Voltage Clamp Technique In Oocytes Research Articles

Neurotoxin Merging: A Strategy Deployed by the Venom of the Spider Cupiennius salei to Potentiate Toxicity on Insects.

The venom of Cupiennius salei is composed of dozens of neurotoxins, with most of them supposed to act on ion channels. Some insecticidal monomeric neurotoxins contain an α-helical part besides their inhibitor cystine knot (ICK) motif (type 1). Other neurotoxins have, besides the ICK motif, an α-helical part of an open loop, resulting in a heterodimeric structure (type 2). Due to their low toxicity, it is difficult to understand the existence of type 2 peptides. Here, we show with the voltage clamp technique in oocytes of Xenopus laevis that a combined application of structural type 1 and type 2 neurotoxins has a much more pronounced cytolytic effect than each of the toxins alone. In biotests with Drosophila melanogaster, the combined effect of both neurotoxins was enhanced by 2 to 3 log units when compared to the components alone. Electrophysiological measurements of a type 2 peptide at 18 ion channel types, expressed in Xenopus laevis oocytes, showed no effect. Microscale thermophoresis data indicate a monomeric/heterodimeric peptide complex formation, thus a direct interaction between type 1 and type 2 peptides, leading to cell death. In conclusion, peptide mergers between both neurotoxins are the main cause for the high cytolytic activity of Cupiennius salei venom.

The Fast Component of hERG Gating Charge: An Interaction between D411 in the S1 and S4 Residues

Kv11.1 (hERG) is a voltage-gated potassium channel that shows very slow ionic current activation kinetics, and an unusual underlying biphasic gating charge movement with fast and slow components that differ greatly in time course. The structural basis and role of the fast component of gating charge (Qfast) is unclear, and its relationship to the slow activation of hERG channels is not understood. In this study we have used the cut-open oocyte voltage-clamp technique to investigate the relationship of fast gating charge movement-to-residue interactions between D411 at the bottom of the S1, and lower S4 domain charged and uncharged residues. Neutralization of D411 or K538 and V535A prevented Qfast and greatly accelerated overall charge movement. Voltage-clamp fluorometry showed a loss of a fast component of S4 fluorescence in D411N, V535A, and K538Q upon depolarization, whereas [2-(trimethyl ammonium) ethyl] methanethiosulfonate chloride modification of I521C in the outer S4 was enhanced at more negative potentials and at earlier times in these same mutants. A functional interaction between these regions during activation was suggested by ΔΔGo values >4.2 kJ/mol obtained from double mutant cycle analysis. The data indicate that interactions of S1 residue D411 with lower S4 residues stabilizes early closed states of the channel, and that disruption of these interactions results in both faster rates of activation gating and an elimination of the fast component of gating charge movement and of fluorescence. We propose that the Qfast charge movement during activation accompanies transitions through early closed states of the hERG activation pathway, and that the weak voltage dependence of these transitions limits the overall activation rate of hERG channels. Disruption of the D411-S4 interactions destabilizes these early closed states, leaving hERG channels able to activate at a rate similar to conventional potassium channels.

BK and CaV Channel Interactions in the Plasma Membrane

Activation of the human voltage- and Ca2+-gated (BK, Slo1) K+ channel is triggered by both depolarization and increases in intracellular Ca2+ concentration. BK channels and different types of voltage gated (CaV) channels associate in the cell membrane such that Ca2+ entering via CaV channels activates BK channels. Protein co-localization and distribution at the plasma membrane level are typically addressed by fluorescence microscopy. We seek to gain insight into the relative proximity and positioning of BK channels and different types of CaV channels (CaV1.2 or CaV2.2) in the membrane by fitting to a mathematical model ionic current recordings obtained from Xenopus oocytes co-expressing BK and CaV channels. Prior to voltage clamp, the oocytes were injected with known concentrations of EGTA or BAPTA. Using the cut-open oocyte voltage-clamp technique, oocytes were subjected to a protocol consisting of two 80 mV pulses, separated by a −70 mV or 0 mV intermediate pulse of variable duration. The magnitude of the BK channel currents, during the second 80 mV pulse, depended on the amplitude and duration of Ca2+ entry during the intermediate pulse, as well as intracellular Ca2+ buffering conditions. Under the same experimental conditions, CaV2.2 channels activated BK channels more efficiently than CaV1.2 types, as suggested by the longer-lasting potentiation of the BK current when CaV2.2 and BK channels were co-expressed. We are interpreting the degree of physical association in view of a mathematical model that computes the activation of CaV and BK channels, as well as intracellular Ca2+ dynamics, and allows for varying density and relative distance between these channels. This model may be useful for predicting the relative distribution of CaV and BK channels in native cells.

