine. In contrast, both nucleotides increased intracellular Ca2+ mobilization in the megakaryoblastic cell lines MO7E and Meg-01, indicating that they contain P2Y receptors or a mixed complement of purinergic receptors. Pharmacological responsiveness of CMK 11-5 cells to nucleotides paralleled those of platelets, in which ADP and ADP-alpha-S are active as agonists and ATP and ATP-alpha-S are inactive as agonists but act as antagonists. [3H]ADP and 35S-ATP-alpha-S bound to CMK 11-5 cells at a high-affinity site (Kd1 and Ki1, 262 and 125 nmol/L, respectively) and a low-affinity site (Kd2 and Ki2, 10,100 and 5400 nmol/L, respectively) with 2 x 10(6) to 6 x 10(6) sites per cell. ADP bound at both sites was competed with ADP, ATP, and ATP-alpha-S with affinities in a rank order similar to that found for platelets (ATP-alpha-S approximately ATP approximately ADP > or = ADP-beta-S approximately adenosine), suggesting the presence of a P2T receptor on CMK 11-5 cells. Photoaffinity labeling of intact CMK 11-5 cells with 35S-ATP-alpha-S resulted in the labeling of the alpha-subunit of GP IIb as found with platelets, although this was confirmed to be independent of ADP receptors. After RNA from CMK 11-5 cells was microinjected into Xenopus oocytes, only ADP and ADP-alpha-S stimulated 45Ca2+ efflux, which was not observed with ATP, 2-methylthio-ATP, alpha, beta-methylene-ATP, ATP-gamma-S, ATP-alpha-S, or adenosine. In addition, incubation of RNA-injected oocytes with ATP or ATP-alpha-S but not adenosine blocked the 45Ca2+ response to ADP. These experiments demonstrate that a nascent receptor that responded specifically to ADP but not to other P1, P2Y, P2X, and P2U agonists was expressed in functional form on Xenopus oocytes.

P2 purinoceptors in rat cortical astrocytes: expression, calcium-imaging and signalling studies.

Extracellular ATP is known to activate intracellular enzymes in astrocytes via P2 purinoceptors that appear to play important physiological and pathological roles in these supporting brain cells. In this study, major P2 purinoceptor subtypes on astrocytes of neonatal rat cerebral cortices were identified in receptor expression experiments, when astrocytic messenger RNA was injected into Xenopus oocytes and recombinant P2 purinoceptors were characterized pharmacologically. In messenger RNA-injected oocytes, ATP evoked inward chloride currents (ICl,Ca) typical of stimulating metabotropic receptors that release intracellular Ca2+. Half-maximal activation with ATP occurred at 40 nM: the Hill coefficient was 0.5, which indicated that ATP stimulated two subtypes of P2 purinoceptor. UTP and 2-methylthioATP were the most active (and equipotent) of a series of nucleotides activating recombinant P2 purinoceptors. These results indicated that the two P2 purinoceptors expressed by astrocytic messenger RNA were of P2U and P2Y subtypes. Responses to ATP were antagonized by the P2 purinoceptor antagonist (suramin) but not by the P1 purinoceptor blocker (sulphophenyltheophylline). Findings in expression studies were confirmed in assays of intracellular signalling systems using primary cultures of rat astrocytes. UTP and 2-methylthioATP stimulated mitogen-activated protein kinase to the same extent as ATP, although UTP was less potent than either ATP or 2-methylthioATP. Both UTP and ATP increased intracellular Ca2+ (as measured by fura-2/AM luminescence) which, in cross-desensitization experiments, indicated the involvement of two subtypes of P2 purinoceptors. In conclusion, rat cortical astrocytes express two major subtypes (P2U and P2Y) of metabotropic ATP receptor which, when activated, raise intracellular Ca2+ and also stimulate mitogen-activated protein kinase.

