Cut-open Voltage Clamp Technique Research Articles

Manipulating Shaker potassium channel gating kinetics with temperature steps

In this work, we studied the effects of rapid temperature steps on the ionic and gating currents of the Shaker potassium channel. Using the endogenous melanin of Xenopus laevis oocytes as a photothermal transducer and a diode laser (445 nm) with a homogenized beam, we can raise the temperature of the oocyte membrane in few milliseconds (9°C in 1.5 ms). In conjunction with the cut-open voltage-clamp technique, this allowed us to have tight control of both voltage and temperature of the oocyte membrane.

Low-Resistance silver bromide electrodes for recording fast ion channel kinetics under voltage clamp conditions

BackgroundTwo-electrode voltage clamp is a widely used technique for studying ionic currents. However, fast activation kinetics of ion channels are disguised by the capacitive transient during voltage clamp of Xenopus oocytes. The limiting factors of clamp performance include, but are not limited to, amplifier gain, membrane capacitance, and micropipette resistance. Previous work has focused on increasing amplifier gain (e.g.; high performing two-electrode amplifiers) or reducing the membrane capacitance (e.g.; the cut-open technique). New methodThe use of an Ag-AgBr electrode with saturated KBr solution to reduce micropipette resistance. ResultsThe conductivity of 4 M KBr was 37 % higher compared to 3 M KCl and the micropipette resistance was reduced by 19 % when 4 M KBr was used, compared to the standard 3 M KCl solution. Micropipette resistances correlated positively with capacitive transient durations. Neither the current-voltage relationship of the voltage-gated sodium channel, Nav1.7, nor Xenopus oocyte stability were affected by bromide ions. Comparison with existing methodsThe de facto standard for two-electrode voltage clamp is 3 M KCl and Ag-AgCl electrodes, which are associated an unnecessarily high micropipette resistance. Elsewise, cut-open voltage clamp techniques are technically demanding and require manipulation of the intracellular environment. ConclusionsThe use of an Ag-AgBr electrode with saturated KBr as micropipette solution reduces the capacitive transient in two-electrode voltage clamp recordings. Moreover, the exchange of chloride against bromide ions does not seem to affect oocyte physiology and ion channel kinetics.

Temperature Sensitivity in a Potassium Channel from Land Plants

The perception of temperature is a crucial sensory mechanism of living organisms and affects cellular and metabolic processes as well as behavior. In animals, changes in environmental temperature are detected by sensory neurons that express temperature sensitive ion channels. Among these, the family of transient receptor potential (TRP) channels plays a major role. In land plants no homologues for TRP channels are found and temperature sensitive ion channels have yet to be uncovered. KAT1 is a hyperpolarization activated potassium channel from Arabidopsis thaliana that regulates stomatal opening. This channel has voltage sensing domain (VSD) and pore domain (PD) similar to depolarization activated potassium channels. KAT1 was expressed in Xenopus laevis oocytes and the cut open voltage clamp technique was used to record the hyperpolarization induced currents. Rapid changes in temperature where achieved as described in the abstract “Probing Ion Channel Thermodynamics With Temperature Jumps In Oocytes”. Dramatic changes in the kinetics of activation and deactivation where observed. Applying a temperature jump before a hyperpolarizing voltage greatly speeds up channel activation with a significant reduction in the delay of the current onset. Changing the bath temperature greatly affects the kinetics of the channel but does not affect the relative conductance versus voltage relation indicating that the voltage dependent transitions are not involved in temperature sensitivity. The activation rate can be well described by two exponentials, each one with a Q10 of about 4. These results indicate that temperature activates KAT1 channels most likely by increasing the open probability trough changes in the coupling between the VSD and PD. The proposed mechanism is different from the allosteric regulation proposed for the TRP family channels. These results provide clues into the evolution of temperature sensitivity in plants. (Support: NIH GM030376)

Hypokalemic Periodic Paralysis Cases with Substitutions from Arginine to Lysine in the Voltage Sensor

Familial hypokalemic periodic paralysis (HypoPP) is a rare skeletal muscle disease caused by the dysregulation of excitability in salcolemma. HypoPP is characterized by repeated episodic paralytic attacks with hypokalemia and several mutations in CACNA1S gene coding Cav1.1 and SCN4A gene coding Nav1.4 have been identified as causative mutations. Most of the mutations are substitution to non-charged amino acid from the positively charged arginine (R) in segment 4 (S4) of the voltage sensor in either Cav1.1 or Nav1.4. Biophysical experiments demonstrated that the loss of the charge in S4 by the arginine-to-non-charged substitution creates a leak current called “gating pore current”. Since the discovery, “the gating pore current theory” has been suggested as the common pathogenic feature in HypoPP. However, the pathological mechanism from generation of “gating pore current” to paralytic attack is still unknown. In this report, we have identified five cases with an arginine(R)-to-lysine (K) substitution in voltage sensor, which retained positive net charge of S4 ; one is a Japanese boy with R219K mutation in SCN4A gene and the other four are one Japanese man, one French man and two French brothers with R897K mutation in CACNA1S gene. All cases presented typical paralytic episodes with hypokalemia and, in four of five cases, significant decrement in the compound action potential was observed with prolonged exercise test. From Nav1.4-R219K mutant channel expressed in vitro, we measured the ionic current by patch-clamp technique and the gating pore current by cut-open voltage clamp technique. Nav1.4-R219K revealed gating pore current, but the size of the conductance was smaller compared to that observed in other mutant HypoPP-Nav1.4 channels previously reported. Our data might provide a new insight in pathogenic mechanism of HypoPP.

