scholarly journals Dynamic Coupling of Voltage Sensor and Gate Involved in Closed-State Inactivation of Kv4.2 Channels

2009 ◽  
Vol 133 (2) ◽  
pp. 205-224 ◽  
Author(s):  
Jan Barghaan ◽  
Robert Bähring
Keyword(s):  
Steady State ◽  
Structural Model ◽  
Low Voltage ◽  
Large Fraction ◽  
Inactivation Kinetics ◽  
Dynamic Coupling ◽  
Alanine Scanning Mutagenesis ◽  
Closed State ◽  
Kv4 Channels ◽  
Steady State Inactivation

Voltage-gated potassium channels related to the Shal gene of Drosophila (Kv4 channels) mediate a subthreshold-activating current (ISA) that controls dendritic excitation and the backpropagation of action potentials in neurons. Kv4 channels also exhibit a prominent low voltage–induced closed-state inactivation, but the underlying molecular mechanism is poorly understood. Here, we examined a structural model in which dynamic coupling between the voltage sensors and the cytoplasmic gate underlies inactivation in Kv4.2 channels. We performed an alanine-scanning mutagenesis in the S4-S5 linker, the initial part of S5, and the distal part of S6 and functionally characterized the mutants under two-electrode voltage clamp in Xenopus oocytes. In a large fraction of the mutants (>80%) normal channel function was preserved, but the mutations influenced the likelihood of the channel to enter the closed-inactivated state. Depending on the site of mutation, low-voltage inactivation kinetics were slowed or accelerated, and the voltage dependence of steady-state inactivation was shifted positive or negative. Still, in some mutants these inactivation parameters remained unaffected. Double mutant cycle analysis based on kinetic and steady-state parameters of low-voltage inactivation revealed that residues known to be critical for voltage-dependent gate opening, including Glu 323 and Val 404, are also critical for Kv4.2 closed-state inactivation. Selective redox modulation of corresponding double-cysteine mutants supported the idea that these residues are involved in a dynamic coupling, which mediates both transient activation and closed-state inactivation in Kv4.2 channels.

scholarly journals Inhibition of Kv4.3 by genistein via a tyrosine phosphorylation-independent mechanism

2011 ◽  
Vol 300 (3) ◽  
pp. C567-C575 ◽  
Author(s):  
Hee Jae Kim ◽  
Hye Sook Ahn ◽  
Bok Hee Choi ◽  
Sang June Hahn
Keyword(s):  
Steady State ◽  
Inactivation Kinetics ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Inactivation Curve ◽  
Voltage Range ◽  
Closed State ◽  
Voltage Dependent ◽  
Dependent Inhibition ◽  
Steady State Inactivation

The effects of genistein, a protein tyrosine kinase (PTK) inhibitor, on voltage-dependent K+ (Kv) 4.3 channel were examined using the whole cell patch-clamp techniques. Genistein inhibited Kv4.3 in a reversible, concentration-dependent manner with an IC50 of 124.78 μM. Other PTK inhibitors (tyrphostin 23, tyrphostin 25, lavendustin A) had no effect on genistein-induced inhibition of Kv4.3. Orthovanadate, an inhibitor of protein phosphatases, did not reverse the inhibition of Kv4.3 by genistein. We also tested the effects of two inactive structural analogs: genistin and daidzein. Whereas Kv4.3 was unaffected by genistin, daidzein inhibited Kv4.3, albeit with a lower potency. Genistein did not affect the activation and inactivation kinetics of Kv4.3. Genistein-induced inhibition of Kv4.3 was voltage dependent with a steep increase over the channel opening voltage range. In the full-activation voltage range positive to +20 mV, no voltage-dependent inhibition was found. Genistein had no significant effect on steady-state activation, but shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. The Ki for the interaction between genistein and the inactivated state of Kv4.3, which was estimated from the concentration-dependent shift in the steady-state inactivation curve, was 1.17 μM. Under control conditions, closed-state inactivation was fitted to a single exponential function, and genistein accelerated closed-state inactivation. Genistein induced a weak use-dependent inhibition. These results suggest that genistein directly inhibits Kv4.3 by interacting with the closed-inactivated state of Kv4.3 channels. This effect is not mediated via inhibition of the PTK activity, because other types of PTK inhibitors could not prevent the inhibitory action of genistein.

