scholarly journals Voltage- and NADPH-dependence of electron currents generated by the phagocytic NADPH oxidase

2005 ◽  
Vol 388 (2) ◽  
pp. 485-491 ◽  
Author(s):  
Gábor L. PETHEŐ ◽  
Nicolas DEMAUREX
Keyword(s):  
Membrane Potential ◽  
Nadph Oxidase ◽  
Voltage Dependence ◽  
Intrinsic Property ◽  
Maximal Activity ◽  
Underlying Mechanism ◽  
Potential Range ◽  
Electron Current ◽  
Steady State Current ◽  
Voltage Dependent

The phagocytic NADPH oxidase generates superoxide by transferring electrons from cytosolic NADPH to extracellular O2. The activity of the oxidase at the plasma membrane can be measured as electron current (Ie), and the voltage dependence of Ie was recently reported to exhibit a strong rectification in human eosinophils, with the currents being nearly voltage independent at negative potentials. To investigate the underlying mechanism, we performed voltage-clamp experiments on inside-out patches from human eosinophils activated with PMA. Electron current was evoked by bath application of different concentrations of NADPH, whereas slow voltage ramps (0.8 mV/ms), ranging from −120 to 200 mV, were applied to obtain ‘steady-state’ current–voltage relationships (I–V). The amplitude of Ie recorded at −40 mV was minimal at 8 μM NADPH and saturated above 1 mM, with half-maximal activity (Km) observed at approx. 110 μM NADPH. Comparison of I–V values obtained at different NADPH concentrations revealed that the voltage-dependence of Ie is strongly influenced by the substrate concentration. Above 0.1 mM NADPH, Ie was markedly voltage-dependent and steeply decreased with depolarization within the physiological membrane potential range (−60 to 60 mV), the I–V curve strongly rectifying only below −100 mV. At lower NADPH concentrations the I–V curve was progressively shifted to more positive potentials and Ie became voltage-independent also within the physiological range. Consequently, the Km of the oxidase decreased by approx. 40% (from 100 to 60 μM) when the membrane potential increased from −60 to 60 mV. We concluded that the oxidase activity depends on both membrane potential and [NADPH], and that the shape of the Ie–V curve is influenced by the concentration of NADPH in the submillimolar range. The surprising voltage-independence of Ie reported in whole-cell perforated patch recordings was most likely due to substrate limitation and is not an intrinsic property of the oxidase.

scholarly journals Amphiphilic Blockers Punch through a Mutant CLC-0 Pore

2008 ◽  
Vol 133 (1) ◽  
pp. 59-68 ◽  
Author(s):  
Xiao-Dong Zhang ◽  
Tsung-Yu Chen
Keyword(s):  
Membrane Potential ◽  
Voltage Dependence ◽  
Reversal Potential ◽  
Dissociation Rate ◽  
Channel Pore ◽  
Apparent Affinity ◽  
Amphiphilic Molecules ◽  
Steady State Current ◽  
Voltage Dependent ◽  
Negative Membrane Potential

Intracellularly applied amphiphilic molecules, such as p-chlorophenoxy acetate (CPA) and octanoate, block various pore-open mutants of CLC-0. The voltage-dependent block of a particular pore-open mutant, E166G, was found to be multiphasic. In symmetrical 140 mM Cl−, the apparent affinity of the blocker in this mutant increased with a negative membrane potential but, paradoxically, decreased when the negative membrane potential was greater than −80 mV, a phenomenon similar to the blocker “punch-through” shown in many blocker studies of cation channels. To provide further evidence of the punch-through of CPA and octanoate, we studied the dissociation rate of the blocker from the pore by measuring the time constant of relief from the block under various voltage and ionic conditions. Consistent with the voltage dependence of the effect on the steady-state current, the rate of CPA dissociation from the E166G pore reached a minimum at −80 mV in symmetrical 140 mM Cl−, and the direction of current recovery suggested that the bound CPA in the pore can dissociate into both intracellular and extracellular solutions. Moreover, the CPA dissociation depends upon the Cl− reversal potential with a minimal dissociation rate at a voltage 80 mV more negative than the Cl− reversal potential. That the shift of the CPA-dissociation rate follows the Cl− gradient across the membrane argues that these blockers can indeed punch through the channel pore. Furthermore, a minimal CPA-dissociation rate at a voltage 80 mV more negative than the Cl− reversal potential suggests that the outward blocker movement through the CLC-0 pore is more difficult than the inward movement.

