Slow Phase Of Inactivation Research Articles

The citrus flavanone hesperetin preferentially inhibits slow-inactivating currents of a long QT syndrome type 3 syndrome Na+ channel mutation.

The citrus flavanone hesperetin has been proposed for the treatment of several human pathologies, but its cardiovascular actions remain largely unexplored. Here, we evaluated the effect of hesperetin on cardiac electrical and contractile activities, on aortic contraction, on the wild-type voltage-gated NaV 1.5 channel, and on a channel mutant (R1623Q) associated with lethal ventricular arrhythmias in the long QT syndrome type 3 (LQT3). We used cardiac surface ECG and contraction force recordings to evaluate the effects of hesperetin in rat isolated hearts and aortic rings. Whole-cell patch clamp was used to record NaV 1.5 currents (INa ) in rat ventricular cardiomyocytes and in HEK293T cells expressing hNaV 1.5 wild-type or mutant channels. Hesperetin increased the QRS interval and heart rate and decreased the corrected QT interval and the cardiac and aortic contraction forces at concentrations equal or higher than 30μmol·L-1 . Hesperetin blocked rat and human NaV 1.5 channels with an effective inhibitory concentration of ≈100μmol·L-1 . This inhibition was enhanced at depolarized holding potentials and higher stimulation frequency and was reduced by the disruption of the binding site for local anaesthetics. Hesperetin increased the rate of inactivation and preferentially inhibited INa during the slow inactivation phase, these effects being more pronounced in the R1623Q mutant. Hesperetin preferentially inhibits the slow inactivation phase of INa , more markedly in the mutant R1623Q. Hesperetin could be used as a template to develop drugs against lethal cardiac arrhythmias in LQT3.

Novel Medications for Epilepsy

Despite the introduction of many second-generation antiepileptic drugs (AEDs) in the last 2 decades, the proportion of individuals with pharmacoresistant epilepsy has not been reduced substantially compared with the late 1960s. All currently available AEDs also have limitations in terms of adverse effects and susceptibility to be involved in clinically important drug-drug interactions. Therefore, the search for potentially more effective and better tolerated agents is continuing. This article reviews the pharmacological and clinical profile of the latest compounds to receive marketing authorization. Since the beginning of 2008, three novel AEDs, lacosamide, eslicarbazepine acetate and retigabine (also known as ezogabine), have become commercially available in Europe, with lacosamide and retigabine also being licensed in the US. All three agents are indicated for the adjunctive treatment of focal seizures in adults. Eslicarbazepine acetate is a produg for eslicarbazepine, which acts by blocking voltage-dependent sodium channels. Lacosamide enhances the slow inactivation phase of voltage-dependent sodium channels, and retigabine potentiates neuronal M-currents by opening Kv 7.2-7.5 potassium channels. All three agents, which are well absorbed from the gastrointestinal tract, exhibit linear pharmacokinetics. Lacosamide is also available as an intravenous formulation intended as replacement therapy for patients temporarily unable to take oral medications. All three drugs are eliminated partly unchanged in urine and partly by biotransformation through glucuronide conjugation (eslicarbazepine, retigabine), N-acetylation (retigabine) and oxidative demethylation (lacosamide). The half-life is in the order of 8-20 hours for eslicarbazepine, 12-16 hours for lacosamide and 6-10 hours for retigabine. Based on the limited information available to date, the ability of these agents to cause pharmacokinetic drug interactions appears to be relatively modest, although eslicarbazepine can cause a significant decrease in the blood levels of ethinylestradiol, levonorgestrel and simvastatin. The approved effective dose ranges are 200-400 mg/day in two divided doses for lacosamide, 800-1200 mg/day once daily for eslicarbazepine acetate, and 600-1200 mg/day in three divided doses for retigabine. In phase III, randomized, double-blind, adjunctive therapy trials, responder rates (proportion of patients with ≥50% reduction in seizure frequency vs baseline) at the highest approved dose were comparable for the three drugs (eslicarbazepine acetate: 37-43% vs 13-20% for placebo; lacosamide: 38-41% vs 18-26% for placebo; retigabine: 33-44% vs 16-18% for placebo). The adverse events most commonly reported with active treatment compared with placebo included dizziness, diplopia and nausea for lacosamide; dizziness, somnolence and nausea for eslicarbazepine acetate; and dizziness, somnolence and fatigue for retigabine. The role of these agents in the treatment algorithm will be increasingly defined as clinical experience accumulates. At present, their use is largely restricted to the adjunctive treatment of focal seizures, with or without secondary generalization, in adults with epilepsy who failed to achieve seizure freedom after having tried two or more first-line agents.

