Ion Channels In Xenopus Oocytes Research Articles

Voltage- and Ca2+-inducible PLC activity for analyzing PI(4,5)P2 sensitivity of ion channels in Xenopus oocytes.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key membrane lipid regulating various ion channel activities. Currently, several molecular tools are used to modulate PIP2 levels, each of which has distinct advantages and drawbacks. In this study, we proposed a novel methodology using heterologous Xenopus oocytes to precisely manipulate PIP2 levels using phospholipase C (PLC)-ζ, which hydrolyzes PIP2. Xenopus oocytes injected with PLCζ exhibited notable hyperpolarization-induced Ca2+ influx driven by the increased driving force of Ca2+. High Ca2+ sensitivity of PLCζ facilitated hyperpolarization-induced PLC activity in Xenopus oocytes that was voltage- and Ca2+-dependent. This study demonstrated the regulatory capacity of PLCζ in modulating PIP2-sensitive ion channels, such as the KCNQ2/3 and GIRK channels, in a voltage- and Ca2+-dependent manner. Moreover, activation pathway of PLCζ only requires a two-electrode voltage clamp setup, making it a convenient molecular tool to manipulate PIP2 levels in combination with a voltage-sensing phosphatase (VSP). PLCζ has distinct characteristics and advantages compared to VSP: (1) Hyperpolarization, but not depolarization, reduced the PIP2 levels, (2) PIP2 levels were decreased without any increase in phosphatidylinositol 4-monophosphate (PIP) levels, and (3) PIP2 levels were reduced by Ca2+ administration. Therefore, PLCζ effectively supports understanding how PIP2 regulates ion channels, alongside VSP. Overall, this study highlights the unique characteristics of PLCζ and its distinct advantages in analyzing ion channel regulation by PIP2 and the PLC pathway in Xenopus oocytes.

Expression and Analysis of Flow-regulated Ion Channels in Xenopus Oocytes.

Mechanically-gated ion channels play key roles in mechanotransduction, a process that translates physical forces into biological signals. Epithelial and endothelial cells are exposed to laminar shear stress (LSS), a tangential force exerted by flowing fluids against the wall of vessels and epithelia. The protocol outlined herein has been used to examine the response of ion channels expressed in Xenopus oocytes to LSS (Hoger et al., 2002; Carattino et al., 2004; Shi et al., 2006). The Xenopus oocyte is a reliable system that allows for the expression and chemical modification of ion channels and regulatory proteins (George et al., 1989; Palmer et al., 1990; Sheng et al., 2001; Carattino et al., 2003). Therefore, this technique is suitable for studying the molecular mechanisms that allow flow-activated channels to respond to LSS.

Screening Spider Toxin Libraries for TRP Channel Inhibitors

Pain caused by activation of pain-sensing peripheral neurons (“nociceptors”) is a major source of human suffering and economic loss. A variety of stimulus- and voltage-gated ion channels in peripheral nociceptors play essential roles in the transduction and transmission of normal and pathological pain signals in human patients, and thus are important targets for pharmacological intervention. Around one hundred inhibitory cystine knot (ICK) peptide toxins were selected from a database of published and unpublished spider toxins. The tethered-toxin (“t-toxin”) method for the heterologous expression of ICK ion channel toxins in membrane tethered form directly in a channel-expressing target cell was applied. Pools of cRNA of t-toxins (∼6 per pool) were co-expressed with ion channels in Xenopus oocytes and the currents were measured by two-electrode voltage clamp 2-4 days after injection. One pool was found to cause ∼50 % suppression of the TRPA1 current induced by 100 μM mustard oil, and subscreening of this pool revealed a single t-toxin responsible for this effect. When co-expressed with TRPV1, this t-toxin suppressed the inward current induced by 20 μM capsaicin ∼80%. The effect of this t-toxin on other ion channels was also investigated. It completely blocked depolarization-activated inward currents of Para, a Drosophila voltage-gated sodium channel. When co-expressed with Nav1.2, it reduced peak currents by 60%. Interestingly, however, it did not affect currents flowing through Kir4.1, an inwardly rectifying potassium channel. These results suggest that this relatively promiscuous toxin acts on a structural feature common to ion channels with six transmembrane domains (Trp channels and voltage-gated channels), but lacking in those that only possess the two pore-spanning transmembrane domains (inward rectifier).