scholarly journals The hyperpolarization-activated current shifts the dynamic range of a voltage-dependent electrical synapse

2021 ◽  
Author(s):  
Wolfgang Stein ◽  
Margaret DeMaegd ◽  
Lena Yolanda Braun ◽  
Andrés G Vidal-Gadea ◽  
Allison L Harris ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Motor Neuron ◽  
Axon Terminal ◽  
Voltage Dependence ◽  
Dynamic Range ◽  
Complex Dynamics ◽  
Ionic Current ◽  
Electrical Synapse ◽  
Resting Membrane Potential ◽  
Voltage Dependent

Like their chemical counterparts, electrical synapses show complex dynamics such as rectification and voltage dependence that interact with other electrical processes in neurons. The consequences arising from these interactions for the electrical behavior of the synapse, and the dynamics they create, remain largely unexplored. Using a voltage-dependent electrical synapse between a descending modulatory projection neuron (MCN1) and a motor neuron (LG) in the crustacean stomatogastric ganglion, we find that the influence of the hyperpolarization-activated inward current (Ih) is critical to the function of the electrical synapse. When we blocked Ih with CsCl, the voltage dependence of the electrical synapse shifted by 18.7 mV to more hyperpolarized voltages, placing the dynamic range of the electrical synapse outside of the range of voltages used by the LG motor neuron (-60.2 mV to -44.9 mV). With dual electrode current- and voltage-clamp recordings, we demonstrate that this voltage shift is due to a sustained effect of Ih on the presynaptic MCN1 axon terminal membrane potential. Ih-induced depolarization of the axon terminal membrane potential increased the electrical postsynaptic potentials and currents. With Ih present, the axon terminal resting membrane potential depolarized, shifting the dynamic range of the electrical synapse towards the functional range of the motor neuron. We thus demonstrate that the function of an electrical synapse is critically influenced by a voltage-dependent ionic current (Ih).

scholarly journals The Role of Na,k-Atpase α Subunit Serine 775 and Glutamate 779 in Determining the Extracellular K+And Membrane Potential–Dependent Properties of the Na,k -Pump

2000 ◽  
Vol 116 (1) ◽  
pp. 47-60 ◽  
Author(s):  
R. Daniel Peluffo ◽  
José M. Argüello ◽  
Joshua R. Berlin
Keyword(s):  
Membrane Potential ◽  
Voltage Dependence ◽  
Protein Structures ◽  
Pump Current ◽  
Transmembrane Segment ◽  
Α Subunit ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
Α1 Subunit

The roles of Ser775 and Glu779, two amino acids in the putative fifth transmembrane segment of the Na,K -ATPase α subunit, in determining the voltage and extracellular K + (K +o) dependence of enzyme-mediated ion transport, were examined in this study. HeLa cells expressing the α1 subunit of sheep Na,K -ATPase were voltage clamped via patch electrodes containing solutions with 115 mM Na+ (37°C). Na,K -pump current produced by the ouabain-resistant control enzyme (RD), containing amino acid substitutions Gln111Arg and Asn122Asp, displayed a membrane potential and K +o dependence similar to wild-type Na,K -ATPase during superfusion with 0 and 148 mM Na+-containing salt solutions. Additional substitution of alanine at Ser775 or Glu779 produced 155- and 15-fold increases, respectively, in the K +o concentration that half-maximally activated Na,K -pump current at 0 mV in extracellular Na+-free solutions. However, the voltage dependence of Na,K -pump current was unchanged in RD and alanine-substituted enzymes. Thus, large changes in apparent K +o affinity could be produced by mutations in the fifth transmembrane segment of the Na,K -ATPase with little effect on voltage-dependent properties of K + transport. One interpretation of these results is that protein structures responsible for the kinetics of K +o binding and/or occlusion may be distinct, at least in part, from those that are responsible for the voltage dependence of K +o binding to the Na,K -ATPase.