Cooperativeness of Orai Cytosolic Domains Tunes Subtype-specific Gating

Activation of immune cells is triggered by the Ca2+ release-activated Ca2+ current, which is mediated via channels of the Orai protein family. A key gating process of the three Orai channel isoforms to prevent Ca2+ overload is fast inactivation, most pronounced in Orai3. A subsequent reactivation is a unique gating characteristic of Orai1 channels, whereas Orai2 and Orai3 currents display a second, slow inactivation phase. Employing a chimeric approach by sequential swapping of respective intra- and extracellular regions between Orai1 and Orai3, we show here that Orai1 specific proline/arginine-rich domains in the N terminus mediate reactivation, whereas the second, intracellular loop modulates fast and slow gating processes. Swapping C-terminal strands lacks a significant impact. However, simultaneous transfer of Orai3 N terminus and its second loop or C terminus in an Orai1 chimera substantially increases fast inactivation centered between wild-type channels. Concomitant swap of all three cytosolic strands from Orai3 onto Orai1 fully conveys Orai3-like gating characteristics, in a strongly cooperative manner. In conclusion, Orai subtype-specific gating requires a cooperative interplay of all three cytosolic domains.

Cooperativeness of Orai Cytosolic Domains Tunes Subtype-Specific Gating

Activation of immune cells is triggered by the Ca2+ release-activated Ca2+ (CRAC) current, which is mediated via channels of the Orai protein family. A key gating process of the three Orai channel isoforms to prevent Ca2+ overload is fast inactivation, most pronounced in Orai3. A subsequent reactivation is a unique gating characteristic of Orai1 channels, while Orai2 and Orai3 currents display a second, slow inactivation phase. Employing a chimeric approach by sequential swapping of respective intra- and extra-cellular regions between Orai1 and Orai3, we show here that Orai1 specific proline-arginine-rich domains in the N-terminus mediate reactivation, while the second, intracellular loop modulates fast and slow gating processes. Swapping C-terminal strands lacks a significant impact. However, simultaneous transfer of Orai3 N-terminus and its second loop or C-terminus in an Orai1 chimera substantially increases fast inactivation centered between wild-type channels. Concomitant swap of all three cytosolic strands from Orai3 onto Orai1 fully conveys Orai3-like gating characteristics, in a strongly cooperative manner. In conclusion Orai subtype-specific gating requires a cooperative interplay of all three cytosolic domains.

Characterization ofBacillus subtilisspore inactivation in low-pressure, low-temperature gas plasma sterilization processes

To identify structural components of Bacillus subtilis spores serving as targets for sterilization with microwave induced low-pressure, low-temperature nitrogen-oxygen plasma. The inactivation of spores followed a biphasic kinetics consisting of a log-linear phase with rapid inactivation followed by a slow inactivation phase. In the course of plasma treatment, damage to DNA, proteins and spore membranes were observed by monitoring the occurrence of auxotrophic mutants, inactivation of catalase (KatX) activity and the leakage of dipicolinic acid, respectively. Spores of the wild-type strain showed the highest resistance to plasma treatment. Spores of mutants defective in nucleotide excision repair (uvrA) and small acid-soluble proteins (Delta sspA Delta sspB) were more sensitive than those defective in the coat protein CotE or spore photoproduct repair (splB). Exclusion of reactive particles and spectral fractions of UV radiation from access to the spores revealed that UV-C radiation is the most effective inactivation agent in the plasma, whereby the splB and Delta cotE mutant spores were equally and slightly less sensitive, respectively, than the wild-type spores. Finally, the extent of damages in the spore DNA determined by quantitative PCR correlated with the spore inactivation. Spore inactivation was efficiently mediated by a combination of DNA damage and protein inactivation. DNA was identified to be the primary target for spore inactivation by UV radiation emitted by the plasma. Coat proteins were found to constitute a protective layer against the action of the plasma. The results provide new evidence to the understanding of plasma sterilization processes. This knowledge supports the identification of useful parameters for novel plasma sterilization equipment to control process safety.