Heterologous expression of C. elegans ion channels in Xenopus oocytes

Physiological methods entered the world of C. elegans, a model system used for many years to study development and a plethora of biological processes mainly employing genetic, molecular and anatomical techniques. One of the methods introduced by physiologists is the use of Xenopus oocytes for expression of C. elegans ion channels. Oocytes of the South African frog Xenopus laevis are used widely for the expression of mammalian channels and transporters contributing to numerous discoveries in these fields. They now promise to aid C. elegans researchers in deciphering mechanisms of channels function and regulation with implications for mammalian patho-physiology. Heterologous cRNA can be easily injected into Xenopus oocytes and translated proteins can be studied using several techniques including electrophysiology, immunocytochemistry and protein biochemistry. This chapter will focus on techniques used for oocyte preparation and injection, and will give a brief overview of specific methods. Limitations of the use of Xenopus oocytes will be also discussed.

Highlights from the Literature

Highlights from the Literature

Differential ion current activation by human 5-HT 1A receptors in Xenopus oocytes: Evidence for agonist-directed trafficking of receptor signalling

The subject of the present study was the functional and pharmacological characterization of human 5-HT 1A receptor regulation of ion channels in Xenopus oocytes. Activation of the heterologously expressed human 5-HT 1A receptor induced two distinct currents in Xenopus oocytes, consisting of a smooth inward current ( I smooth) and an oscillatory calcium-activated chloride current, I Cl(Ca). 5-HT 1A receptor coupling to both ionic responses as well as to co-expressed inward rectifier potassium (GIRK) channels was pharmacologically characterized using 5-HT 1A receptor agonists. The relative order of efficacy for activation of GIRK current was 5-HT ≈ F13714 ≈ L694,247 ≈ LY228,729 > flesinoxan ≈ (±)8-OH-DPAT. In contrast, flesinoxan and (±)8-OH-DPAT typically failed to activate I Cl(Ca). The other ligands behaved as full or partial agonists, exhibiting an efficacy rank order of 5-HT ≈ L694,247 > F13714 ≈ LY228,729. The pharmacological profile of I smooth activation was completely distinct: flesinoxan and F13714 were inactive and rather exhibited an inhibition of this current. I smooth was activated by the other agonists with an efficacy order of L694,247 > 5-HT ≈ LY228,729 > (±)8-OH-DPAT. Moreover, activation of I smooth was not affected by application of pertussis toxin or the non-hydrolyzable GDP-analogue, guanosine-5′- O-(2-thio)-diphosphate (GDPβS), suggesting a GTP binding protein-independent pathway. Together, these results suggest the existence of distinct and agonist-specific signalling states of this receptor.

Teleost Fh14-3-3a protein protects Xenopus oocytes from hyperosmolality.

We have previously cloned and characterized a novel 14-3-3 gene from the euryhaline telost Fundulus heteroclitus, Fh14-3-3a (Kültz et al., 2001). The corresponding gene product is osmoregulated and most highly expressed in gill epithelium of this fish. In the present study we have expressed Fh14-3-3a cRNA in Xenopus laevis oocytes and investigated the survival and electrophysiological parameters of Xenopus oocytes in isosmotic and various hyperosmotic media. Xenopus oocytes expressing Fh14-3-3a show no mortality after a 16 hour exposure to hyperosmolality in the form of elevating medium K(+), Na(+), polyethylene glycol, or sorbitol concentrations up to 444 mosmol/kg. In contrast, 16 hours of the same hyperosmolality caused 100% mortality in control Xenopus oocytes injected with water. As a result of hyperosmolality the Xenopus oocyte membrane potential decreased between 10 and 70% in oocytes expressing Fh14-3-3a whereas it was completely abolished in control oocytes. We report that one potential cause for the osmoprotective effect of Fh14-3-3a on Xenopus oocytes could be its inhibition of an endogenous chloride current. Hyperosmotic urea was not as harmful to Xenopus oocytes as hypertonicity and maybe acting through a different mechanism. Coexpression of Fh14-3-3a with a human calcium channel in Xenopus oocytes did not affect the electrophysiological properties of this exogenous channel. Thus, the osmoprotective effect of Fh14-3-3a may prove a valuable tool for the characterization of exogenous ion channels in Xenopus oocytes exposed to hyperosmotic conditions.