Biophysical Characterization of Genetically Encoded Voltage Sensor ASAP1: Dynamic Range Improvement

Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. This indicator is based on the Gallus gallus voltage-sensitive phosphatase with the phosphatase domain removed and a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Using the cut-open voltage clamp technique, we have simultaneously recorded fluorescence signals and gating currents from Xenopus laevis oocytes expressing ASAP1. Gating charge movement and fluorescence kinetics track closely with each other, although ASAP1 gating currents are significantly faster than those of Ciona intestinalis voltage-sensitive phosphatase. Altering the residue before the first gating charge removes a split in the ASAP1 QV curve, but preserves the accelerated kinetics that allow for the faithful tracking of action potentials in neurons.

Biophysical Characterization of ASAP1

Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. ASAP1 is based on the Gallus gallus voltage sensitive phosphatase (GgVSP) with a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Expressing this molecule in Xenopus oocytes and recording fluorescence simultaneously with gating current using the cut-open voltage clamp technique, our studies show that the gating currents of ASAP1 are significantly faster than CiVSP.

Characterization of a Fast Voltage-Sensing Protein using Voltage-Clamp Fluorometry

Voltage-sensing domains in voltage-activated ion channels and other voltage-sensing proteins contain well-conserved S4 helices containing basic residues capable of sensing changes in membrane potential to trigger opening or closing of ion-selective pores or phosphatase activity in voltage-sensitive phosphatases. Here we report the discovery of a protein which contains a voltage-sensing domain capable of rapid and slow rearrangements in response to changes in voltage. In place of a pore domain, this voltage sensor has large N- and C-termini predicted to contain structured and disordered domains for interaction with intracellular proteins. Our working hypothesis is that this protein, which we name Coupled Voltage Sensor (CVS), functions as a voltage sensor that couples to intracellular signaling pathways (as yet undefined). Our goal in the present experiments was to demonstrate that CVS undergoes voltage-dependent conformation changes and to characterize those using site-specific voltage-clamp fluorometry. We identified several positions at the external end of S4 where introduced and fluorophore-labeled Cys residues produce changes in fluorescence as a function of membrane potential. Several positions give complex fluorescence responses, starting with a rapid fluorescence increase (dequenching), followed by slower fluorescence decrease (quenching). Interestingly the rapid component is as fast as the clamp speed when using the cut-open voltage clamp technique (tau ∼ 200 μs). To exclude the possibility of Stark effect we examined the effects of a series of soluble quenchers and observed enhanced quenching with membrane depolarization, consistent with an outward movement of the S4 helix. Overall, our results support a model in which CVS undergoes two temporally distinct conformation rearrangements in response to membrane depolarization.

Exploring Molecular Mechanisms of the Functional Interaction between Kv1.3 and Nav Beta1

Voltage gated ion channels (VGICs) play crucial roles in the propagation electrical signals in excitable cells. In contrast, several VGICs have been also known to express in non-excitable cells and participate in keeping cell homeostasis. The voltage-gated potassium channel subfamily A member 3 (Kv1.3) is one of the Shaker-related channels expressed mainly on T lymphocytes. Many reports have indicated that Kv1.3 is associated with several diseases including autoimmune diseases, inflammatory diseases and obesity. On the other hand, the voltage-gated sodium channel (Nav) Beta 1 is known as a subunit modulating kinetics and expression of Nav, but several studies have shown that NavBeta1 also participates in brain development and cell signaling. These ‘non-canonical’ functions in VGICs have been focused upon recently and an exciting report showed the functional interaction between murine Kv1.3 (mKv1.3) and rat Navbeta1 (rBeta1) (Nguyan HM et al. PNAS 2012). The authors demonstrated that the activation of mKv1.3 ionic current is accelerated by rBeta1. Here, we explore the molecular mechanisms of the acceleration in Kv1.3 activation through gating currents of human Kv1.3 (hKv1.3) with and without rBeta1 using the cut-open voltage clamp technique. We found that the N-terminus of hKv1.3 is associated with trafficking efficiency of the protein to the membrane in Xenopus oocytes. Our data indicated that rBeta1 did not accelerate the gating current activation of hKv1.3, but shifted the Q-V in the depolarizing direction and slowed their kinetics. Because there are several differences in the amino acid sequences of mKv1.3 and hKv1.3, the differences between the previous report and our results may give clues on the molecular mechanisms of the functional interaction between Kv1.3 and rBeta1. Support: 13POST14800031 (AHA), U54GM087519 and R01GM030376