Modulation of L-type Ca2+ channel current density and inactivation by β-adrenergic stimulation during murine cardiac embryogenesis

L-type Ca(2+) current (I (CaL)) is a key regulatory and functional element during early embryonic cardiomyogenesis. As the embryonic heart underlies hormonal modulation, e.g. catecholamines, we aimed at studying effects of beta-adrenergic stimulation on I (CaL) densities and inactivation kinetics during murine heart development. I (CaL) was recorded applying the whole-cell patch-clamp technique in ventricular myocytes of early embryonic (EDS, E9.5-11.5) and late developmental, fetal (LDS, E16.5-18.5) stages as well as adult cardiomyocytes. To distinguish between Ca(2+)-(CDI) and voltage-dependent inactivation (VDI), Ca(2+) was replaced with Ba(2+) in the extracellular recording solution. The beta-adrenergic signaling pathway was simulated by applying isoproterenol (Iso). Basal current densities showed an increase of I (CaL) during development (EDS: -9.61 +/- 1.97 pA/pF, n = 9; LDS: -13.2 +/- 4.26 pA/pF, n = 12; adult: -16.1 +/- 4.63 pA/pF, n = 5). Iso (1 microM) enhanced I (CaL) density with low effects at EDS (17.1 +/- 3.58%, n = 8, P > 0.05) but strong effects at LDS (74.1 +/- 3.77%, n = 8, P < 0.01) and in adults (90.6 +/- 7.01%, n = 6, P < 0.001). The current availability was significantly higher at LDS as compared to EDS and elevated after application of Iso. In the presence of Ca(2+), the fast phase of I (CaL) inactivation (tau(f)) was significantly enhanced by Iso at LDS. The slow phase of inactivation (tau(s)) was unaltered at both developmental stages. However, VDI was significantly reduced under Iso in LDS and adult cardiomyocytes. These results imply that beta-adrenergic modulation becomes of importance especially during fetal heart development. CDI and VDI of I (CaL) are modulated by beta-adrenergic stimulation in fetal but not in early embryonic mouse cardiomyocytes. Furthermore our data suggest important changes of the L-type Ca(2+) channel protein, and/or maturation of the Ca(2+)-induced Ca(2+) release (CICR) machinery.

Kinetic properties of Kv4.3 and their modulation by KChIP2b

KChIPs are a family of Kv4 K + channel ancillary subunits whose effects usually include slowing of inactivation, speeding of recovery from inactivation, and increasing channel surface expression. We compared the effects of the 270 amino acid KChIP2b on Kv4.3 and a Kv4.3 inner pore mutant [V(399, 401)I]. Kv4.3 showed fast inactivation with a bi-exponential time course in which the fast time constant predominated. KChIP2b expressed with wild-type Kv4.3 slowed the fast time constant of inactivation; however, the overall rate of inactivation was faster due to reduction of the contribution of the slow inactivation phase. Introduction of [V(399, 401)I] slowed both time constants of inactivation less than 2-fold. Inactivation was incomplete after 20 s pulse durations. Co-expression of KChIP2b with Kv4.3 [V(399, 401)I] slowed inactivation dramatically. KChIP2b increased the rate of recovery from inactivation 7.6-fold in the wild-type channel and 5.7-fold in Kv4.3 [V(399,401)I]. These data suggest that inner pore structure is an important factor in the modulatory effects of KChIP2b on Kv4.3 K + channels.

Beta-Adrenergic stimulation modulates Ca2+- and voltage-dependent inactivation of L-type Ca2+ channel currents in guinea-pig ventricular myocytes.

