Cut-open Oocyte Voltage Clamp Technique Research Articles

Modulation of KV7.1 by NaVβ1 Subunit

Several small proteins, referred to as “β-subunits”, have been shown to modulate the activity of potassium-selective, voltage-gated (KV) channels, rendering K+-conductance with different voltage dependence and kinetics. The physiological role of these interactions has been identified in some case. For instance, the interaction of KV7.1 and KCNE1 produces the cardiac K+-current known as IKS, which has a slower kinetics and negatively shifted voltage dependence for activation with respect to KV7.1 alone. In contrast, KCNE3 renders a channel that is open at physiological potentials. This characteristic of KV7.1 makes it into a versatile “α-subunit” with activity that can be physiologically customized by β-subunits. Recent studies have shown that NaVβ1, which is regarded as a β-subunit of sodium-selective, voltage-gated (NaV) channels, can modulate the activity of several KV channels. Since both KV7.1 and NaVβ1 are expressed in the hippocampus and cerebellum of mice, we wonder whether these two proteins could functionally interact. Thus, we recorded K+-currents from Xenopus oocytes co-expressing KV7.1 and NaVβ1 using the Cut-open Oocyte Voltage Clamp technique. We found that the expression of NaVβ1 caused KV7.1 to display a slow inactivating-like behavior, decreasing the K+-conductance over 20% after depolarization to −10 mV and above. Further, the deactivation rate of the K+-current decreased, displaying at least three phases, namely, a partial recovery from inactivation, a fast deactivating phase, and a slower deactivating phase. The rate of the initial fast deactivation was similar to the deactivating rate of the KV7.1 expressed alone. In contrast, the rate of the slow deactivation was 2-fold slower than the rate of deactivation of KV7.1 alone. Furthermore, our results indicate that NaVβ1 was able to modulate the voltage dependence for activation KV7.1 showing an apparent displacement of −20 mV. The physiological significance of this interaction is yet to be determined.

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.

High Affinity Nav Channel Binder with Specific Conjugation Site Derived from Scorpion Toxin with Attenuated Toxicity

Voltage-gated sodium channels (Nav) play a key role in generating action potential which leads to physiological signaling in excitable cells. However, the availability of probes for functional study of mammalian Nav is limited. Based on the beta-scorpion toxin Ts1, we have chemically synthesized a novel biologically friendly binder [S14R, W50Pra]Ts1 (Ts1-S14R) for Nav with high affinity and extremely low dissociation rate. This Ts1 analog has a S14R point mutation, corresponding to E15R in Css4, another beta-scorpion toxin, and also has a propargylglycine (Pra) mutation from W50 for bioconjugation. We identified the pharmacological properties of Ts1-S14R on rat skeletal muscle Nav channel (Nav1.4) using the cut-open oocyte voltage clamp technique and confirmed that its toxicity has been dramatically reduced. Chemical synthesis of this binder enabled us to conjugate with molecules of interest for various studies. Using the fluorescent-dye, BODIPY, conjugated binder, we examined its binding location in Nav1.4 using Lanthanide-based Resonance Energy Transfer technique (LRET). Our LRET study showed Ts1-S14R-BODIPY binds to Nav1.4 at virtually identical location as that of Ts1-BODIPY. In addition, LRET data showed that, once Ts1-S14R-BODIPY binds to Nav1.4, it stays in place for at least 40 min in toxin-free solution, indicating that Ts1-S14R-BODIPY can be employed as a stable binder for Nav1.4. Moreover, we generated Ts1-S14R conjugated with gold nanoparticle (Ts1-S14R-AuNP). We demonstrated this conjugate can induce action potentials by visible light stimulation through the opto-thermal-capacitive effect in rat dorsal root ganglia (DRG) neurons similarly to that previously reported (Carvalho-de-Souza JL et al. Neuron 2015). Ts1-S14R is therefore a novel probe with great potential for wider applications in Nav-related neuroscience research. Support: U54GM087519 and GM030376.

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.

Controlling the Activity of a Phosphatase and Tensin Homolog (PTEN) by Membrane Potential

The recently discovered voltage-sensitive phosphatases (VSPs) hydrolyze phosphoinositides upon depolarization of the membrane potential, thus representing a novel principle for the transduction of electrical activity into biochemical signals. Here, we demonstrate the possibility to confer voltage sensitivity to cytosolic enzymes. By fusing the tumor suppressor PTEN to the voltage sensor of the prototypic VSP from Ciona intestinalis, Ci-VSP, we generated chimeric proteins that are voltage-sensitive and display PTEN-like enzymatic activity in a strictly depolarization-dependent manner in vivo. Functional coupling of the exogenous enzymatic activity to the voltage sensor is mediated by a phospholipid-binding motif at the interface between voltage sensor and catalytic domains. Our findings reveal that the main domains of VSPs and related phosphoinositide phosphatases are intrinsically modular and define structural requirements for coupling of enzymatic activity to a voltage sensor domain. A key feature of this prototype of novel engineered voltage-sensitive enzymes, termed Ci-VSPTEN, is the novel ability to switch enzymatic activity of PTEN rapidly and reversibly. We demonstrate that experimental control of Ci-VSPTEN can be obtained either by electrophysiological techniques or more general techniques, using potassium-induced depolarization of intact cells. Thus, Ci-VSPTEN provides a novel approach for studying the complex mechanism of activation, cellular control, and pharmacology of this important tumor suppressor. Moreover, by inducing temporally precise perturbation of phosphoinositide concentrations, Ci-VSPTEN will be useful for probing the role and specificity of these messengers in many cellular processes and to analyze the timing of phosphoinositide signaling.

