Blockade of HERG channels expressed in Xenopus laevis oocytes by external divalent cations

We have investigated actions of various divalent cations (Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Zn2+) on human ether-a-go-go related gene (HERG) channels expressed in Xenopus laevis oocytes using the voltage clamp technique. All divalent cations inhibited HERG current dose-dependently in a voltage-dependent manner. The concentration for half-maximum inhibition (Ki) decreased at more negative potentials, indicating block is facilitated by hyperpolarization. Ki at 0 mV for Zn2+, Ni2+, Co2+, Ba2+, Mn2+, and Sr2+ was 0.19, 0.36, 0. 50, 0.58, 2.36, and 6.47 mM, respectively. The effects were manifested in four ways: 1) right shift of voltage dependence of activation, 2) decrease of maximum conductance, 3) acceleration of current decay, and 4) slowing of activation. However, each parameter was not affected by each cation to the same extent. The potency for the shift of voltage dependence of activation was in the order Zn2+ > Ni2+ >/= Co2+ > Ba2+ > Mn2+ > Sr2+, whereas the potency for the decrease of maximum conductance was Zn2+ > Ba2+ > Sr2+ > Co2+ > Mn2+. The kinetics of activation and deactivation were also affected, but the two parameters are not affected to the same extent. Slowing of activation by Ba2+ was most distinct, causing a marked initial delay of current onset. From these results we concluded that HERG channels are nonselectively blocked by most divalent cations from the external side, and several different mechanism are involved in their actions. There exist at least two distinct binding sites for their action: one for the voltage-dependent effect and the other for reducing maximum conductance.     

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.     

Modulation of cardiac Ca2+ channels in Xenopus oocytes by protein kinase C.

L-Type calcium channel was expressed in Xenopus laevis oocytes injected with RNAs coding for different cardiac Ca2+ channel subunits, or with total heart RNA. The effects of activation of protein kinase C (PKC) by the phorbol ester PMA (4 beta-phorbol 12-myristate 13-acetate) were studied. Currents through channels composed of the main (alpha 1) subunit alone were initially increased and then decreased by PMA. A similar biphasic modulation was observed when the alpha 1 subunit was expressed in combination with alpha 2/delta, beta and/or gamma subunits, and when the channels were expressed following injection of total rat heart RNA. No effects on the voltage dependence of activation were observed. The effects of PMA were blocked by staurosporine, a protein kinase inhibitor. beta subunit moderate the enhancement caused by PMA. We conclude that both enhancement and inhibition of cardiac L-type Ca2+ currents by PKC are mediated via an effect on the alpha 1 subunit, while the beta subunit may play a mild modulatory role.     

Cyclic AMP synthesis in Xenopus laevis oocytes: inhibition by progesterone.

[alpha-32P]ATP was microinjected into Xenopus oocyte and neosynthesized cyclic AMP was isolated. Cholera toxin inhibited progesterone-induced maturation and stimulated after 3 h of preincubation the amount of neosynthesized cyclic AMP. Progesterone decreased the neosynthesis of cyclic AMP during the first hour following addition of the hormone.     

Cyclic AMP synthesis in Xenopus laevis oocytes inhibition by progesterone

[α-³²P] ATP was microinjected into Xenopus oocyte and neosynthesized cyclic AMP was isolated. Cholera toxin inhibited progesterone-induced maturation and stimulated after 3 h of preincubation the amount of neosynthesized cyclic AMP. Progesterone decreased the neosynthesis of cyclic AMP during the first hour following addition of the hormone.     

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.     

Gating of I(sK) channels expressed in Xenopus oocytes.

The channel underlying the slow component of the voltage-dependent delayed outward rectifier K+ current, I(Ks), in heart is composed of the minK and KvLQT1 proteins. Expression of the minK protein in Xenopus oocytes results in I(Ks)-like currents, I(sK), due to coassembly with the endogenous XKvLQT1. The kinetics and voltage-dependent characteristics of I(sK) suggest a distinct mechanism for voltage-dependent gating. Currents recorded at 40 mV from holding potentials between -60 and -120 mV showed an unusual "cross-over," with the currents obtained from more depolarized holding potentials activating more slowly and deviating from the Cole-Moore prediction. Analysis of the current traces revealed two components with fast and slow kinetics that were not affected by the holding potential. Rather, the relative contribution of the fast component decreased with depolarized holding potentials. Deactivation and reactivation, after a short period of repolarization (100 ms), was markedly faster than the fast component of activation. These gating properties suggest a physiological mechanism by which cardiac I(Ks) may suppress premature action potentials.     