The objective of this study was to examine the effect of beta-adrenergic stimulation upon voltage- and Ca2+-induced inactivation of native cardiac L-type Ca2+ channels. Whole-cell currents were recorded from guinea-pig isolated ventricular myocytes. Total and voltage-dependent inactivation was separated by replacing extracellular Ca2+ with Mg2+. L-type Ca2+ channel behaviour was monitored with outward Ca2+ channel currents. First, the voltage dependence of inactivation was studied at fixed times (50 and 1000 ms) after activation. This showed that under control conditions Ca2+ contributed little to inactivation. In isoproterenol (isoprenaline), voltage-dependent inactivation was markedly reduced and Ca2+ contributed largely to total inactivation. Second, the time dependence of inactivation was studied at a fixed voltage (+10 mV). In control conditions the fast phase of inactivation (tau(f) approximately 15 ms) was reduced to the same extent by ryanodine (tau(f) approximately 30 ms) and the absence of Ca2+ (tau(f) approximately 30 ms) while the slow phase of inactivation (tau(s) approximately 70 ms) was reduced by ryanodine (tau(s) approximately 160 ms) and further reduced in the absence of Ca2+ (tau(s) approximately 300 ms). In isoproterenol, biphasic inactivation of Ca2+ currents (tau(f) approximately 4 ms, tau(s) approximately 60 ms) was replaced by a single slow (tau approximately 450 ms) phase of inactivation in the absence of Ca2+. It is concluded that, under control conditions Ca2+ channel current decay is largely dominated by rapid voltage-dependent inactivation, while in isoproterenol this is replaced by Ca2+-induced inactivation.

Inactivation of betaine aldehyde dehydrogenase from amaranth leaves by pyridoxal 5′-phosphate

Pyridoxal 5′-phosphate (PLP) inactivated betaine aldehyde dehydrogenase (BADH) from amaranth leaves in a time-dependent reversible process with biphasic reaction kinetics. The initial rapid phase, which we could not resolve in time, was followed by a slower phase with a second-order forward rate constant of 11.2 M −1 s −1 and a first-order reverse rate constant of 0.0012 s −1. The second, slower phase of inactivation was first order with respect to PLP and the rate increased linearly with PLP concentrations, suggesting that over the concentration range used no significant E-PLP complex accumulates during inactivation. The pH dependence of this second, slower phase of the modification shows a p K of 8.0±0.1. Correlation studies between the remaining activity and mol of PLP residues incorporated per mol of enzyme subunit after reduction with NaBH 3CN suggested that there are two critical lysyl groups for enzyme monomer. Both nucleotides, NAD + and NADH, provided significant protection against the slow phase of inactivation, but only NADH protected the enzyme against the first fast phase. The PLP-modified enzyme was not able to bind to an AMP-Sepharose column, suggesting that at least one of the PLP-modified lysyl residues is involved in the binding of the nucleotides.

Functional properties of a slowly inactivating potassium current in guinea pig dorsal lateral geniculate relay neurons

1. The time- and voltage-dependent properties of a slowly inactivating and depolarization-activated potassium current and the functional consequences of its activation was investigated with current and single-electrode voltage-clamp techniques applied to guinea pig dorsal lateral geniculate neurons maintained as a slice in vitro. 2. In current clamp, application of a step depolarization to near firing threshold resulted in a slowly rising membrane potential that took up to 10 s to reach steady state and firing threshold. In voltage clamp, step depolarization of the membrane potential to values positive to approximately -65 mV resulted in the rapid activation followed by slow inactivation of an outward current. In both cases the sudden depolarization was associated with a large increase in membrane conductance, which gradually lessened in parallel with the slow depolarization in current clamp or with the decrease in outward current in voltage clamp. 3. The time course of inactivation of the outward current, which we refer to as IAs, was well fitted by a two-exponential function with time constants of 96 and 2,255 ms, suggesting the presence of a fast and slow phase of inactivation. The activation threshold for IAs was about -65 to -60 mV, whereas inactivation was incomplete even at -50 mV, suggesting the presence of a substantial "window" current. The time course of removal of inactivation of IAs at -85 to -100 mV was well fitted by a single exponential function with time constant of 91 ms. 4. IAs appears to be mediated by K+. Increasing [K+]o from 2.5 to 10 mM resulted in a reduction in amplitude of IAs, whereas changing from 10 to 2.5 mM [K+]o enhanced this current. Intracellular injection of Cs+ resulted in an abolition of IAs, whereas extracellular application of Ba2+ resulted in a large decrease in the apparent input conductance but relatively little reduction of IAs. 5. Both phases of inactivation of the transient outward current were completely blocked by low doses (100 microM) of 4-aminopyridine (4-AP), but not by extracellular application of Cs+, tetraethylammonium (TEA), tetrodotoxin (TTX), or after block of transmembrane Ca2+ currents. Local application of 4-AP to neurons depolarized to near firing threshold resulted in depolarization associated with a decrease in apparent input conductance, thereby confirming the presence of a window current.4+ this bias against depolarizing inputs.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation and inactivation of excitation‐contraction coupling in rat soleus muscle.