P2 purinoceptors in rat cortical astrocytes: expression, calcium-imaging and signalling studies

Extracellular ATP is known to activate intracellular enzymes in astrocytes via P2 purinoceptors that appear to play important physiological and pathological roles in these supporting brain cells. In this study, major P2 purinoceptor subtypes on astrocytes of neonatal rat cerebral cortices were identified in receptor expression experiments, when astrocytic messenger RNA was injected into Xenopus oocytes and recombinant P2 purinoceptors were characterized pharmacologically. In messenger RNA-injected oocytes, ATP evoked inward chloride currents (ICl,Ca) typical of stimulating metabotropic receptors that release intracellular Ca2+. Half-maximal activation with ATP occurred at 40 nM: the Hill coefficient was 0.5, which indicated that ATP stimulated two subtypes of P2 purinoceptor. UTP and 2-methylthioATP were the most active (and equipotent) of a series of nucleotides activating recombinant P2 purinoceptors. These results indicated that the two P2 purinoceptors expressed by astrocytic messenger RNA were of P2U and P2Y subtypes. Responses to ATP were antagonized by the P2 purinoceptor antagonist (suramin) but not by the P1 purinoceptor blocker (sulphophenyltheophylline). Findings in expression studies were confirmed in assays of intracellular signalling systems using primary cultures of rat astrocytes. UTP and 2-methylthioATP stimulated mitogen-activated protein kinase to the same extent as ATP, although UTP was less potent than either ATP or 2-methylthioATP. Both UTP and ATP increased intracellular Ca2+ (as measured by fura-2/AM luminescence) which, in cross-desensitization experiments, indicated the involvement of two subtypes of P2 purinoceptors. In conclusion, rat cortical astrocytes express two major subtypes (P2U and P2Y) of metabotropic ATP receptor which, when activated, raise intracellular Ca2+ and also stimulate mitogen-activated protein kinase.

Expression of taste reception response of fleshfly in Xenopus oocytes.

Functional expression of the gustatory sensitivity to amino acids and sugars was investigated in Xenopus oocytes injected with poly(A+) RNA extracted from fleshfly labellar taste organs. The current induced by the application of amino acids and sugars that stimulate fleshfly sugar receptor cells was recorded under voltage clamp conditions. L-Phenylalanine and L-valine induced a large transient inward current in poly(A+) RNA-injected oocytes but only small responses in uninjected control oocytes. Glucose and fructose also produced inward currents in the RNA-injected oocytes. N-methylated compounds or D-isomers of these amino acids induced either no response or a much smaller response than L-amino acids as they did in the fleshfly sugar receptor cells. The oocyte expression system is a useful tool for characterizing the taste transduction mechanism.

Injection of rat hepatocyte poly(A) + RNA to Xenopus laevis oocytes leads to expression of a constitutively-active divalent cation channel distinguishable from endogenous receptor-activated channels