scholarly journals EPSPs in rat neocortical neurons in vitro. II. Involvement of N-methyl-D-aspartate receptors in the generation of EPSPs

1989 ◽  
Vol 61 (3) ◽  
pp. 621-634 ◽  
Author(s):  
B. Sutor ◽  
J. J. Hablitz
Keyword(s):  
Electrical Stimulation ◽  
Membrane Potential ◽  
Action Potentials ◽  
Voltage Dependence ◽  
Negative Slope ◽  
Inward Rectification ◽  
Potential Range ◽  
Bathing Solution ◽  
Postsynaptic Potentials ◽  
Neocortical Neurons

1. Intracellular recordings were obtained from neurons in layer II/III of rat frontal cortex. Single-electrode current- and voltage-clamp techniques were employed to compare the sensitivity of excitatory postsynaptic potentials (EPSPs) and iontophoretically evoked responses to N-methyl-D-aspartate (NMDA) to the selective NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-2-APV). The voltage dependence of the amplitudes of the EPSPs before and after pharmacologic changes in the neuron's current-voltage relationship was also examined. 2. NMDA depolarized the membrane potential, increased the neuron's apparent input resistance (RN), and evoked bursts of action potentials. The NMDA-induced membrane current (INMDA) gradually increased with depolarization from -80 to -40 mV. The relationship between INMDA and membrane potential displayed a region of negative slope conductance in the potential range between -70 and -40 mV which was sufficient to explain the apparent increase in RN and the burst discharges during the NMDA-induced depolarization. 3. Short-latency EPSPs (eEPSPs) were evoked by low-intensity electrical stimulation of cortical layer IV. Changes in the eEPSP waveform following membrane depolarization and hyperpolarization resembled those of NMDA-mediated responses. However, the eEPSP was insensitive to D-2-APV applied at concentrations (up to 20 microM) that blocked NMDA responses. 4. EPSPs with latencies between 10 and 40 ms [late EPSPs (lEPSPs)] were evoked by electrical stimulation using intensities just subthreshold to the activation of IPSPs. The amplitude of the lEPSP increased with hyperpolarization and decreased with depolarization. 5. The lidocaine derivative QX-314, injected intracellularly, suppressed sodium-dependent action potentials and depolarizing inward rectification. Simultaneously, the amplitude of the eEPSP significantly decreased with depolarization. Neither the amplitude of a long-latency EPSP nor the amplitude of inhibitory postsynaptic potentials (IPSPs) was significantly affected by QX-314. 6. Cesium ions (0.5-2.0 mM) added to the bathing solution reduced or blocked hyperpolarizing inward rectification. Under these conditions, the amplitude of the eEPSP increased with hyperpolarization. The amplitude of the lEPSP was unaltered or enhanced. 7. The lEPSP was reversibly blocked by D-2-APV (5-20 microM), although the voltage-dependence of its amplitude did not resemble the action of NMDA on neocortical neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Physiological role of Ca(2+)-activated and voltage-dependent K+ currents in rabbit coronary myocytes

1994 ◽  
Vol 266 (6) ◽  
pp. C1523-C1537 ◽  
Author(s):  
N. Leblanc ◽  
X. Wan ◽  
P. M. Leung
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Physiological Role ◽  
Cell Voltage ◽  
Resting Membrane Potential ◽  
Physiological Level ◽  
Steady State Current ◽  
Voltage Dependent ◽  
A Minor ◽  
And Function