1. Potassium (K+) contractures have been used to characterize the processes of activation and inactivation of excitation-contraction coupling during prolonged depolarization of fibres in small bundles dissected from rat soleus muscles at 23 degrees C. 2. The smallest measurable K+ contracture tension was recorded with depolarization to -40 mV in 30 mM-K+ and maximum tension was achieved between -26 mV in 80 mM-K+ and -19 mV in 120 mM-K+. 3. The rate of inactivation of K+ contracture tension was voltage dependent. Tension decayed from 80 to 20% of the peak amplitude within 44.0 +/- 2.2 s at -26 mV (in 80 mM-K+), compared with 66.7 +/- 4.8 s at -35 mV (in 40 mM-K+). Results are given as mean +/- 1 S.E.M. 4. The effect of inactivation on maximum tension was determined using a two pulse protocol in which a 'conditioning' depolarization in solutions containing 20-120 mM-K+ was applied for 0.5-10 min before a 'test' depolarization to -8 mV in 200 mM-K+. The amplitude of the test contracture was compared with the mean amplitude of 'control' 200 mM-K+ contractures elicited in normally polarized fibres immediately before and after the two pulse protocol. Conditioning depolarization to -47 mV (in 20 mM-K+) did not reduce test 200 mM-K+ contracture tension. Significant inactivation was seen with further conditioning depolarization to more positive potentials: after 10 min at -40 mV (in 30 mM-K+), or -35 mV (in 40 mM-K+), test 200 mM-K+ contracture tension was reduced by 33 and 70% respectively. 5. In contrast to amphibian muscle, where maximum tension falls to zero within a few minutes of depolarization to potentials positive to -50 mV, test 200 mM-K+ contracture tension in rat soleus fibres fell initially rapidly and then slowly, but was not reduced to zero, even after 10 min at -19 mV in 120 mM-K+. 6. The fast phase of inactivation of test 200 mM-K+ contracture tension occurred during the decay of the conditioning K+ contracture. The slow phase of inactivation reached completion after 10 min of conditioning depolarization and occurred during the period when conditioning tension was reduced to zero or to a plateau level. Both phases of inactivation in rat soleus fibres are slow compared with fast and slow inactivation times of 5-100 s respectively reported for amphibian muscle. 7. When repolarized after prolonged depolarization, the muscle fibres were initially refractory, i.e. unable to produce tension in response to electrical stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

The calcium antagonist D600 inhibits calcium-independent transient outward current in isolated rat ventricular myocytes.

1. The whole-cell voltage-clamp technique was applied to isolated rat ventricular myocytes to investigate the effects of D600 (10(-9)-10(-3) M) on the intracellular calcium-independent component of transient outward current. I(lo), recorded in a sodium-free medium containing 0.5 x 10(-3) M-cadmium and 10(-6) M-ryanodine. 2. Externally applied D600 reduced Ilo in a dose-dependent, reversible manner, and accelerated the decay of the current. 3. Current-voltage relationships and corresponding activation curves (determined assuming I(lo) to be a pure potassium current) were shifted towards positive potentials in the presence of 10(-3) M but not 10(-5) M-D600. Steady-state inactivation curves were not affected by either low or high concentrations of D600. 4. Under control conditions, the inactivation of I(lo) is composed of a fast and a slow component. The amplitude of the slow component was more strongly reduced by D600 than that of the fast one. In the presence of 10(-3) M-D600, the slow component was entirely suppressed. 5. Both the time to peak Ilo and the time constant of the fast component of inactivation were markedly reduced at all potentials by D600. The time constant of the slow component was less sensitive to the drug. 6. When the relative quantity of charge carried by each kinetic component of Ilo was plotted versus the concentration of D600, the data could be fitted by two distinctly separate dose-response curves with an almost 100-fold difference between the two apparent dissociation constants, of which the values were 2.88 x 10(-6) M for the slow phase of inactivation and 2.07 x 10(-4) M for the fast one, with Hill coefficients of 0.68 and 0.73 respectively. 7. The inhibition of I(lo) by D600 displayed little or no use dependence, one of the major characteristics of the effects of phenylalkylamines on the cardiac calcium current ICa. 8. Our results show that at least part of I(lo) is sensitive to D600 in the same range of concentrations as ICa. Although the effects of D600 on the two currents differ in several points, this observation raises the possibility that, besides clear differences, certain similarities may exist between the channels responsible for I(lo) and ICa.