scholarly journals Arachidonic acid modulation of α1H, a cloned human T-type calcium channel

2000 ◽  
Vol 278 (1) ◽  
pp. H184-H193 ◽  
Author(s):  
Yi Zhang ◽  
Leanne L. Cribbs ◽  
Jonathan Satin
Keyword(s):  
Arachidonic Acid ◽  
Steady State ◽  
Low Voltage ◽  
Specific Inhibitor ◽  
Nordihydroguaiaretic Acid ◽  
Ca Channel ◽  
Lipoxygenase Inhibitor ◽  
Inactivation Curve ◽  
Steady State Inactivation ◽  
Window Current

Arachidonic acid (AA) and the products of its metabolism are central mediators of changes in cellular excitability. We show that the recently cloned and expressed T-type or low-voltage-activated Ca channel, α1H, is modulated by external AA. AA (10 μM) causes a slow, time-dependent attenuation of α1H current. At a holding potential of −80 mV, 10 μM AA reduces peak inward α1H current by 15% in 15 min and 70% in 30 min and shifts the steady-state inactivation curve −25 mV. AA inhibition was not affected by applying the cyclooxygenase inhibitor indomethacin or the lipoxygenase inhibitor nordihydroguaiaretic acid. The epoxygenase inhibitor octadecynoic acid partially antagonized AA attenuation of α1H. The epoxygenase metabolite epoxyeicosatrienoic acid (8,9-EET) mimicked the inhibitory effect of AA on α1H peak current. A protein kinase C (PKC)-specific inhibitor (peptide fragment 19–36) only partially antagonized the AA-induced reduction of peak α1H current and the shift of the steady-state inactivation curve but had no effect on 8,9-EET-induced attenuation of current. In contrast, PKA has no role in the modulation of α1H. These results suggest that AA attenuation and shift of α1H may be mediated directly by AA. The heterologous expression of T-type Ca channels allows us to study for the first time properties of this important class of ion channel in isolation. There is a significant overlap of the steady-state activation and inactivation curves, which implies a substantial window current. The selective shift of the steady-state inactivation curve by AA reduces peak Ca current and eliminates the window current. We conclude that AA may partly mediate physiological effects such as vasodilatation via the attenuation of T-type Ca channel current and the elimination of a T-type channel steady window current.

scholarly journals The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. II. Closed state reverse use-dependent block by 4-aminopyridine.

1993 ◽  
Vol 101 (4) ◽  
pp. 603-626 ◽  
Author(s):  
D L Campbell ◽  
Y Qu ◽  
R L Rasmusson ◽  
H C Strauss
Keyword(s):  
Steady State ◽  
Right Ventricular ◽  
Ventricular Myocytes ◽  
Patch Clamp Technique ◽  
Time Constants ◽  
Closed State ◽  
Whole Cell Patch Clamp ◽  
Dependent Behavior ◽  
Steady State Inactivation ◽  
K Current