Gating Current of the KCNQ1 Voltage-Gated Potassium Channel

KCNQ1 pore-forming alpha-subunits are crucial to physiology, operating in vivo with KCNE1 and KCNE3 beta-subunits in the heart and stomach, respectively. Recently, the gating currents of KCNQ4 and KCNQ5 channels were characterized (Miceli, Channels, 2009); here, equivalent measurements are described for KCNQ1 channels formed in the absence of beta-subunits. Human KCNQ1 was studied in Xenopus oocytes with the cut-open oocyte voltage clamp technique. To record gating currents, cells were depleted of internal potassium ions and residual ionic current was blocked with tetraethyammonium and barium. At room temperature, gating currents were too small to resolve. At 28°C, ON gating current gave rise to peak charge movement at +40 mV of ∼0.7 nC / µA of ionic current. The total charge movement at each test potential was conserved in the OFF-gating currents. Analysis of the charge-voltage (QV) relationship and conductance-voltage (GV) relationship showed a 10 mV hyperpolarizing shift of the half-maximal voltage of activation of the normalized QV curve with respect to the normalized GV curve. KCNQ1 ON-gating current decay constants were 4-fold slower than in KCNQ4 and required longer test pulses to fully resolve. Overlays of gating and ionic currents revealed that, as for KCNQ4, gating charges were still moving even after KCNQ1 channels started to open, indicating that charge movement was a rate-limiting factor in channel opening. These first characterizations of KCNQ1 gating currents and are an important step towards understanding mutations that lead to cardiac arrhythmias, such as long-QT syndrome, and the effects of beta-subunits on channel function. Supported by NIH GM030376, University of Chicago MSTP, and The Paul and Daisy Soros Fellowship for New Americans.

The Src Homology 3 Domain of the β-Subunit of Voltage-gated Calcium Channels Promotes Endocytosis via Dynamin Interaction

High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary beta-subunit to the alpha-interaction-domain in the pore-forming alpha1-subunit is required to form functional channels. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the alpha-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated beta-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the alpha1-subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing alpha1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length beta-subunit also down-regulates voltage-gated calcium channels but only when lacking the alpha-interaction domain. Moreover, isolated Src homology 3 domain and the full-length beta-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the beta-subunit regulates the turnover of voltage-gated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the beta-subunit to the alpha1-pore-forming subunit. Our findings define a novel function for the beta-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.

Folding of Active Calcium Channel β1b -Subunit by Size-exclusion Chromatography and Its Role on Channel Function

Voltage-gated calcium channels mediate the influx of Ca(2+) ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming alpha(1)-subunit and the auxiliary beta- and alpha(2)delta-subunits. The beta-subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity. Four beta-subunit genes have been cloned, beta(1-4), with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The beta(1b)-subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming alpha(1)- and the regulatory beta-subunit. However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria. We applied an on-column refolding procedure based on size exclusion chromatography to fold the beta(1b)-subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The beta(1b)-subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the alpha(1)-subunit (the alpha-interaction domain). Using the cut-open oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac alpha(1)-subunit (alpha(1C)) co-injected with folded-beta(1b)-protein or beta(1b)-cRNA. We demonstrate that the co-expression of the alpha(1C)-subunit with either folded-beta(1b)-protein or beta(1b)-cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the beta(1b)-subunit primarily modulates channel activity, rather than expression.

Gating currents associated with intramembrane charge displacement in HERG potassium channels.

HERG (human ether-a-go-go-related gene) encodes a delayed rectifier K+ channel vital to normal repolarization of cardiac action potentials. Attenuation of repolarizing K+ current caused by mutations in HERG or channel block by common medications prolongs ventricular action potentials and increases the risk of arrhythmia and sudden death. The critical role of HERG in maintenance of normal cardiac electrical activity derives from its unusual gating properties. Opposite to other voltage-gated K+ channels, the rate of HERG channel inactivation is faster than activation and appears to be intrinsically voltage dependent. To investigate voltage sensor movement associated with slow activation and fast inactivation, we characterized HERG gating currents. When the cut-open oocyte voltage clamp technique was used, membrane depolarization elicited gating current with fast and slow components that differed 100-fold in their kinetics. Unlike previously studied voltage-gated K+ channels, the bulk of charge movement in HERG was protracted, consistent with the slow rate of ionic current activation. Despite similar kinetic features, fast inactivation was not derived from the fast gating component. Analysis of an inactivation-deficient mutant HERG channel and a Markov kinetic model suggest that HERG inactivation is coupled to activation.

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.

Transitions Near the Open State in Shaker K+-channel: Probing with Temperature

Ionic currents from non-inactivating Shaker K+-channels were expressed in Xenopus oocytes and recorded with the cut-open oocyte voltage clamp technique. Kinetic properties of ionic currents were studied between 1.4 and 19°C. The forward step of the last transition (…C → O) of the activation pathway, just before the channel opening, was studied by the recovery of the ionic current after a short interval (< 500 μsec) at a hyperpolarized potential (− 90 mV) between two identical depolarizing pulses (0, 40 or 100 mV). Ionic currents under these experimental conditions are described by at least three kinetic components. The predominant kinetic component (corresponding to the …C → O transition) has a low temperature dependence (activation energy, Ea, around −5 kcal/mol) and it is voltage independent, suggesting that this step does not carry charge and has a mainly entropic energy barrier. The backward step (O → C…) was studied from ionic tail currents at −120 mV after pulsing to 40 mV. The closure of the channel has a high temperature dependence (Q10 > 4), having an Ea of 24–25 kcal/mol. The closure of the channel also is voltage dependent, having a valence times fraction of the electric field, zβ of 0.5 eo. We conclude that the transition from the closed to the opened state has a net decrease in entropy and enthalpy, suggesting that the channel gets more ordered when it is open. Copyright © 1996 Elsevier Science Ltd

Gating of Shaker K+ channels: I. Ionic and gating currents

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).