Analysis of CorA-Catalyzed Mg2+ Selective Currents in Xenopus Oocytes

In prokaryotes, Magnesium homeostasis is tightly controlled through the workings of three distinct transport systems, with CorA being the primary source of Mg2+ uptake. The recent structures of CorA from the thermophilic eubacteria Thermotoga maritima (TmCorA) have provided an excellent model for a molecular understanding of Mg2+ transport. Yet, a detailed characterization of CorA function has been conspicuously missing so far, as only in-vivo bulk assays have been available to evaluate Mg2+ translocation leaving unanswered fundamental questions related to CorA ion selectivity and specificity, gating mechanism and regulation. Here we have developed a system for high-level expression of TmCorA in Xenopus oocytes that allows for an accurate functional readout of Mg2+ transport through several electrophysiological techniques. Mg2+ currents from TmCorA in the order of 1 to 10 μA were routinely obtained with the cut-open voltage clamp technique and two electrodes Voltage clamp.We demonstrate that CorA act as a strong inward rectifier, an observation in agreement with its physiological role as the primary source of Mg2+ uptake. As expected, Mg2+ currents are blocked by Co(III) hexamine, a structural analog of a hydrated Mg2+. CorA selectivity towards Magnesium vs. other divalent and monovalents was characterized using a perfusion TEV setup. The divalent selectivity series for CorA was determined as Co2+>=Mg2+>Ni2+>Zn2+>>>Ca2+ = Ba2+. Macroscopic CorA currents record from oocytes macropatches show that Mg2+-binding to the cytoplasm domain act as a gating factor. Single channel transitions are still below our detection capabilities and efforts are ongoing to characterize the conductance of TmCorA through noise analysis. The ability to evaluate CorA function by means of electrophysiological methods will allow for a detailed analysis of structure/function relationships in CorA.

PH Modulation of the Cardiac Voltage Gated Sodium Channel, Nav1.5

Alterations in the function of the cardiac voltage-gated sodium channel (NaV1.5) are a known cause of cardiac disease and arrhythmia. Elevated concentrations of protons decrease conductance and depolarize the voltage dependence of activation and steady-state fast inactivation (SSFI) of NaV1.5 channels (Zhang & Siegelbaum, 1991, Khan et al., 2006). A complete analysis of the effects of low pH on NaV1.5 channel kinetics has not previously been reported. We sought to characterize the effects of low pH on NaV1.5 kinetics. NaV1.5 was co-expressed in Xenopus laevis oocytes with the β1 subunit, and currents were recorded at 20 °C using the cut-open voltage clamp technique with the extracellular solution titrated to either pH 7.4 (control) or pH 6.0. Application of solution at pH 6.0 significantly depolarized the voltage dependence of activation and SSFI; −34.4 ± 0.3mV to −25.2 ± 0.2mV and −76.4 ± 0.1mV to −72.7 ± 0.2mV, respectively. The apparent valences of activation and SSFI were significantly decreased; from 3.4 ± 0.12e to 2.5 ± 0.04e, and from −4.6 ± 0.07e to −4.1 ± 0.09e, respectively. At pH 6.0, the fast time constant of use-dependent inactivation was significantly increased and the use dependent current reduction was decreased from 40.6 ± 0.12% to 34.8 ± 0.05%. The rates of open-state fast inactivation onset were significantly decreased at potentials between −30mV and +30mV, and the rates of recovery at −90mV and −80mV were significantly increased. There was also a visible increase in window current. All effects were reversible upon reperfusion of solution at pH 7.4. Taken together, these data suggest that lowering extracellular pH from 7.4 to 6.0 destabilizes the fast-inactivated state of NaV1.5 channels, an effect that could act as an arrhythmogenic trigger during ischemic events.

Cav2.3 Calcium Channels Inactivate from the Open State with Partial Charge Immobilization and Altered Deactivation Kinetic

Voltage-dependent ion channels undergo inactivation following activation. In the sodium and potassium channels, the molecular determinants that govern the mechanism of inactivation involve pore blocking by a cytoplasmic particle. In calcium channels the consensus model is that the intracellular loop joiningthe first two homology domains of the pore forming subunit contributes to the inactivation gate or hinged-lid. To investigate key features of this model, we expressed CaV2.3 channel in Xenopus oocytes without auxiliary subunits and recorded ionic and gating currents using the cut-open voltage-clamp technique. Ionic current were recorded in either Ba2+ or Ca2+. Here we report that consistent with a hinge-lead mechanisms, charge movement at the end of a depolarizing pulse decreases up to a 50% with channel inactivation. In contrast with a previous report by Patil et al (1998, Neuron 20:1020) that co-expresses auxiliary subunit with CaV2.3, trains of pulse elicited minimal inactivation. It appears then that when expressed alone, CaV2.3 channels inactivate mostly from the open state. We also found that as channels inactivate, a slow component emerges in tail-current recordings. This component contributes to about 20% of the tail currents in 80% inactivated currents and can be accounted for with a classic allosterically-coupled model provided that channels can re-open multiple times from the last closed state and that some closed-inactive channels re-open during membrane repolarization.Supported by REFD-24 and FONDECYT 1980635 to AN and a CONICYTFellowship to GC.