Block of the calcium-independent transient outward K+ current, I(to), by 4-aminopyridine (4-AP) was studied in ferret right ventricular myocytes using the whole cell patch clamp technique. 4-AP reduces I(to) through a closed state blocking mechanism displaying "reverse use-dependent" behavior that was inferred from: (a) development of tonic block at hyperpolarized potentials; (b) inhibition of development of tonic block at depolarized potentials; (c) appearance of "crossover phenomena" in which the peak current is delayed in the presence of 4-AP at depolarized potentials; (d) relief of block at depolarized potentials which is concentration dependent and parallels steady-state inactivation for low 4-AP concentrations (V1/2 approximately -10 mV in 0.1 mM 4-AP) and steady-state activation at higher concentrations (V1/2 = +7 mV in 1 mM 4-AP, +15 mV in 10 mM 4-AP); and (e) reassociation of 4-AP at hyperpolarized potentials. No evidence for interaction of 4-AP with either the open or inactivated state of the I(to) channel was obtained from measurements of kinetics of recovery and deactivation in the presence of 0.5-1.0 mM 4-AP. At hyperpolarized potentials (-30 to -90 mV) 10 mM 4-AP associates slowly (time constants ranging from approximately 800 to 1,300 ms) with the closed states of the channel (apparent Kd approximately 0.2 mM). From -90 to -20 mV the affinity of the I(to) channel for 4-AP appears to be voltage insensitive; however, at depolarized potentials (+20 to +100 mV) 4-AP dissociates with time constants ranging from approximately 350 to 150 ms. Consequently, the properties of 4-AP binding to the I(to) channel undergo a transition in the range of potentials over which channel activation and inactivation occurs (-30 to +20 mV). We propose a closed state model of I(to) channel gating and 4-AP binding kinetics, in which 4-AP binds to three closed states. In this model 4-AP has a progressively lower affinity as the channel approaches the open state, but has no intrinsic voltage dependence of binding.

Aging-associated changes in whole cell K+ and L-type Ca2+ currents in rat ventricular myocytes

2000 ◽  
Vol 279 (3) ◽  
pp. H889-H900 ◽  
Author(s):  
Shi J. Liu ◽  
Richard P. Wyeth ◽  
Russell B. Melchert ◽  
Richard H. Kennedy
Keyword(s):  
Steady State ◽  
Young Adult ◽  
Action Potential ◽  
Action Potential Duration ◽  
Ventricular Myocytes ◽  
Inactivation Kinetics ◽  
Whole Cell ◽  
Rat Ventricular Myocytes ◽  
Whole Cell Patch Clamp ◽  
Steady State Inactivation

The effect of aging on cardiac membrane currents remains unclear. This study examined the inward rectifier K+ current ( I K1), the transient outward K+current ( I to), and the L-type Ca2+ channel current ( I Ca,L) in ventricular myocytes isolated from young adult (6 mo) and aged (>27 mo) Fischer 344 rats using whole cell patch-clamp techniques. Along with an increase in the cell size and membrane capacitance, aged myocytes had the same magnitude of peak I K1 with a greater slope conductance but displayed smaller steady-state I K1. Aged myocytes also had a greater I to with an increased rate of activation, but the I to inactivation kinetics, steady-state inactivation, and responsiveness to l-phenylephrine, an α1-adrenergic agonist, were unaltered. The magnitude of peak I Ca,L in aged myocytes was decreased and accompanied by a slower inactivation, but the I Ca,L steady-state inactivation was unaltered. Action potential duration in aged myocytes was prolonged only at 90% of full repolarization (APD90) when compared with the action potential duration of young adult myocytes. Aged myocytes from Long-Evans rats showed similar changes in I toand I Ca,L but an increased I K1. These results demonstrate aging-associated changes in action potential, in morphology, and in I K1, I to, and I Ca,L of rat ventricular myocytes that possibly contribute to the decreased cardiac function of aged hearts.

Transient outward current in opossum esophageal circular muscle

1995 ◽  
Vol 268 (6) ◽  
pp. G979-G987 ◽  
Author(s):  
H. I. Akbarali ◽  
N. Hatakeyama ◽  
Q. Wang ◽  
R. K. Goyal
Keyword(s):  
Steady State ◽  
Action Potential ◽  
Circular Muscle ◽  
Outward Current ◽  
Resting Membrane Potential ◽  
Inactivation Kinetics ◽  
Transient Outward Current ◽  
Potential Range ◽  
Patch Clamp Technique ◽  
Steady State Inactivation