A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes

The Xenopus laevis oocyte is widely used to express exogenous channels and transporters and is well suited for functional measurements including currents, electrolyte and nonelectrolyte fluxes, water permeability and even enzymatic activity. It is difficult, however, to transform functional measurements recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here a method of estimating the number of exogenously expressed channels and transporters inserted in the plasma membrane of oocytes. The method is based on the facts that the P (protoplasmic) face in water-injected control oocytes exhibit an extremely low density of endogenous particles (212±48 particles/m², mean, sd) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/m²) only on the P face. The utility and generality of the method were demonstrated by estimating the gating charge per particle of the Na⁺/ glucose cotransporter (SGLT1) and a nonconducting mutant of the Shaker K⁺ channel proteins, and the single molecule water permeability of CHIP (Channel-like Intramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a gating charge of 3.5 electronic charges for SGLT1 and 9 for the mutant Shaker K⁺ channel from the ratio of Q _max to density of particles measured on the same oocytes. The gating charges were 3-fold larger than the effective valences calculated by fitting a Boltzmann equation to the same charge transfer data suggesting that the charge movement in the channel and cotransporter occur in several steps. Single molecule water permeabilities (p _f s) of 1.4 × 10^–14 cm³/ sec for CHIP and of 1.5 × 10^–16 cm³/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (P _f ) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit 100-fold less effective than CHIP.We thank Manoli Contreras for preparation and injection of oocytes. This work was supported by NIH Grants EY-04110 (to GAZ), DK-19567 (to EW), GM30376 (to F.B.) and EY-05661 (to J.E.H.).     

Heteropolymeric potassium channels expressed in Xenopus oocytes from cloned subunits

Voltage-dependent potassium currents were measured in Xenopus oocytes previously injected with RNAs generated in vitro from each of three cloned cDNAs (RBK1, RBK2, and RGK5). The currents differed in their sensitivities to blockade by tetraethylammonium (TEA; respective KDs 0.3, greater than 100, and 10 mM) and in their inactivation during a depolarizing pulse. Injections of RNA combinations (RBK1/RBK2 and RBK1/RGK5) caused currents that had TEA sensitivities different from those expected from the sum, in any proportion, of the two native channels. It is concluded that novel potassium channels are formed by the oocytes injected with two RNAs, presumably by heteropolymerization of subunits; such heteropolymerization would contribute functional diversity to voltage-dependent potassium channels in addition to that provided by a large gene family.     

Cyclic AMP-dependent phosphorylation and regulation of the cardiac dihydropyridine-sensitive Ca channel.

A polyclonal antibody, CR2, prepared using the C-terminal peptide of the alpha 1 subunit of the rabbit cardiac DHP-sensitive Ca channel, specifically immunoprecipitated the [3H]PN200-110-labeled Ca channel solubilized from cardiac microsomes. The antibody recognized 250 and 200-kDa cardiac microsomal proteins as determined by immunoblotting, and cAMP-dependent protein kinase phosphorylated the 250-kDa, but not the 200-kDa protein in vitro. CHO cells, transfected with the cardiac alpha 1 subunit cDNA carried by an expression vector, synthesized a 250-kDa protein which was recognized by CR2. Adding db-cAMP or forskolin to the transformed CHO cells induced phosphorylation of the 250-kDa protein and stimulated the DHP-sensitive Ba current under patch-clamp conditions. These results suggested that the cardiac DHP-sensitive Ca channel was regulated by cAMP-dependent phosphorylation of the alpha 1 subunit.     

Human voltage-dependent anion-selective channel expressed in the plasmalemma of Xenopus laevis oocytes

Recent studies indicate a plasmalemmal localisation of eukaryotic porin, i.e. voltage-dependent anion-selective channel (VDAC), and there is evidence that the channel in this cell compartment is engaged in cell volume regulation. Until recently, others and we have used immuno-topochemical and biochemical methods to demonstrate the integration of the channel into the cell membrane and endoplasmic reticulum of vertebrate cells. In the present study, we used molecular biological methods to induce the heterologous expression of tagged human type-1 porin in oocytes of Xenopus laevis and to illustrate its appearance at the plasma membrane of these cells. Applying confocal fluorescent microscopy, green fluorescent protein attached to the C-terminus of porin could clearly be recorded at the cell surface. N-terminal green fluorescent protein-porin fusion proteins remained in the cytoplasm, indicating a strong influence of the porin N-terminus on protein trafficking to the plasma membrane. FLAG-tagged porin was also expressed in frog oocytes. Here, plasmalemmal expression was observed using anti-FLAG M2 monoclonal antibodies and gold-conjugated secondary antibodies, followed by silver enhancement through scanning electron microscopy. In contrast to the EGFP-porin fusion protein, the influence of the small FLAG-epitope (8 amino acids) did not prevent plasmalemmal expression of N-terminally tagged porin. These results indicate the definite expression of human type-1 porin in the plasma membrane of Xenopus oocytes. They thus corroborate our early data on the extra-mitochondrial expression of the eukaryotic porin channel and are essential for future electrophysiological studies on the channel.     

