N-bromoacetamide Research Articles

Two types of modified cardiac Na+ channels after cytosolic interventions at the alpha-subunit capable of removing Na+ inactivation.

Failure of inactivation is the typical response of voltage-gated Na+ channels to the cytosolic presence of proteolytic enzymes, protein reagents such as N-bromoacetamide (NBA) or iodate, and antibodies directed against the linker between domains III and IV of the alpha-subunit. The present patch clamp experiments with cardiac Na+ channels aimed to test the hypothesis that these interventions may provoke the occurrence of non-inactivating Na+ channels with distinct kinetic properties. A site-directed polyclonal antibody (anti-SLP2, target sequence 1481-1496 of the cardiac Na+ channel alpha-subunit) eliminated fast Na+ inactivation to induce burst activity which was accompanied by the occurrence of two open states. A deactivation process terminated channel activity during membrane depolarization proceeding with time constants of close to 40 ms (at -40 mV). NBA-modified and iodatemodified Na+ channels were kinetically indistinguishable from the anti-SLP2-modified type since they likewise deactivate and, thus, attain an only moderate Po of close to 20%. This is fundamentally different from the behaviour of enzymatically-modified Na+ channels: after cytosolic proteolysis with alpha-chymotrypsin, trypsin or pronase, mean Po during membrane depolarization amounted to approximately 40% because deactivation operated extremely slowly and less efficiently (time constants 100-200 ms at -40 mV, as a minimum) or was virtually non-operating. Invitro cleavage of the synthetic linker sequence 1481-1496 confirmed that this part of the alpha-subunit provides a substrate for these peptidases or reactants for NBA but cannot be chemically modified by iodate. This iodate resistance indicates that iodate-modified Na+ channels are based on a structural alteration of still another region which is also involved in Na+ inactivation, besides the linker between domains III and IV of the alpha-subunit. Endogenous peptidases such as calpain did not affect Na+ inactivation. This stresses the stochastic nature of a kinetic peculiarity of cardiac Na+ channels, mode-switching to a non-inactivating mode.

Studies on the oxidation of ?-amino acids by N-Bromo oxidants: Kinetics of the reaction of bromide ion with N-Bromoacetamide

The rate of oxidation of amino acids (AA) by N-Bromoacetamide (NBA) was studied in aqueous buffered medium at 35°C. The rate of disappearance of [NBA] is catalyzed by the Br− produced from the reduction of NBA. Analysis of the autocatalyzed reaction gives the kinetic data for the oxidation of bromide ion by NBA. The results suggest that the protonated NBA reacts with Br− to form Br2 which rapidly oxidizes amino acids. The rate constant for the reaction between protonated NBA and Br− at 35°C is estimated. © 1996 John Wiley & Sons, Inc.

Mechanism of iridium(III) catalysis in the oxidation of diols by N-bromoacetamide

The kinetics of iridium(III)-catalysed oxidation of 1,2-ethanediol and 1,4-butanediol by N-bromoacetamide (NBA) in HClO4 in the presence of [Hg(OAc)2] as a scavenger for Br− have been investigated. The reactions are zero-order with respect to both diols, and first-order in NBA at low NBA concentrations, tending to zero order at high concentrations. The order in IrIII decreases from unity to zero at high iridium(III) concentrations. A positive effect on the oxidation rate is observed for [H+] and [HgII] whereas a negative effect is observed for acetamide and [Cl−]. Ionic strength does not influence the oxidation rate. (H2OBr)+ is postulated as the oxidizing species. A mechanism consistent with the observed kinetic data is proposed.

Quantification of exponential Na+ current activation in N-bromoacetamide-treated cardiac myocytes of guinea-pig.