The whole cell patch-clamp technique was used to record a transient outward K+ current (ITO) from single smooth muscle cells isolated from opossum esophageal circular muscle. The threshold for its activation was -50 mV from holding potentials negative to -70 mV. The current peaked within 10 ms and decayed completely in 200 ms between test depolarization of -40 and -10 mV. ITO was recorded at room temperature in the presence of 5 mM internal ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid. Both activation and inactivation kinetics of ITO were markedly changed when recordings were made at higher temperatures (32 degrees C). 4-Amino-pyridine (4-AP, 3 mM) abolished the fast component of the outward current. Tetraethylammonium ion (TEA, 1-30 mM) reduced the sustained component but did not affect ITO. In the presence of TEA and nifedipine, the voltage dependence of the steady-state inactivation data was well fitted by a Boltzmann distribution with a half-inactivation potential of -57 mV. The half-inactivation potential was shifted to a more positive potential in the presence of Cd2+ (-35 mV). The steady-state inactivation and activation data overlap between -50 and -30 mV, suggesting the presence of a "window" current in this potential range. In current-clamp mode, 4-AP depolarized single esophageal cells by approximately 8 mV and shifted the upstroke of the action potential to the left. These results indicate that, in the esophageal circular muscle, ITO is involved in the resting membrane potential and modulation of the onset of action potential.

Cloning and characterization of an Ito-like potassium channel from ferret ventricle

1994 ◽  
Vol 267 (4) ◽  
pp. H1383-H1395 ◽  
Author(s):  
M. B. Comer ◽  
D. L. Campbell ◽  
R. L. Rasmusson ◽  
D. R. Lamson ◽  
M. J. Morales ◽  
...  
Keyword(s):  
Steady State ◽  
Outward Current ◽  
Fourth Power ◽  
Inactivation Kinetics ◽  
Transient Outward Current ◽  
Fast Time ◽  
Recovery From Inactivation ◽  
Acid Protein ◽  
Steady State Inactivation ◽  
K Concentration

FK1, a ferret ventricular full-length cDNA clone, encodes a 654-amino acid protein with 98% identity to human K+ transient outward current (Ito)-like HK1 (Tamkun et al. FASEB J.5: 331-337, 1991). FK1 is detectable in ferret brain, atrium, left and right ventricle, and kidney but not in skeletal muscle, endothelial cells, aorta, and liver. In Xenopus oocytes, FK1 cRNA gives rise to a rapidly activating and inactivating Ito-like current, which is highly K+ selective (Na(+)-to-K+ permeability ratio = 0.003). Activation occurs over an approximately 50-mV range (-40 to +10 mV) and displays a sigmoid delay in onset with potential-dependent time constants that decrease with depolarization. Steady-state activation can be described with either a simple Boltzmann relationship [half-activation potential (V1/2) = -25 mV, slope (k) = 10 mV] or a Boltzmann relationship raised to either the third or fourth power (alpha 3: V1/2 = -43 mV, kappa = 13.1 mV; alpha 4: V1/2 = -48 mV, kappa = 13.6 mV, where alpha is the activation variable). Inactivation kinetics are biexponential, with the main fast time constant becoming independent of membrane potential depolarized to 0 mV. Steady-state inactivation can be described with a single Boltzmann relationship (V1/2 = -57 mV, kappa = 5.0 mV). Fast inactivation is removed by NH2-terminal deletions. Recovery from inactivation (-90 mV) is quite slow (half-time = 4.8 +/- 2.5 s). In 2 mM extracellular K+ concentration ([K+]o), FK1 tail currents display conventional deactivation behavior; however, in 98 mM [K+]o the tail currents display "reopening" behavior. These results suggest a molecular basis for the electrophysiological similarities between ferret and human ventricular Ito (Campbell et al. J. Gen. Physiol. 101: 571-601, 1993; Nabauer et al. Circ. Res. 73: 386-394, 1993).

A fast transient potassium current in thalamic relay neurons: kinetics of activation and inactivation

1991 ◽  
Vol 66 (4) ◽  
pp. 1304-1315 ◽  
Author(s):  
J. R. Huguenard ◽  
D. A. Coulter ◽  
D. A. Prince
Keyword(s):  
Steady State ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Inactivation Kinetics ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
K Current ◽  
K Currents ◽  
Relay Neurons