29] High-level expression and detection of ion channels in Xenopus oocytes

Publisher Summary Because the initial demonstration by Miledi and co-workers that ion channels and neural receptors can be functionally expressed in Xenopus oocytes, this system has become a standard for demonstrating that a specific cloned cDNA encodes a functional channel or receptor. Many different ion channels and receptors have been expressed in oocytes for functional analysis, and oocytes have been used for functional cloning of receptors, as described by Ltibbert et al. and Julius et al. RNA for injection into oocytes can be isolated from the appropriate tissue sample or cell line, or it can be synthesized in vitro from a cDNA clone. This chapter discusses some of the approaches that are used for high-level expression and analysis of cloned ion channels in Xenopus oocytes. This expression system has the advantage that channels can be expressed and analyzed by electrophysiologic, biochemical, and physical approaches. Most of the electrophysiologic recording techniques described here on electrophysiology can be used for analysis of ion channels in oocytes. In addition, the system can be used effectively as an assay for the functional cloning of channels that have only been identified by their electrophysiologic properties. Expression of ion channels in oocytes is an excellent system for correlating structure with function using a combination of molecular biological, biochemical, and electrophysiologic techniques.

Follicular tissues reduce drug effects on ion channels in oocytes of Xenopus laevis.

The influence of follicular tissues on drug effects on ion channels in Xenopus oocytes was tested by investigating the pharmacological properties of a cloned potassium channel in oocytes with and without follicular tissues. The data show that the efficacy of blocking agents (ranging from metal ions to peptides) is drastically reduced by the follicular tissues (reductions by as much as 90% and increases of the IC50 values up to 30-fold). Furthermore, the time course of the blocking effect was slowed down by the tissues (increases of the t50 values up to 40-fold). The described impairment could be mitigated, but not abolished by partial removal of the follicular tissues (so-called defolliculation, leaving only the vitelline envelope and part of the follicle cells on the oocyte surface). The results indicate that the follicular tissues can induce significant errors in pharmacological measurements on membrane proteins in Xenopus oocytes.

A new technique for evaluating volume sensitivity of ion channels.

A new technique is described for evaluating the volume sensitivity of both endogenous and expressed ion channels in Xenopus oocytes. The technique utilizes vesicles derived from inside-out, excised patches and permits evaluation of volume sensitivity at the single-channel level, which is not possible with whole oocytes since removal of the vitelline membrane renders oocytes too fragile to withstand osmotic stress. Using this method, the volume sensitivity of the normally occurring oocyte mechanosensitive (SA-cat) channel was assessed. In 12 experiments, osmotic swelling increased vesicle volume by an average of 60 +/- 8% and increased the surface area of the inner side of the vesicle by 47 +/- 10%. This was associated with an increase in mean number of open channels (NPo) from zero (unswollen) to 0.1 +/-0.02 (after 5 min). Hence, oocyte SA-cat channels are volume as well as stretch sensitive, where the volume sensitivity may involve cytoskeletal elements adhering to the membrane but probably does not involve freely diffusible components of the original oocyte.