The properties and function of Ca(2+)-activated K+ (KCa) and voltage-dependent K+ (IK) currents of rabbit coronary myocytes were studied under whole cell voltage-clamp conditions (22 degrees C). Inhibition of KCa by tetraethylammonium chloride (1-10 mM) or charybdotoxin (50-100 nM) suppressed noisy outward rectifying current elicited by 5-s voltage steps or ramp at potentials > 0 mV, reduced the hump of the biphasic ramp current-voltage relation, and shifted by less than +5 mV the potential at which no net steady-state current is recorded (Enet; index of resting membrane potential). Inhibition of steady-state inward Ca2+ currents [ICa(L)] by nifedipine (1 microM) displaced Enet by -11 mV. Analysis of steady-state voltage dependence of IK supported the existence of a "window" current between -50 and 0 mV. 4-Aminopyridine (2 mM) blocked a noninactivating component of IK evoked between -30 and -40 mV, abolished the hump current during ramps, and shifted Enet by more than +15 mV; hump current persisted during 2-min ramp depolarizations and peaked near the maximum overlap of the steady-state activation and inactivation curves of IK (about -22 mV). A threefold rise in extracellular Ca2+ concentration (1.8-5.4 mM) enhanced time-dependent outward K+ current (6.7-fold at +40 mV) and shifted Enet by -30 mV. It is concluded that, under steady-state conditions, IK and ICa(L) play a major role in regulating resting membrane potential at a physiological level of intracellular Ca2+ concentration, with a minor contribution from KCa. However, elevation of intracellular Ca2+ concentration enhances KCa and hyperpolarizes the myocyte to limit Ca2+ entry through ICa(L).

Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone

1990 ◽  
Vol 259 (1) ◽  
pp. C3-C18 ◽  
Author(s):  
M. T. Nelson ◽  
J. B. Patlak ◽  
J. F. Worley ◽  
N. B. Standen
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Katp Channel ◽  
Voltage Dependence ◽  
Muscle Tone ◽  
Ca Channels ◽  
Resistance Arteries ◽  
Arterial Smooth Muscle ◽  
Membrane Hyperpolarization ◽  
Voltage Dependent

Resistance arteries exist in a maintained contracted state from which they can dilate or constrict depending on need. In many cases, these arteries constrict to membrane depolarization and dilate to membrane hyperpolarization and Ca-channel blockers. We discuss recent information on the regulation of arterial smooth muscle voltage-dependent Ca channels by membrane potential and vasoconstrictors and on the regulation of membrane potential and K channels by vasodilators. We show that voltage-dependent Ca channels in the steady state can be open and very sensitive to membrane potential changes in a range that occurs in resistance arteries with tone. Many synthetic and endogenous vasodilators act, at least in part, through membrane hyperpolarization caused by opening K channels. We discuss evidence that these vasodilators act on a common target, the ATP-sensitive K (KATP) channel that is inhibited by sulfonylurea drugs. We propose the following hypotheses that presently explain these findings: 1) arterial smooth muscle tone is regulated by membrane potential primarily through the voltage dependence of Ca channels; 2) many vasoconstrictors act, in part, by opening voltage-dependent Ca channels through membrane depolarization and activation by second messengers; and 3) many vasodilators work, in part, through membrane hyperpolarization caused by KATP channel activation.

scholarly journals Structural mechanisms of phospholipid activation of the human TPC2 channel

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ji She ◽  
Weizhong Zeng ◽  
Jiangtao Guo ◽  
Qingfeng Chen ◽  
Xiao-chen Bai ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Membrane Potential ◽  
Voltage Dependence ◽  
Structural Comparison ◽  
Closed Conformation ◽  
Voltage Dependent ◽  
Selective Channel ◽  
Form Features ◽  
Structural Mechanisms ◽  
Pore Lining

Mammalian two-pore channels (TPCs) regulate the physiological functions of the endolysosome. Here we present cryo-EM structures of human TPC2 (HsTPC2), a phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-activated, Na+ selective channel, in the ligand-bound and apo states. The apo structure captures the closed conformation, while the ligand-bound form features the channel in both open and closed conformations. Combined with functional analysis, these structures provide insights into the mechanism of PI(3,5)P2-regulated gating of TPC2, which is distinct from that of TPC1. Specifically, the endolysosome-specific PI(3,5)P2 binds at the first 6-TM and activates the channel – independently of the membrane potential – by inducing a structural change at the pore-lining inner helix (IS6), which forms a continuous helix in the open state but breaks into two segments at Gly317 in the closed state. Additionally, structural comparison to the voltage-dependent TPC1 structure allowed us to identify Ile551 as being responsible for the loss of voltage dependence in TPC2.