1. Whole-cell voltage-clamp techniques were used to record K+ currents in relay neurons (RNs) that had been acutely isolated from rat thalamic ventrobasal complex and maintained at 23 degrees C in vitro. Tetrodoxin (TTX; 0.5 microM) was used to block Na+ currents, and reduced extracellular levels of Ca2+ (1 mM) were used to minimize contributions from Ca2+ current (ICa). 2. In RNs, depolarizing commands activate K+ currents characterized by a substantial rapidly inactivating (time constant approximately 20 ms) component, the features of which correspond to those of the transient K+ current (IA) in other preparations, and by a smaller, more slowly activating K+ current, "IK". IA was reversibly blocked by 4-aminopyridine (4-AP, 5 mM), and the reversal potential varied with [K+]o as predicted by the Nernst equation. 3. IA was relatively insensitive to blockade by tetraethylammonium [TEA; 50%-inhibitory concentration (IC50) much much greater than 20 mM]; however, two components of IK were blocked with IC50S of 30 microM and 3 mM. Because 20 mM TEA blocked 90% of the sustained current while reducing IA by less than 10%, this concentration was routinely used in experiments in which IA was isolated and characterized. To further minimize contamination by other conductances, 4-AP was added to TEA-containing solutions and the 4-AP-sensitive current was obtained by subtraction. 4. Voltage-dependent steady-state inactivation of peak IA was described by a Boltzman function with a slope factor (k) of -6.5 and half-inactivation (V1/2) occurring at -75 mV. Activation of IA was characterized by a Boltzman curve with V1/2 = -35 mV and k = 10.8. 5. IA activation and inactivation kinetics were best fitted by the Hodgkin-Huxley m4h formalism. The rate of activation was voltage dependent, with tau m decreasing from 2.3 ms at -40 mV to 0.5 ms at +50 mV. Inactivation was relatively voltage independent and nonexponential. The rate of inactivation was described by two exponential decay processes with time constants (tau h1 and tau h2) of 20 and 60 ms. Both components were steady-state inactivated with similar voltage dependence. 6. Temperature increases within the range of 23-35 degrees C caused IA activation and inactivation rates to become faster, with temperature coefficient (Q10) values averaging 2.8. IA amplitude also increased as a function of temperature, albeit with a somewhat lower Q10 of 1.6. 7. Several voltage-dependent properties of IA closely resemble those of the transient inward Ca2+ current, IT. (ABSTRACT TRUNCATED AT 400 WORDS)

Effect of intracellular and extracellular acidosis on sodium current in ventricular myocytes

1995 ◽  
Vol 268 (4) ◽  
pp. H1749-H1756 ◽  
Author(s):  
C. L. Watson ◽  
M. R. Gold
Keyword(s):  
Steady State ◽  
Time Course ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Essential Element ◽  
Neonatal Rat ◽  
Cardiac Conduction ◽  
Inactivation Kinetics ◽  
Extracellular Acidosis ◽  
Steady State Inactivation

Conduction slowing is an essential element in the generation of ischemic ventricular arrhythmias and is determined in part by the inward Na+ current (INa). Because intracellular acidosis is an early consequence of ischemia, we hypothesized that lowering intracellular pH (pHi) would reduce or kinetically modulate INa and thus affect cardiac conduction. To test this hypothesis, the whole cell patch-clamp method was used to measure INa in neonatal rat ventricular myocytes exposed to varying extracellular pH (pHo 6.4–7.4), while perfusing the cells with acidic solutions (pHi 6.2–7.2). With simultaneous acidification of pHo and pHi there was a progressive increase in time to peak current, a 31% decrease in peak INa (298 +/- 18 to 206 +/- 16 pA/pF), and a complex slowing of inactivation kinetics. At the most extreme levels of acidification, there was a 5-mV hyperpolarizing shift in steady-state inactivation and a 6-mV depolarizing shift in activation. Independent changes of pHo and pHi indicate that the reduction of peak INa is a function of pHo. However, steady-state inactivation is modulated by pHi. The time course of activation and inactivation appears to depend on both pHo and pHi. We conclude that both intracellular and extracellular acidosis are significant but distinct modulators of INa amplitude and kinetics in cardiac myocytes.