Functional and morphological correlates of connexin50 expressed in Xenopus laevis oocytes

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 +/- 3 helices, (b) when their density reached 300-400/microm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1-2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in approximately 0.1-microm diameter "coated" and in larger 0.2-0.5-microm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5-40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80, 000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60-500 microm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in approximately 24 h.     

Reconstitution of expressed KCa channels from Xenopus oocytes to lipid bilayers.

Reconstitution of large conductance calcium-activated potassium (KCa) channels from native cell membranes into planar lipid bilayers provides a powerful method to study single channel properties, including ion conduction, pharmacology, and gating. Recently, KCa channels derived from the Drosophila Slowpoke (Slo) gene have been cloned and heterologously expressed in Xenopus oocytes. In this report, we describe the reconstitution of cloned and expressed Slo KCa channels from Xenopus oocyte membranes into lipid bilayers. The reconstituted channels demonstrate functional properties characteristic of native KCa channels. They possess a mean unitary conductance of approximately 260 pS in symmetrical potassium (250 mM), and they are voltage- and calcium-sensitive. At 50 microM Ca2+, their half-activation potential was near -20 mV; and their affinity for calcium is in the micromolar range. Reconstituted Slo KCa channels were insensitive to external charybdotoxin (40-500 nM) and sensitive to micromolar concentrations of external tetraethylammonium (KD = 158 microM, at 0 mV) and internal Ba2+ (KD = 76 microM, at 40 mV). In addition, they were blocked by internally applied "ball" inactivating peptide (KD = 480 microM, at 40 mV). These results demonstrate that cloned KCa channels expressed in Xenopus oocytes can be readily incorporated into lipid bilayers where detailed mechanistic studies can be performed under controlled internal and external experimental conditions.     

Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange

Pendred syndrome,characterized by congenital sensorineural hearing loss and goiter, isone of the most common forms of syndromic deafness. The gene causingPendred syndrome (PDS) encodes a protein designated pendrin,which is expressed in the thyroid, kidney, and fetal cochlea. Pendrinfunctions as an iodide and chloride transporter, but its role in thedevelopment of hearing loss and goiter is unknown. In this study, weexamined the mechanism of pendrin-mediated anion transport inXenopus laevis oocytes. Unlabeled formate added to the uptakemedium inhibited pendrin-mediated ³⁶Cl uptake in X. laevis oocytes. In addition, the uptake of[¹⁴C]formate was stimulated in oocytes injected with PDScRNA compared with water-injected controls. These results indicate thatformate is a substrate for pendrin. Furthermore, chloride stimulatedthe efflux of [¹⁴C]formate and formatestimulated the efflux of ³⁶Cl in oocytes expressingpendrin, results consistent with pendrin-mediated chloride/formateexchange. These data demonstrate that pendrin is functionally similarto the renal chloride/formate exchanger, which serves as an importantmechanism of chloride transport in the proximal tubule. A similarprocess could participate in the development of ion gradients withinthe inner ear.

     

Amino acid uptake in Xenopus laevis oocytes.

The isolated oocytes from Xenopus laevis are able to take up radioactive amino acids from the exogenous medium. Most amino acids tested are taken up to reach concentrations higher than the extracellular medium. The initial uptake velocities vary with the external amino acid concentration in a Michaelis-Menten fashion. Aspartic acid requires concentrations an order of magnitude higher than the five other amino acids tested to reach half the maximal uptake velocity. The uptake mechanism seems to be specific for groups of analogous amino acids, as can be determined by competition studies. The amino acid groups for which there is some evidence of uptake specificity would be aromatic, aliphatic, acidic and basic. Amino acid pools of oocytes show that these cells can concentrate amino acids from Xenopus blood, as well as from artificial media.     