PVP-containing solutions for analysis of divalent cation-dependent NMDA responses in Xenopus oocytes

The electrophysiological analysis of Ca 2+-conducting ion channels in Xenopus oocytes is difficult due to secondary intracellular effects induced by Ca 2+. In the presence of polyvinylpyrrolidone (PVP) membrane currents can be recorded in nominally divalent cation-free solutions. The Ca 2+-permeable recombinant NMDA receptors of the NR1/NR2A subtype were used as assay system and the results show that PVP has no effect on NMDA receptor-induced currents. Ca 2+ and Ba 2+ depress NMDA-induced currents at submillimolar concentrations probably by interfering with the Na +/K + flux. This block is fully reversible as also observed for Mg 2+ but shows in contrast no pronounced voltage dependence. PVP-containing solutions may be useful for the analysis of divalent cation-dependent ion channels.

Structure-Function Relations in Ligand-Gated Ion Channels: Reconstitution in Lipid Bilayers and Heterologous Expression in Xenopus Oocytes

Ligand-gated ion channels play a fundamental role in cell-to-cell communication. In response to a chemical stimulus, these membrane proteins undergo a conformational change from a closed nonconductive state to an open conductive state, allowing the selective flow of ions into and out of the cell. The molecular mechanisms that underlie such a precise task are not yet fully understood. Reconstitution of purified proteins in lipid bilayers and heterologous expression of cDNAs encoding neurotransmitter-activated ion channels in Xenopus oocytes are powerful and sensitive approaches to gaining fundamental information about structure-function relationships of this superfamily of membrane receptors.

16] Preparation of RNA for injection into Xenopus oocytes

Publisher Summary The most important step involved in the expression of receptors and ion channels in Xenopus oocytes is preparation of RNA, because the quality of the mRNA dictates the number of neurotransmitter receptors and voltage-activated ion channels expressed in the oocytes. This chapter describes the procedures for isolation of total mRNA from the brain, size fractionation of mRNA, and synthesis of mRNA by in vitro transcription of cloned cDNAs. For tissues with high RNase levels, the guanidinium thiocyanate method is generally most effective in obtaining good quality of the RNA. However, for fresh and frozen brains the phenol–chloroform method—as a protein denaturant and as a means of separating the proteins from the nucleic acids—consistently provides with active mRNA coding for neurotransmitter receptors and voltage-activated ion channels. Functional mRNA can be prepared by in vitro transcription of cloned cDNAs with bacteriophage RNA polymerase in the presence of 5 ' cap analog for expression in Xenopus oocytes.

18] In Vitro synthesis of RNA for expression of ion channels in Xenopus oocytes

Xenopus oocytes are widely used for the heterologous expression of cDNA clones encoding ion channels, and the widespread applicability of this system is because of the ability of the oocyte to translate RNAs encoding channels from a variety of sources. This chapter describes the methods for in vitro synthesis of RNAs suitable for expression in Xenopus oocytes. In vitro synthesis of RNA is most easily carried out by cloning a cDNA encoding the channel into a vector containing a bacteriophage promoter and then specifically transcribing that sequence by using an RNA polymerase that recognizes the phage promoter. Direct in vitro transcription of ion channel cDNAs synthesized using the polymerase chain reaction (PCR) enables the rapid amplification and expression of ion channels encoded by low abundance mRNAs. However, functional expression of a channel cannot be detected in oocytes, even though the RNA is properly sized and readily translated in vitro.

Characterization of stretch‐activated ion channels in Xenopus oocytes.