Effects of Ropivacaine on a Potassium Channel (hKv1.5) Cloned from Human Ventricle

1997 ◽  
Vol 86 (3) ◽  
pp. 718-728 ◽  
Author(s):  
Carmen Valenzuela ◽  
Eva Delpon ◽  
Laura Franqueza ◽  
Pilar Gay ◽  
Dirk J. Snyders ◽  
...  
Keyword(s):  
Time Course ◽  
Underlying Mechanism ◽  
K Channel ◽  
Activation Time ◽  
Patch Clamp Technique ◽  
Steady State Current ◽  
Voltage Dependent ◽  
Initial Activation ◽  
Ion Pore ◽  
Cytoplasmic Side

Background Ropivacaine, a new amide local anesthetic agent chemically related to bupivacaine, is able to induce early after depolarizations in isolated cardiac preparations. The underlying mechanism by which ropivacaine induces this effect has not been explored, but it is likely to involve K+ channel block. Methods Cloned human cardiac K+ channels (hKv1.5) were stably transfected in Ltk cells, and the effects of ropivacaine on the expressed hKv1.5 currents were assessed using the whole-cell configuration of the patch-clamp technique. Results Ropivacaine (100 microM) did not modify the initial activation time course of the current, but induced a fast subsequent decline to a lower steady-state current level with a time constant of 12.2 +/- 0.6 ms. Ropivacaine inhibited hKv1.5 with an apparent KD of 80 +/- 4 microM. Block displayed an intrinsic voltage-dependent, consistent with an electrical distance for the binding site of 0.153 +/- 0.007 (n = 6) (from the cytoplasmic side). Ropivacaine reduced the tail current amplitude recorded at -40 mV, and slowed the deactivation time course, resulting in a "crossover" phenomenon when control and ropivacaine tail currents were superimposed. Conclusions These results indicate that: (1) ropivacaine is an open channel blocker of hKv1.5; (2) binding occurs in the internal mouth of the ion pore; and (3) unbinding is required before the channel can close. These effects explain the ropivacaine availability of induction early after depolarizations and could be clinically relevant.

Characterization of K+ Currents Underlying Pacemaker Potentials of Fish Gonadotropin-Releasing Hormone Cells

1999 ◽  
Vol 81 (2) ◽  
pp. 643-653 ◽  
Author(s):  
Hideki Abe ◽  
Yoshitaka Oka
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Gonadotropin Releasing Hormone ◽  
Resting Membrane Potential ◽  
Potential Range ◽  
Releasing Hormone ◽  
Voltage Dependent ◽  
Pacemaker Potentials ◽  
Low Threshold