The expression of hepatocyte plasma membrane receptor-activated divalent cation channels in immature (stages V and VI) Xenopus laevis oocytes and the properties which allow these channels to be distinguished from endogenous receptor-activated divalent cation channels were investigated. Divalent cation inflow to oocytes housed in a multiwell plate was measured using the fluorescent dyes Fluo-3 and Fura-2. In control oocytes, ionomycin, cholera toxin, thapsigargin, 3-fluoro-inositol 1,4,5-trisphosphate (InsP 3F) and guanosine 5′-[γ-thio]triphosphate (GTPγS) stimulated Ca 2+ and Mn 2+ inflow following addition of these ions to the oocytes. lonomycin-, cholera-toxin-, thapsigargin-and InsP 3F-stimulated Ca 2+ inflow was inhibited by Gd 3+ (half maximal inhibition at less than 5 μM Gd 3+ for InsP 3F-stimulated Ca 2+ inflow). GTPγS-stimulated Ca 2+ inflow was insensitive to 50 μM Gd 3+ and to SK&F 96365. These results indicate that at least three types of endogenous receptor-activated Ca 2+ channels can be detected in Xenopus oocytes using Ca 2+-sensitive fluorescent dyes: lanthanide-sensitive divalent cation channels activated by intracellular Ca 2+ store depletion, lanthanide-sensitive divalent cation channels activated by cholera toxin, and lanthanide-insensitive divalent cation channels activated by an unknown trimeric G-protein. Oocytes microinjected with rat hepatocyte poly(A) + RNA exhibited greater rates of Ca 2+ and Mn 2+ inflow in the basal (no agonist) state, greater rates of Ca 2+ inflow in the presence of vasopressin or InsP 3F and greater rates of Ba 2+ inflow in the presence of InsP 3F, when compared with ‘mock’-injected oocytes. In poly(A) + RNA-injected oocytes, vasopressin- and InsP 3F-stimulated Ca 2+ inflow, but not basal Ca 2+ inflow, was inhibited by Gd 3+. It is concluded that at least one type of hepatocyte plasma membrane divalent cation channel, which admits Mn 2+ as well as Ca 2+ and is lanthanide-insensitive, can be expressed and detected in Xenopus oocytes.

Amiloride-sensitive sodium channels in confluent M-1 mouse cortical collecting duct cells

Confluent M-1 cells show electrogenic Na+ absorption and possess an amiloride-sensitive Na(+)-conductance (Korbmacher et al., J. Gen. Physiol. 102:761-793, 1993). In the present study, we further characterized this conductance and identified the underlying single channels using conventional patch clamp technique. Moreover, we isolated poly(A)+ RNA from M-1 cells to express the channels in Xenopus laevis oocytes, and to check for the presence of transcripts related to the epithelial Na+ channel recently cloned from rat colon (Canessa et al., Nature 361:467-470, 1993). Patch clamp experiments were performed in 6-13-day-old confluent M-1 cells at 37 degrees C. In whole-cell experiments application of 10(-5) M amiloride caused a hyperpolarization of 24.9, SEM +/- 2.2 mV (n = 35) and a reduction of the inward current by 107 +/- 10 pA (n = 51) at a holding potential of -60 mV. Complete removal of bath Na+ had similar effects, indicating that the amiloride-sensitive component of the inward current is a Na+ current. The effect of amiloride was concentration-dependent with half-inhibition at 0.22 microM. The Na+ current saturated with increasing extracellular Na+ concentrations with an apparent Km of 24 mM. Na+ replacement for Li+ demonstrated a higher apical membrane conductance for Li+ than for Na+. In excised inside-out (i/o) or outside-out (o/o) patches from the apical membrane, we observed single-channels which showed slow kinetics and were reversibly inhibited by amiloride. Their average conductance for Na+ was 6.8 +/- 0.5 pS (n = 15) and for Li+ 11.2 +/- 1.0 pS (n = 14). They had no measurable conductance for K+. In o/o patches, channel activity was slightly voltage dependent with an open probability (NPo) of 0.46 +/- 0.14 and 0.16 +/- 0.05 at a holding potential of -100 and 0 mV, respectively (n = 8, P < 0.05). Using the two-microelectrode voltage-clamp technique, we assayed defolliculated stage V-VI Xenopus oocytes for an amiloride-sensitive inward current 1-6 days after injection with H2O or with 20-50 ng of M-1 poly(A)+ RNA. In poly(A)+ RNA-injected oocytes held at -60 or -100 mV application of amiloride (2 microM) reduced the Na-inward current by 25.5 +/- 4.6 nA (n = 25) while it had no effect in H2O-injected oocytes (n = 19). Northern blot analysis of M-1 poly(A+) RNA revealed the presence of transcripts related to the three known subunits of the rat colon Na+ channel (Canessa et al., Nature 367:463-467, 1994). We conclude that the channel in M-1 cells is closely related to the amiloride-sensitive epithelial Na+ channel in the rat colon and that the M-1 cell line provides a useful tool to investigate the biophysical and molecular properties of the corresponding channel in the cortical collecting duct.