1. The gating and permeation properties of endogenous stretch-activated (SA) ion channels in Xenopus oocytes have been studied using the patch-clamp single channel recording technique. 2. As estimated from the probability of being open (Po), SA channels were equally sensitive to suction or pressure. The Po was also weakly sensitive to voltage, increasing with depolarization. Channel activation did not require Ca2+. 3. Kinetic analysis of single-channel records indicated that there are three closed states and one open state. Among three closed-time distributions, the longest was the most sensitive to both pipette pressure and membrane voltage. The open time was independent of both pressure and voltage under a wide variety of ionic conditions, but was sensitive to the species of extracellular ion as follows: Na+ greater than Cs+ greater than K+ greater than Rb+ greater than Li+. The open time had a monotonic mole fraction relationship in mixtures of Li+ and K+. 4. The SA channels were cation-selective inward rectifiers. The selectivity for permeation, based on slope conductance, was: K+ greater than NH4+ greater than Cs+ greater than Rb+ greater than Na+ greater than Li+ greater than Ca2+. 5. Tetraethylammonium (TEA+) was impermeable but was not a channel blocker. 6.Open-channel current amplitude saturated with increasing extracellular K+, and was a monotonic function of the mole fraction of Li+ and K+ in mixtures of the two ions. 7. The channel has at least two separate ion binding sites: an intra-channel site suggested by the permeation data, and an allosteric site suggested by the voltage-independent effects of permeant ions on open time. A symmetric two-barrier, one-site model can quantitatively describe the permeation data. A kinetic model is proposed to quantify the gating kinetics and the effect of ion binding at the allosteric site.

Cloning of a novel glutamate receptor subunit, GluR5: Expression in the nervous system during development

We have isolated cDNAs encoding a glutamate receptor subunit, designated GluR5, displaying 40%–41% amino acid identity with the kainate/AMPA receptor subunits GluR1, GIuR2, GIuR3, and GIuR4. This level of sequence similarity is significantly below the approximately 70% intersubunit identity characteristic of kainate/AMPA receptors. The GIuR5 protein forms homomeric ion channels in Xenopus oocytes that are weakly responsive to L-glutamate. The GIuR5 gene is expressed in subsets of neurons throughout the developing and adult central and peripheral nervous systems. During embryogenesis, GluR5 transcripts are detected in areas of neuronal differentiation and synapse formation.

Molecular Cloning and Functional Expression of Glutamate Receptor Subunit Genes

Three closely related genes, GluR1, GluR2, and GluR3, encode receptor subunits for the excitatory neurotransmitter glutamate. The proteins encoded by the individual genes form homomeric ion channels in Xenopus oocytes that are sensitive to glutamatergic agonists such as kainate and quisqualate but not to N-methyl-D-aspartate, indicating that binding sites for kainate and quisqualate exist on single receptor polypeptides. In addition, kainate-evoked conductances are potentiated in oocytes expressing two or more of the cloned receptor subunits. Electrophysiological responses obtained with certain subunit combinations show agonist profiles and current-voltage relations that are similar to those obtained in vivo. Finally, in situ hybridization histochemistry reveals that these genes are transcribed in shared neuroanatomical loci. Thus, as with gamma-aminobutyric acid, glycine, and nicotinic acetylcholine receptors, native kainate-quisqualate-sensitive glutamate receptors form a family of heteromeric proteins.

Cloning by functional expression of a member of the glutamate receptor family

We have isolated a complementary DNA clone by screening a rat brain cDNA library for expression of kainate-gated ion channels in Xenopus oocytes. The cDNA encodes a single protein of relative molecular mass (Mr) 99,800 which on expression in oocytes forms a functional ion channel possessing the electrophysiological and pharmacological properties of the kainate subtype of the glutamate receptor family in the mammalian central nervous system.

Block of Stretch-Activated Ion Channels in Xenopus Oocytes by Gadolinium and Calcium Ions

Gadolinium ions produce three distinct kinds of block of the stretch-activated (SA) ion channels in Xenopus oocytes: a concentration-dependent reduction in channel open time, a concentration-dependent reduction in open channel current, and a unique, steeply concentration-dependent, reversible inhibition of channel opening. This last effect reduces the probability of a channel being open from about 10(-1) at 5 microM to less than 10(-5) at 10 microM gadolinium. Calcium has effects on open time and current similar to that of gadolinium, but this channel is permeable to calcium and calcium does not completely inhibit channel activity. The availability of a blocker for SA ion channels may help to define their physiological function, and will simplify the use of oocytes as an expression system for ion channels.