1. The activation kinetics of the Na+ current was investigated in single ventricular cells of the guinea-pig heart using an improved oil-gap voltage clamp method. The inactivation of the current was removed by an intracellular application of N-bromoacetamide (NBA) for less than 1 min. Although the NBA treatment slightly decreased the peak amplitudes (81.7 +/- 13.4% of control, n = 15), the Na+ current remained stable after the removal of inactivation. 2. On depolarization, the activation of Na+ current took an exponential time course after the capacitive current decreased to 5% of its peak amplitude (40-100 microseconds after the pulse onset). The time course of deactivation, recorded on repolarization from 1.2 ms depolarization, was also a single exponential. 3. The time constants of activation and deactivation were almost identical when compared at a given test potential within a range of -50 to -30 mV. These findings indicate that the cardiac Na+ current activation is determined by m1 kinetics, or one rate-limiting step. 4. At potentials negative to -60 mV, the deactivation was complete, and its time constant decreased e-fold per 20.3 +/- 1.8 mV hyperpolarization (n = 7). 5. The degree of steady-state activation (m(infinity)) was fitted to a Boltzmann equation with a slope factor of 7.4 +/- 0.3 mV and a half-maximum potential of -33.3 +/- 0.8 mV (n = 8). 6. Rate constants for the rate-limiting activation step between a closed state and an open state (alpha m, beta m), were determined from m(infinity) and tau m over a potential range between -100 and +50 mV. On a logarithmic scale, beta m-1 was a linear function of the membrane potential over the range -100 and -30 mV. 7. Fitting the newly determined activation kinetics to the rising phase of the action potential indicated that the activation kinetics in the present study is relevant to the physiological action potential. The density of the Na+ channels thus obtained was 1075 +/- 186 pF-1 (n = 6). 8. The measurements in the NBA-treated Na+ current were compared with those obtained without treatment.

Kinetics of the oxidation of phosphinic, phenylphosphinic, and phosphorous acids by N‐bromoacetamide

AbstractThe oxidation of phosphinic, phenylphosphinic, and phosphorous acids by N‐bromoacetamide (NBA) in acid solution, results in the formation of corresponding higher oxyacids of phosphorus. The reaction is first order with respect to NBA, second order in the oxyacid and inverse first in hydrogen ions. The oxidation of deuteriated phosphorus oxyacids showed the presence of a substantial primary kinetic isotope effect. The reaction failed to induce polymerization of acrylonitrile. Added acetamide has no effect on the reaction rate. It has been shown that the ‘inactive’ tautomer of the phosphorus oxyacids, RHP(O)OH, participates in the oxidation process. A rate‐determining step involving transfer of a hydride ion from the PH bond to the oxidant has been proposed.

Kinetics and mechanism of oxidation of some hydroxy acids by N-bromoacetamide

Studies of the kinetics of oxidation of citric acid and tartaric acid by N-bromoacetamide (NBA) in the presence of perchloric acid and iridium trichloride are reported. The reactions follow identical kinetics, being zero order in substrate and first order in each NBA, Ir(III) and mercuric acetate. A negative effect of hydrogen ions, acetamide and Cl − is observed, while ionic strength has no effect on reaction velocity. Various activation parameters were calculated and recorded. A suitable mechanism consistent with the above observations is proposed.

Kinetics and mechanism of oxidation of carbohydrazide and thiocarbo-hydrazide by N-bromoacetamide

Kinetics of oxidation of carbobydrazide (ch) by N-bromoacetamide (NBA) in aqueous perchloric acid medium and thiocarbohydrazide (tch) in the free state and in its metal complex by NBA both in the presence and absence of added bromide ion have been investigated in aqueous perchloric acid medium. Oxidation of carbohydrazide showed first order kinetics in [NBA], nearly first order in [ch] and inverse fractional order in [H+]. The rate slightly decreased with increase in ionic strength of the medium. Addition of either the reduced product of the oxidant or bromide had no significant effect on the rate of CH oxidation. Oxidation of thiocarbohydrazide in the free and metal-bound states both in the presence and absence of added bromide showed first order kinetics in [NBA] and fractional order in both [tch] and [H+]. But the fractional order in [H+] for the oxidation of tch in the presence of Br¯ was almost twice that in its absence. The rate increased with increase in [Br¯] with a fractional order of about 0–3. Variation in ionic strength of the medium had very negligible effects. Addition of acetamide, the reduced product of the oxidant, had no effect on the rate. Michaelis-Menten type mechanisms have been considered to explain the observed results. Activation parameters have been computed for all the oxidations by measuring rates at different temperatures. Detailed mechanisms of oxidations have also been suggested.