Identification of amino acid residues involved in substrate recognition by the catalytic subunit of bovine cyclic AMP dependent protein kinase: peptide-based affinity labels.

Two peptide-based affinity inactivators Ac-Leu-(BrAc)Orn-Arg-Ala-Ser-Leu-Gly (4) and Ac-Leu-Arg-(BrAc)Orn-Ala-Ser-Leu-Gly (5) were prepared as probes for the study of the nature of the active-site residues in the catalytic subunit of cyclic AMP dependent protein kinase. Under conditions of inhibitor in excess, both peptides inactivated the catalytic subunit by an apparent biphasic process. A fast phase, which inactivated the protein by approximately 40%, was followed by a slow phase that accounted for the loss of the remaining enzyme activity. Protection experiments with the kinase substrates showed that the slow phase of inactivation was active site directed, while the fast phase was not. Studies with radioactively labeled peptides 4 and 5 indicated incorporation of two peptide residues per molecule of the catalytic subunit upon complete inactivation. This observation is consistent with the occurrence of one alkylation event in each phase of the inactivation. The protein was proteolyzed subsequent to its modification with radioactive peptides. High-performance liquid chromatography afforded two radioactive peptide fragments in each case, which were sequenced by Edman degradation. Peptide 4 alkylated Thr-197 and Glu-346, while peptide 5 modified Cys-199 and also Glu-346. Data are presented to support the conclusion that Thr-197 and Cys-199 are located at or near the active site.

Intracellular N-methyl-d-glucamine modifies the kinetics and voltage-dependence of the calcium current in guinea pig ventricular heart cells

The effects of internal substitution of the impermeant cation N-methyl-D-glucamine (NMG) for Cs ion on the properties of the Ca-current (L-type channel) were examined in single guinea pig cardiac myocytes with the whole-cell clamp technique. The properties of the cobalt-sensitive Ca current recorded in the presence of internal NMG or Cs were compared and the results were as follows. (1) The overall duration of the Ca-dependent slow action potential was markedly increased in the presence of internal NMG (6-fold at 0 mV) when compared to action potentials recorded with internal Cs. (2) The cobalt-sensitive Ca currents recorded with internal NMG or Cs had similar reversal potentials. However, in the presence of internal NMG, the maximum current density of the cobalt-sensitive Ca current was decreased and both the threshold and potential at which maximum current occurred were negatively shifted. (3) Voltage-dependence of steady-state activation, but not inactivation, of the cobalt-sensitive Ca current was shifted by -11.8 mV with internal NMG. (4) NMG increased the half-time of activation and inactivation of the cobalt-sensitive Ca current. The voltage-dependence of the half-time of inactivation was shifted by about -30 mV between 0 and +60 mV. Time constants measurements showed that NMG affected more the slow phase of inactivation of the Ca current. (5) When Ba was the charge carrier, NMG removed most of the inactivation of the current, suggesting a slowing of the voltage-dependent process of inactivation. (6) The results are consistent with a modification of the properties of the Ca channel by internal NMG.

The kinetics of recovery and development of potassium channel inactivation in perfused squid (Loligo pealei) giant axons.