Extracts from plants used in Mexican traditional medicine activate Ca(2+)-dependent chloride channels in Xenopus laevis oocytes.

The two-electrode voltage-clamp technique was employed to investigate the effects of chloroform-methanol (1:1) extracts derived from five medicinal plants on Xenopus laevis oocytes. When evaluated at concentrations of 1 to 500 microg/ml, the extracts prepared from the aerial parts of Baccharis heterophylla H.B.K (Asteraceae), Chenopodium murale L. (Chenopodiaceae), Desmodium grahami Gray (Leguminosae) and Solanum rostratum Dun (Solanaceae) produced concentration-dependent oscillatory inward currents in the oocytes, while the extract of Gentiana spathacea did not induce any response. The reversal potential of the currents elicited by the active extracts was -17 +/- 2 mV and was similar to the chloride equilibrium potential in oocytes. These ionic responses were independent of extracellular calcium. However, they were eliminated by overnight incubation with BAPTA-AM (10 microM), suggesting that the currents were dependent on intracellular Ca2+ increase. Thus the plant extracts activate the typical oscillatory Ca(2+)-dependent Cl- currents generated in the Xenopus oocyte membrane more probably via a mechanism that involves release of Ca2+ from intracellular reservoirs. These observations suggest that Xenopus oocyte electrophysiological recording constitutes a suitable assay for the study of the mechanisms of action of herbal medicines.     

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.     

Activation by acidic pH of CLC-7 expressed in oocytes from Xenopus laevis

ClC chloride channels are important in diverse physiological functions such as transepithelial transport, cell volume regulation, excitability, and acidification of intracellular organelles. We have investigated the expression of CLC-7 in oocytes from Xenopus laevis with the two electrode voltage clamp technique and Western blot analysis. Using a specific antibody against CLC-7, we found an approximately 80 kDa protein in oocytes, previously injected with CLC-7-cRNA. In voltage clamp experiments on ClC-7-cRNA-injected oocytes, no current changes were detected at normal pH (7.4). However, acidification of the Ringer solution to pH values between 6 and 4 revealed strong currents which reversed at about -15 mV (30 mV positive to the normal resting potential) and showed strong outward rectification. We therefore suggest that ClC-7 in oocytes is a functional chloride current at acidic pH. Since ClC-7 is also found in neuronal tissues and was upregulated in a rat pain model, we suggest a role of CLC-7 also for nociception and pain.     

Surface Charges of K channels. Effects of strontium on five cloned channels expressed in Xenopus oocytes

The effects of strontium (Sr2+; 7-50 mM) on five different cloned rat K channels (Kv1.1, Kv1.5, Kv1.6, Kv2.1, and Kv3.4), expressed in oocytes of Xenopus laevis, were investigated with a two-electrode voltage clamp technique. The main effect was a shift of the Gk(V) curve along the potential axis, different in size for the different channels. Kv1.1 was shifted most and Kv3.4 least, 21 and 8 mV, respectively, at 50 mM. The effect was interpreted in terms of screening of fixed surface charges. The estimated charge densities ranged from -0.37 (Kv1.1) to -0.11 (Kv3.4) e nm-2 and showed good correlation with the total net charge of the extracellularly located amino acid residues of the channel as well as with the charge of a specific region (the loop between the S5 segment and the pore forming segment). The estimated surface potentials were found to be linearly related to the activation midpoint potential, suggesting a functional role for the surface charges.     

The plasma membrane of Xenopus laevis oocytes contains voltage-dependent anion-selective porin channels

Recent patch-clamp studies have shown that anti-porin antibodies, applied to the external side of excised plasma membrane patches of mammalian astrocytes, close chloride channels that are thought to be engaged in cell volume regulation. Frog oocytes are often used to study this basic cell function. Here we document the localisation of endogenous porin voltage-dependent anion-selective channels in Xenopus laevis oocyte plasma membranes. In confocal laser microscopy images a disjunctive pattern of fluorescing spots appear about 10 microm apart. Labelling was prevented by preabsorption of the antibodies with synthetic peptides comprising the epitope of the antigen. Immuno-gold marking of oocyte surfaces followed by silver enhancement of the gold particles lead to a plasma membrane labelling corresponding to that obtained by the confocal laser approach. The data suggests the presence of voltage-dependent, anion-selective channels in oocyte plasma membranes. This data should be borne in mind when frog oocytes are used to study the characteristics of endogenous or heterologously expressed ion channels or regulatory proteins.