Temperature dependence of early and late currents in human cardiac wild-type and long Q-T ΔKPQ Na+ channels

1998 ◽  
Vol 275 (6) ◽  
pp. H2016-H2024 ◽  
Author(s):  
Toshihisa Nagatomo ◽  
Zheng Fan ◽  
Bin Ye ◽  
Gayle S. Tonkovich ◽  
Craig T. January ◽  
...  
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Inactivation Kinetics ◽  
Wild Type ◽  
Positive Direction ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation ◽  
293 Cells ◽  
Steady State Inactivation

Na+current ( I Na) through wild-type human heart Na+channels (hH1) is important for normal cardiac excitability and conduction, and it participates in the control of repolarization and refractoriness. I Na kinetics depend strongly on temperature, but I Na for hH1 has been studied previously only at room temperature. We characterized early I Na (the peak and initial decay) and late I Na of the wild-type hH1 channel and a mutant channel (ΔKPQ) associated with congenital long Q-T syndrome. Channels were stably transfected in HEK-293 cells and studied at 23 and 33°C using whole cell patch clamp. Activation and inactivation kinetics for early I Na were twofold faster at higher temperature for both channels and shifted activation and steady-state inactivation in the positive direction, especially for ΔKPQ. For early I Na (<24 ms), ΔKPQ decayed faster than the wild type for voltages negative to −20 mV but slower for more positive voltages, suggesting a reduced voltage dependence of fast inactivation. Late I Na at 240 ms was significantly greater for ΔKPQ than for the wild type at both temperatures. The majority of late I Na for ΔKPQ was not persistent; rather, it decayed slowly, and this late component exhibited slower recovery from inactivation compared with peak I Na. Additional kinetic changes for early and peak I Na for ΔKPQ compared with the wild type at both temperatures were 1) reduced voltage dependence of steady-state inactivation with no difference in midpoint, 2) positive shift for activation kinetics, and 3) more rapid recovery from inactivation. This study represents the first description of human Na+ channel kinetics near physiological temperature and also demonstrates complex gating changes in the ΔKPQ that are present at 33°C and that may underlie the electrophysiological and clinical phenotype of congenital long Q-T Na+ channel syndromes.

Low-threshold calcium current in isolated Purkinje cell bodies of rat cerebellum

1990 ◽  
Vol 63 (5) ◽  
pp. 1046-1051 ◽  
Author(s):  
M. Kaneda ◽  
M. Wakamori ◽  
C. Ito ◽  
N. Akaike
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Purkinje Cell ◽  
Time Constant ◽  
Inactivation Kinetics ◽  
High Threshold ◽  
Internal Perfusion ◽  
Time To Peak ◽  
Steady State Inactivation ◽  
Low Threshold

1. The low- and high-threshold Ca2+ currents were observed in Purkinje cell bodies isolated from the cerebellum of newborn (2 wk old) and adult (8 wk old) rats under whole-cell clamp. A transient Ca2+ current (low-threshold or "T-type" ICa) was elicited by depolarizing step pulses to -60 mV or more positive potentials from a holding potential (VH) of -100 mV. In cells dissociated from newborn rats, a long-lasting Ca2+ current (high-threshold or "L-type" ICa) was also elicited by depolarizing command pulses beyond -30 mV. 2. The low-threshold ICa was resistant to the "washout" effect during the internal perfusion, whereas the high-threshold ICa faded gradually with time during the continuous internal perfusion. 3. In the current-voltage (I-V) relationship, the low-threshold ICa had a threshold potential around -60 mV and reached the maximum inward current around -20 mV. The activation and inactivation kinetics of the current depended on membrane potential: for a test-potential change from -60 to +40 mV, the time to peak of the current (activation) decreased from 31.9 to 5.0 ms, and the time constant of current decay (inactivation) decreased from 78.5 to 22.9 ms. 4. Steady-state inactivation of low-threshold ICa was membrane-potential dependent, and the inactivation of the 50% level was -79 mV. Recovery time constant from steady-state inactivation varied depending on the membrane potential. The time constants were 3.3 and 2.5 s at VHs of -100 and -120 mV, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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