Aminopyridines block an inactivating potassium current having slow recovery kinetics

The blocking action of aminopyridines on an inactivating K current (lKi) in GH3 pituitary cells was studied before and after altering the macroscopic decay of the current with N-bromoacetamide (NBA). The first depolarizing pulse delivered either seconds or minutes after beginning 4-aminopyridine (4AP) application, elicited a current with both a more rapid decay and a reduced peak amplitude. The rapid decay (or time-dependent block) was especially prominent in NBA-treated cells. With continued drug application, subsequent test pulses revealed a stable block of peak current, greater in NBA-treated than control cells. Recovery from block was enhanced by hyperpolarizing holding potentials and by the first depolarizing pulse delivered after prolonged recovery intervals. Unlike aminopyridine block of other K currents, there was no convincing evidence for voltage shifts in activation or inactivation, or for voltage and frequency-dependent unblock. Increasing the open probability of the channels did, however, facilitate the block. Although the behavior of currents in 4AP was suggestive of "open channel block," the block was not produced by 4-aminopyridine methiodide, a positively charged aminopyridine. Moreover, because partial block and recovery occurred without opening the channels we suggest that aminopyridines bind to, or near, this K channel, that this binding is enhanced by opening the channel, and that a conformational change is induced which mimics inactivation. Because recovery from block is enhanced by negative potentials, we suggest that aminopyridine molecules may become "trapped" by inactivation awaiting the slow process of reactivation to escape their binding sites.

Reconstituted voltage-sensitive sodium channels from eel electroplax: Activation of permeability by quaternary lidocaine, N-bromoacetamide, and N-bromosuccinimide

We have investigated the ion permeability properties of sodium channels purified from eel electroplax and reconstituted into liposomes. Under the influence of a depolarizing diffusion potential, these channels appear capable of occasional spontaneous openings. Fluxes which result from these openings are sodium selective and blocked (from opposite sides of the membrane) by tetrodotoxin (TTX) and moderate concentrations of the lidocaine analogue QX-314. Low concentrations of QX-314 paradoxically enhance this channel-mediated flux. N-bromoacetamide (NBA) and N-bromosuccinimide (NBS), reagents which remove inactivation gating in physiological preparations, transiently stimulate the sodium permeability of inside-out facing channels to high levels. The rise and subsequent fall of permeability appear to result from consecutive covalent modifications of the protein. Titration of the protein with the more reactive NBS can be used to produce stable, chronically active forms of the protein. Low concentrations of QX-314 produce a net facilitation of channel activation by NBA, while higher concentrations produce block of conductance. This suggests that rates of modifications by NBA which lead to the activation of permeability are influenced by conformational changes induced by QX-314 binding.

The inactivating K+ current in GH3 pituitary cells and its modification by chemical reagents.

1. Whole-cell and single-channel recording techniques were applied to the study of the permeability and gating of inactivating K+ channels from clonal pituitary cells. 2. The cation selectivity sequence (measured from reversal potentials) for the channels underlying the inactivating K+ current was Tl+ greater than K+ greater than Rb+ greater than NH4+. The conductance sequence (determined from current amplitudes) was K+ = Tl+ greater than Rb+ greater than NH4+. 3. The inactivating current (IK(i] which was blocked by 4-aminopyridine (4-AP), activated at voltages more positive than -40 mV and half-inactivated at that voltage. Inactivation proceeded as the sum of two exponentials with mean time constants of 21 and 82 ms. Deactivation followed a single-exponential time course. 4. Recovery from inactivation was slow, voltage dependent and multi-exponential, taking more than 50 s near the cell's resting potential. 5. The magnitudes of outward current and of slope conductance increased as the concentration of external K+ was increased. 6. On-cell and outside-out membrane patches revealed minicurrents with gating and pharmacological properties identical to whole-cell currents. Single channels with inactivating characteristics, while rarely observed, had an average slope conductance of 6-8 pS. 7. Intracellular application of the disulphonic stilbene derivative, SITS, and the protein-modifying reagent, N-bromoacetamide (NBA), at concentrations of 0.2-1 mM for several tens of minutes dramatically slowed the decay (inactivation) of K+ currents and caused coincident increases in the magnitude of outward IK(i). 8. Extracellular application of NBA at much lower concentrations (1-100 microM) and much shorter exposure times (1-30 s) also slowed inactivation. This effect was reversible for brief applications at low doses, but became irreversible after longer exposures. 9. Both internal and external NBA shifted the steady-state inactivation-voltage relation by +10 mV and reduced inactivation at voltages more positive than 0 mV. 10. The efficacy of external NBA was independent of holding potential between -80 and 0 mV. 11. Potassium minicurrents and single channels recorded from on-cell membrane patches were not affected by application of NBA to the extrapatch membrane. In contrast, NBA reversibly slowed the decay, increased the magnitude of minicurrents and prolonged the open times of single K+ channels recorded from outside-out patches. The single-channel conductance was unchanged by NBA.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of Ru(III) catalysis in N-bromoacetamide oxidation of some glycols in perchloric acid media