オピオイドレセプターの情報伝達系による脱感作の違い

RNA-injected Xenopus oocytes were used for studying homologous desensitization of opioid receptors to G-protein-coupled intracellular signaling pathways. In the oocytes expressing K opioid receptor, voltage-dependent Ca2+ channel α2+ and β subunits, the κ agonist U50488H (1 μM) inhibited Ca2+ channel current in a reversible manner, and the inhibition was diminished by sustained agonist exposure (homologous desensitization). More than 10 min was required to halve the inhibition of BI- or Q-type Ca2+ channels by the κ agonist, however, the t½ for U50488H-induced inhibition of BIII- or N-type channels was only 6±1 min. Moreover, in the oocytes expressing κ opioid receptor, EP4 prostaglandin receptor and CFTR channel, no apparent desensitization was observed in the κ receptor-mediated potentiation of the cyclic AMP production by prostaglandin E2 even when the oocytes were treated with U50488H for 2 hours or more. The time-course of desensitization of μ opioid receptor was also different in the effector molecules. These observations suggest that rate of homologous desensitization of opioid receptor is not only dependent on the receptor molecule itself but also on its intracellular signaling systems.

Characterization of the platelet-activating factor acetylhydrolase from human plasma by heterologous expression in Xenopus laevis oocytes.

Platelet-activating factor (PAF) has been implicated as a mediator of inflammation and atherosclerosis. A specific degradative enzyme found in plasma, PAF acetylhydrolase, plays important roles in various pathophysiological events induced by PAF. Human macrophages and Hep G2 cells secrete PAF acetylhydrolase with characteristics identical to the plasma activity. Other investigators reported that apolipoprotein B may possess phospholipase A2 activity, which suggested that apolipoprotein B might be a zymogen for PAF acetylhydrolase. However, while macrophages express PAF acetylhydrolase activity, we did not detect cDNAs for apolipoprotein B in a cDNA library from these cells, indicating that macrophages do not express this protein. In contrast, Hep G2 cells had high levels of cDNA for apolipoprotein B, as expected. We next injected Xenopus laevis oocytes with poly(A)+ RNA extracted from cultured human macrophages and Hep G2 cells. Twenty-five to 50% of Xenopus oocytes injected with poly(A)+ RNA from macrophages or Hep G2 cells secreted a PAF acetylhydrolase activity (1.0-7.8 nmol/ml per h) that also utilized a synthetic oxidized phospholipid as substrate. The activity secreted by poly(A)+ RNA-injected oocytes associated with lipoproteins and transferred between the particles in a pH-dependent manner, much like the plasma activity. These experiments establish that the properties of the enzyme released from poly(A)+ RNA-injected oocytes are identical to those of the plasma form of PAF acetylhydrolase and that the activity detected is not the expression of a domain in apolipoprotein B.

Expression of the choroid plexus sodium-nucleoside cotransporter (N3) in Xenopus laevis oocytes

In this study, we determined the expression of the Na +nucleoside cotransporter (N3) in Xenopus laevis oocytes injected with poly(A) + RNA isolated from rabbit choroid plexus. The Na +-dependent thymidine uptake in poly(A) + RNA-injected oocytes (maximum 4–5 days after injection) increased proportionally with the injected dose of poly(A) + RNA. Uptake was enhanced 4- to 5-fold in oocytes injected with 40 ng poly (A) + RNA in comparison to water-injected oocytes. Consistent with the N3 Na +-nucleoside cotransporter, Na +-dependent thymidine uptake in poly(A) + RNA-injected oocytes was inhibited significantly by both purine and pyrimidine nucleosides, but not by dideoxycytidine, a nucleoside analog modified on the ribose ring. These data suggest for the first time that the N3 Na +-nucleoside cotransporter in rabbit choroid plexus can be expressed in X. laevis oocytes.