K+ currents were studied at a normal (-69 mV) and at a depolarized (-49 mV) membrane potential in voltage-clamped squid giant axons perfused with 350 mM-K+ and bathed in K+-free artificial sea water containing tetrodotoxin to block the Na+ channels. Steady-state and instantaneous K+ currents were reduced by over 50% at corresponding voltages at the depolarized membrane potential. Instantaneous chord conductance-voltage curves showed that the depolarized membrane potential caused a uniform reduction of K+ conductance across the voltage range under study. The driving force for K+ ions was comparable at both membrane potentials when a short (2 ms) pre-pulse was used to open the K+ channels. When a longer (7.5 ms) pre-pulse was used, the driving force was actually larger at the depolarized membrane potential. The depolarized membrane potential did drive some K+ ions into the periaxonal space. The amount of K+ ions driven into the periaxonal space was estimated by two independent methods, with similar results. The resulting increase of K+ ions in the periaxonal space (10 mM) was about 40 times too small to account for the large reduction in currents in terms of a reduced driving force for K+ ions. The kinetics of recovery and development of inactivation were monitored by repeatedly applying a 7.5 ms test pulse followed by a long conditioning potential. Both recovery and development of inactivation, from the depolarized membrane potential, were described by the sum of two exponential terms plus a constant. The time constant-voltage curves for both phases of inactivation peaked at about -54 mV at 10 degrees C. The time constant of the slow phase of inactivation at -54 mV was about 12.4 s, while the corresponding time constant for the fast phase was about 2.3 s. The slow relaxation had an apparent plateau of about 11 s at more depolarized membrane potentials. Recovery from inactivation was rapid at hyperpolarized membrane potentials. The steady-state inactivation curve of the K+ channel was incomplete in the depolarizing region; and apparent plateau was reached with about 75% of the K+ current inactivated. The temperature sensitivity of both phases of inactivation corresponded to a Q10 of about 3. Elevated external concentrations of K+ ions did not block either phase of the inactivation process, although the kinetics of recovery from inactivation were slightly faster under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Ionic currents in cultured dorsal root ganglion cells from adult guinea pigs.

Ionic currents in cultured dorsal root ganglion (DRG) neurons from adult guinea pigs were analyzed by voltage-clamp techniques. The Na+ inward current had a reversal potential at +33 mV, and revealed activation and inactivation kinetics similar to those of squid giant axons. A typical value for the maximum Na+ conductance was 178 mS/cm2 and the peak current was 2.5 mA/cm2. The delayed K+ outward current showed a fast and a slow phase of inactivation and was sensitive to tetraethylammonium (TEA; approximately 130 mM) and 4-aminopyridine (approximately 2 mM). The maximum K+ conductance was 26 +/- 9 (mean +/- SD) mS/cm2. The slow Ca2+ inward current was identified in Na+-free, TEA-containing solution. Its peak value was increased by 1.7-fold when [Ca2+]o was increased from 5 to 10 mM. The current was blocked by Co2+ but not by tetrodotoxin. Sr2+ and Ba2+ could substitute in carrying this current. The maximum peak of the Ca2+ current was 0.22 +/- 0.14 mA/cm2. At potentials positive to 0 mV, the Ca2+ current was often followed by a slowly developing outward current, which was also sensitive to Co2+, suggesting a Ca2+-activated outward current. It is concluded that the action potential of the adult guinea pig DRG neuron is mediated by Ca2+ as well as by Na+ and K+ currents. The current densities of these ionic channels are considered to be different from embryonic neurons and from nodes of Ranvier.

Development of Na inactivation in motor and sensory myelinated nerve fibres of Rana esculenta.

Development of Na permeability inactivation was investigated in myelinated motor (N = 12) and sensory (N = 12) nerve fibres of Rana esculenta at 20 degrees C. The K currents were blocked by 10 mM tetraethylammonium chloride, added to the superfusing solution. Additionally, in 4 fibres of each group internal CsCl was applied by diffusion from the cut internodes. Development of Na inactivation was approximated by the sum of two exponentials. The time constants of the fast and slow inactivation phase (tau h1 and tau h2) were dependent on membrane potential (E) with similar values in both fibre types for a given E. In contrast, significant differences were found in the contribution of both phases. In motor nerve fibres the amplitude of the fast phase was g = 0.70 (mean value of 12 fibres) throughout the potential range investigated (-30 mV less than or equal to E less than or equal to 40 mV). In sensory fibres g was potential dependent, increasing from 0.76 (mean value of 12 fibres) at E = -30 mV to unity at E greater than 30 mV. This difference in Na permeability inactivation is a further distinguishing property between motor and sensory nerve fibres.