The kinetics of ruthenium(III) chloride catalysis in the oxidation of diethylene glycol (DG) and methyl diethylene glycol (MDG) by N-bromo-acetamide (NBA) in perchloric acid media are reported. The reactions follow identical kinetics, showing zero-order dependence on NBA, but first-order on each of H +, Ru(III) and Cl − ions. The first-order kinetics with respect to glycol at low concentrations shifts to zero-order at higher concentrations. A negative effect of ionic strength is observed, while successive addition of acetamide, D 2O and mercuric acetate shows zero effect on the reaction rate. Various activation parameters have been computed. The products of the reactions were identified as the corresponding acids. A suitable mechanism consistent with the kinetic observations has been proposed.

Mechanistic Study of the Oxidation of Substituted Benzylamines by N-Bromoacetamide

Abstract The oxidation of ortho-, meta-, and para-substituted benzylamines by N-bromoacetamide (NBA), to the corresponding benzaldehydes, is first order with respect to NBA and the amine. The pH dependence of the reaction rate suggests that the unprotonated benzylamine is the reductant. There is no effect of added acetamide. NBA itself has been postulated as the reactive oxidizing species. The oxidation of benzylamine-α,α-d2 exhibited a substantial primary kinetic isotope effect (kH⁄kD=5.81). The rates of the oxidation of the meta- and para-substituted benzylamines were separately correlated in Taft’s and Swain’s dual substituent parameter equations. For the para-substituted compounds, the best correlation is obtained with σI and σR+ values, while meta-substituted compounds correlated best with σI and σR° values. The reaction constants have negative values. The oxidation rates of the ortho-substituted compounds yield excellent correlation in a triparametric equation involving Taft’s σI and σR+ values and Charton’s steric parameter, V. A mechanism involving transfer of a hydride ion from the amine to NBA, in the rate-determining step has been proposed.

Ru(III) catalysis in N‐bromoacetamide oxidation of ethylene glycol and glycerol: A kinetic and mechanistic study

AbstractKinetics of oxidation of ethylene glycol and glycerol by acidic solution of N‐bromoacetamide (NBA) in the presence of ruthenium (III) chloride as a homogeneous catalyst and mercuric acetate as scavenger in the temperature range of 30–50°C have been reported. The reactions follow identical kinetics, being zero‐order in substrate and first‐order in Ru(III). First order dependence of the reaction on NBA at its low concentrations tends to zero order in the higher concentration range. Positive effect of [H−] and [Cl−] has been observed. A negative effect of acetamide and ionic strength of the medium is observed while D2O and mercuric acetate show zero effect on the reaction velocity. Various activation parameters have been computed. The main product of the oxidation is corresponding acid. (H2OBr)+ has been postulated as the oxidizing species. A suitable mechanism in conformity with the kinetic data has been proposed.

Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide

Ca2+-activated K+ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca2+ activation site was modified using N-bromoacetamide (NBA). 1 mM NBA in the bath solution, at 2.5 microM Ca2+ reduces the open probability, Po, of the channel to less than 0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca2+-sensitive, requiring 25 mM Ca2+ to raise Po to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca2+ (1 mM) protects the channels from modification. Also presented is a second class of Ca2+-activated K+ channels which are normally present in MTAL cells which open infrequently at 10 microM Ca2+ (Po = 0.01) but have a Po of 0.08 at 1 mM Ca2+. We can conclude (i) that NBA modifies the channel by shifting Ca2+-sensitivity to very high Ca2+, (ii) that NBA acts on a site involved in Ca2+ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.

Kinetics and Mechanism of Bromination of Toluene and Substituted Toluenes by N-Bromo Acetamide (NBA)

Article Kinetics and Mechanism of Bromination of Toluene and Substituted Toluenes by N-Bromo Acetamide (NBA) was published on January 1, 1987 in the journal Zeitschrift für Physikalische Chemie (volume 268O, issue 1).