Characterization of K+ currents underlying pacemaker potentials of fish gonadotropin-releasing hormone cells. Endogenous pacemaker activities are important for the putative neuromodulator functions of the gonadotropin-releasing hormone (GnRH)-immunoreactive terminal nerve (TN) cells. We analyzed several types of voltage-dependent K+ currents to investigate the ionic mechanisms underlying the repolarizing phase of pacemaker potentials of TN-GnRH cells by using the whole brain in vitro preparation of fish (dwarf gourami, Colisa lalia). TN-GnRH cells have at least four types of voltage-dependent K+currents: 1) 4-aminopyridine (4AP)-sensitive K+current, 2) tetraethylammonium (TEA)-sensitive K+ current, and 3) and 4) two types of TEA- and 4AP-resistant K+ currents. A transient, low-threshold K+ current, which was 4AP sensitive and showed significant steady-state inactivation in the physiological membrane potential range (−40 to −60 mV), was evoked from a holding potential of −100 mV. This current thus cannot contribute to the repolarizing phase of pacemaker potentials. TEA-sensitive K+ current evoked from a holding potential of −100 mV was slowly activating, long lasting, and showed comparatively low threshold of activation. This current was only partially inactivated at steady state of −60 to −40 mV, which is equivalent to the resting membrane potential. TEA- and 4AP-resistant sustained K+ currents were evoked from a holding potential of −100 mV and were suggested to consist of two types, based on the analysis of activation curves. From the inactivation and activation curves, it was suggested that one of them with low threshold of activation may be partly involved in the repolarizing phase of pacemaker potentials. Bath application of TEA together with tetrodotoxin reversibly blocked the pacemaker potentials in current-clamp recordings. We conclude that the TEA-sensitive K+ current is the most likely candidate that contributes to the repolarizing phase of the pacemaker potentials of TN-GnRH cells.

Participation of a Chloride Conductance in the Subthreshold Behavior of the Rat Sympathetic Neuron

1999 ◽  
Vol 82 (4) ◽  
pp. 1662-1675 ◽  
Author(s):  
Oscar Sacchi ◽  
Maria Lisa Rossi ◽  
Rita Canella ◽  
Riccardo Fesce
Keyword(s):  
Membrane Potential ◽  
Sympathetic Neuron ◽  
Chloride Current ◽  
Equilibrium Potential ◽  
Potential Range ◽  
Charge Movement ◽  
Channel Blockers ◽  
Voltage Range ◽  
Voltage Dependent ◽  
Input Conductance

The presence of a novel voltage-dependent chloride current, active in the subthreshold range of membrane potential, was detected in the mature and intact rat sympathetic neuron in vitro by using the two-microelectrode voltage-clamp technique. Hyperpolarizing voltage steps applied to a neuron held at −40/−50 mV elicited inward currents, whose initial magnitude displayed a linear instantaneous current-voltage ( I-V) relationship; afterward, the currents decayed exponentially with a single voltage-dependent time constant (63.5 s at −40 mV; 10.8 s at −130 mV). The cell input conductance decreased during the command step with the same time course as the current. On returning to the holding potential, the ensuing outward currents were accompanied by a slow increase in input conductance toward the initial values; the inward charge movement during the transient onresponse (a mean of 76 nC in 8 neurons stepped from −50 to −90 mV) was completely balanced by outward charge displacement during theoff response. The chloride movements accompanying voltage modifications were studied by estimating the chloride equilibrium potential ( E Cl) at different holding potentials from the reversal of GABA evoked currents. [Cl−]i was strongly affected by membrane potential, and at steady state it was systematically higher than expected from passive ion distribution. The transient current was blocked by substitution of isethionate for chloride and by Cl− channel blockers (9AC and DIDS). It proved insensitive to K+ channel blockers, external Cd2+, intracellular Ca2+ chelators [bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid (BAPTA)] and reduction of [Na+]e. It is concluded that membrane potential shifts elicit a chloride current that reflects readjustment of [Cl−]i. The cell input conductance was measured over the −40/−120-mV voltage range, in control medium, and under conditions in which either the chloride or the potassium current was blocked. A mix of chloride, potassium, and leakage conductances was detected at all potentials. The leakage component was voltage independent and constant at ∼14 nS. Conversely, gCl decreased with hyperpolarization (80 nS at −40 mV, undetectable below −110 mV), whereas gK displayed a maximum at −80 mV (55.3 nS). Thus the ratio gCl/gK continuously varied with membrane polarization (2.72 at −50 mV; 0.33 at −110 mV). These data were forced in a model of the three current components here described, which accurately simulates the behavior observed in the “resting” neuron during membrane migrations in the subthreshold potential range, thereby confirming that active K and Cl conductances contribute to the genesis of membrane potential and possibly to the control of neuronal excitability.

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