Evidence for an active site arginine in UDP-glucuronyltransferase.

2,3-Butanedione inactivates the pure form of UDP-glucuronyltransferase used in these experiments (GT2P) (EC 2.4.1.17) purified from pig liver microsomes. The kinetics of the reaction indicates that 2,3-butanedione reacts with two amino acids that affect activity. A rapid, partial inactivation is followed by a slower rate of inactivation that leads eventually to completely inactive enzyme. UDP-glucuronic acid and glucuronic acid, as compared with UDP, are effective as protectors against the slow, secondary phase of inactivation; no ligand tested protected against the rapid phase of inactivation. The lipid environment of GT2P was a determinant of the pseudo-first order rate constant for the slow phase of inactivation, but did not affect the rate of the rapid phase of inactivation. The data suggest that GT2P contains an active site arginine that interacts with the -COO- at C-6 of the glucuronic acid moiety of UDP-glucuronic acid.

Activation and inactivation kinetics of Torpedo californica acetylcholine receptor in reconstituted membranes.

By use of a quench-flow technique to measure tracer ion flux rates in a physiologically significant time domain, the kinetics of activation and inactivation of purified reconstituted acetylcholine receptor (AChR) were investigated. After solubilization in sodium cholate, purification by affinity chromatography, and reconstitution into soybean lipids, the AChR from Torpedo californica displayed a characteristically fast rate of ion influx measured with 86Rb+. At 4 degrees C 1 mM carbamoylcholine (Carb) stimulated a fast (t1/2 = 7 ms) first-order filling of vesicle internal volume that presented a 10(4)-fold stimulation of ion flux rate by Carb. The concentration dependence of activation was sigmoidal with a half-maximal value at 3 X 10(-4) M Carb. In the presence of Carb, the purified AChR also underwent a two-step inactivation (desensitization) process. Inactivation was measured by preincubating AChR with Carb for various times (milliseconds to minutes) and then measuring the 86Rb+ influx rate. The two inactivation processes were each characterized by a distinct maximum rate (5.3 and 0.10 s-1) and by a different dependence on Carb concentration. The slow phase of inactivation gave a half-maximal rate at 2.5 X 10(-4) M Carb, and the fast inactivation was half-maximal at 1.3 X 10(-3) M Carb. The concentration dependence curves for both inactivation processes were approximately hyperbolic. The results are discussed in terms of models that describe the relationship between ligand binding and the processes of channel activation and desensitization.

Studies on (K + + H +)-ATPase : II. Role of sulfhydryl groups in its reaction mechanism

1. 1. Preincubation of a microsomal membrane preparation from pig gastric mucosa with 5,5′-dithiobis(2-nitrobenzoic acid) inhibits the (K + + H +)-ATPase and K +-stimulated 4-nitrophenylphosphatase activities of the preparation. 2. 2. The activating monovalent cation, K +, in the presence of CDTA, increases the rate of inactivation of (K + + H +)-ATPase by 5,5′-dithiobis(2-nitrobenzoic acid), indicating a conformational change in the presence of K +. 3. 3. In the absence of added Mg 2+ or in the presence of CDTA, the semilogarithmic plot of ATPase activity vs. reaction time is linear. Upon addition of Mg 2+, a fast and a slow phase become discernible. 4. 4. The nucleotides ATP, ADP and dATP in 0.5 mM concentration strongly protect the enzyme against inactivation by the modifying agent in the presence of CDTA. The ATP analogues AMPPNP and AMPPCP exert moderate protection, whilst other nucleotides like GTP, ITP and CTP show only minor protection. 5. 5. Analysis of the protective effect of ATP indicates that in the presence of 2 mM CDTA, 5,5′-dithiobis(2-nitrobenzoic acid) and ATP do not compete for the same site, suggesting that the essential sulfhydryl groups are not located in the ATP binding site. 6. 6. The slow inactivation phase, which occurs in the presence of Mg 2+, is completely blocked by ATP, and its non-phosphorylating analogue AMPPNP now appears to be equally potent.