Gating by Voltage and Ca2+ in Human Connexin (cx26) Hemichannels

Opening of connexin hemichannels permits the release of small metabolites, such as ATP and glutamate, which play an important autocrine/paracrine signaling in a variety of cell types. The recently solved crystal structure of the Cx26 gap junction channel allows us to explore in greater detail the relationship between the structure and function of both, hemichannels and gap junction channels. Here, we begin by revisiting the activation mechanisms of human Cx26 (hCx26) hemichannels by voltage and Ca2+. Using the two electrode oocyte voltage-clamp technique, we found that depolarization up to +60 mV induced activation of hemichannel currents, and repolarization produced large tail-currents with slow deactivation (τ ∼10 s). Interestingly, the magnitudes of the tail-currents were dependent on the lengths of the depolarizating pulses rather than the magnitudes of the currents activated during the pulses. Strikingly, cell-attached single channel recordings showed that depolarizating pulses (≤40 mV) stimulated only infrequent and brief openings of hCx26 hemichannels. However, the repolarizating pulses induced opening of single hemichannel currents (likely corresponding to the tail currents) with smaller conductance and longer mean open times. In addition, we found that low Ca2+ (500 μM) increased macroscopic hemichannel currents at positive potentials and slowed deactivation of the tail currents. We are currently performing single channel recordings to elucidate how Ca2+ modulates channel activation and deactivation kinetics in response to depolarizing and repolarizing voltages. Our results indicate that while depolarization causes opening of some hCx26 hemichannels, it mostly shifts the hemichannels to a non-conductive state that will likely open after repolarization; and that Ca2+ ions may play a role by regulating the energetics of these transitions. Supported by The Grass Foundation.

Gating kinetics of Shaker K+ channels are differentially modified by general anesthetics.

The ShakerB K+ channel was used as a model voltage-gated channel to probe the interaction of volatile general anesthetics with gating mechanisms. The effects of three anesthetics, chloroform (CHCl3), isoflurane, and halothane, were studied using recombinant native and mutant Shaker channels expressed in Xenopus oocytes. Gating currents and macroscopic ionic currents were recorded with the cut-open oocyte voltage-clamp technique. The effects of CHCl3 and isoflurane on gating kinetics of noninactivating mutants were opposite, whereas halothane had no effect. The effects on ionic currents were also agent dependent: CHCl3 and halothane produced a reduction of the macroscopic conductance, whereas isoflurane increased it. The results indicate that the gating machinery of the channel is mostly insensitive to the anesthetics during activation until near the open state. The effects on the conductance are mainly due to changes in the transitions in and out of the open state. The data give support to direct protein-anesthetic interactions. The magnitude and nature of the effects invite reconsideration of Shaker-like K+ channels as important sites of action of general anesthetics.

17] Cut-open oocyte voltage-clamp technique

In this article, we described the use and limitations of the COVG technique. The advantages of the present method as follows: 1. High-frequency response and low noise recording (24-microseconds time constant and 1.2-nA rms at 5 kHz). This allows the accurate description of small gating currents and fast activation or deactivation of ionic currents to be adequately resolved. Currents up to 20-30 microA can be adequately clamped. 2. Stable recording conditions lasting for several hours. Even though this technique has internal access, rundown is minimized compared to excised patches. 3. Access to the cell interior. The intracellular medium can be exchanged with various solutions. This was of invaluable help in recording K+ gating currents, an experimental condition that required the complete blockade of all ionic currents. This advantage will make the present method suitable for the study of channel modulation by second messengers and drugs. In addition, channel selectivity properties can be defined by ion substitution experiments. 4. Channel localization. A study of the membrane compartmentalization of channels can be easily obtained with this method, since it allows the voltage clamping of different regions of the oocyte surface.