Saxitoxin and tetrodotoxin. Electrostatic effects on sodium channel gating current in crayfish axons

The effects of extracellular saxitoxin (STX) and tetrodotoxin (TTX) on gating current (IgON) were studied in voltage clamped crayfish giant axons. At a holding potential (VH) of -90 mV, integrated gating charge (QON) was found to be 56% suppressed when 200 nM STX was added to the external solution, and 75% suppressed following the addition of 200 nM TTX. These concentrations of toxin are sufficiently high to block greater than 99% of sodium channels. A smaller suppression of IgON was observed when 1 nM STX was used (KD = 1-2 nM STX). The suppression of IgON by external toxin was found to be hold potential dependent, with only minimal suppression observed at the most hyperpolarized hold potentials, -140 to -120 mV. The maximal effect of these toxins on IgON was observed at hold potentials where the QON vs. VH plot was found to be steepest, -100 to -80 mV. The suppression of IgON induced by TTX is partially relieved following the removal of fast inactivation by intracellular treatment with N-bromoacetamide (NBA). The effect of STX and TTX on IgON is equivalent to a hyperpolarizing shift in the steady state inactivation curve, with 200 nM STX and 200 nM TTX inducing shifts of 4.9 +/- 1.7 mV and 10.0 +/- 2.1 mV, respectively. Our results are consistent with a model where the binding of toxin displaces a divalent cation from a negatively charged site near the external opening of the sodium channel, thereby producing a voltage offset sensed by the channel gating apparatus.

Kinetics and mechanism of oxidation of substituted phenyl methyl sulphides by n-bromoacetamide

The kinetics of oxidation of several substituted phenyl methyl sulphides by N-bromoacetamide (NBA) in 80%acetonitrile-20%water (v/v) mixture are presented. The reaction is first order with respect to both sulphide and NBA and second order in Hg(II). The rate of the reaction exhibits inverse dependence on both [acetamide] and [NBA]. Electron-releasing substituents present in the phenyl ring of the sulphides accelerate the rate while electron-withdrawing ones retard it. The results are in accord with a mechanism involving the reaction between NBA-Hg(II) and sulphide-Hg(II) complexes in the slow rate-limiting step affording a sulphoniumion which hydrolyses in a subsequent fast step to the sulphoxide.

Kinetics and mechansim of oxidation of some ketones by [formula omitted]-bromoacetamide

The kinetics of oxidation of n-butyl methyl ketone (n-BMK) and i-butyl methyl ketone (i-BMK) by N -bromoacetamide (NBA) is studied in perchloric acid media in the presence of mercuric acetate. The main product of the oxidation is the corresponding 1,2-dicarbonyl compound. The reaction order with respect to NBA is zero while with respect to ketone and H + it is unity. Mercuric acetate, acetamide and sodium perchlorate have negligible effect on the reaction rate, while the dielectric effect is negative. A solvent isotope effect (k o(D 2O)/k o(H 2O) = 1.80–2.05 and 1.70–2.06 for n-BMK and 1-BMK, respectively) at 35°C is observed. On the basis of the available evidence a suitable mechanism consistent with the experimental results is proposed in which it is suggested that the mechanistic route for NBA oxidation is through the enol form of the ketone in an acidic medium.

Dynamics of strychnine block of single sodium channels in bovine chromaffin cells.

The kinetics of strychnine block of single Na channels in bovine chromaffin cells were studied using the gigohm seal, patch-clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Strychnine, applied to the cytoplasmic face of Na channels at concentrations ranging from 25 to 100 microM, caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single-channel current. Average currents in the presence of strychnine accompanied 'hooked' tail currents upon repolarization. The histograms for blocked times and the histograms for open times could be fitted with a single-exponential function. The mean open time (to) became shorter as the drug concentration was increased, while the mean blocked time (tb) was concentration independent. The association (blocking) rate constant, k, calculated from the slope of the curve relating the reciprocal mean open time to strychnine concentration was 7.6 X 10(6) M-1 S-1 at -40 mV in a typical experiment. to-1 and tb-1 had opposite voltage dependences: tb-1 became larger as the membrane was hyperpolarized whereas to-1 became smaller. The voltage dependence suggests that a first-order blocking site is located 35% of the way through the membrane electric field from the cytoplasmic surface. An increase in the external Na+ concentration greatly decreased to-1 without affecting tb-1. The voltage dependence of both to-1 and tb-1 did not change when the external Na concentration was changed. It is suggested that the strychnine block of single Na channels is not 'current-dependent'. All of the features of strychnine block of single Na channels are compatible with the sequential model, in which strychnine molecules block open Na channels, and the blocked channels could not close until strychnine molecules had left the blocking site in the channels.

N-bromoacetamide removes a calcium-dependent component of channel opening from calcium-activated potassium channels in rat skeletal muscle.

Calcium-activated potassium channels from cultured rat skeletal muscle were treated with the protein-modifying reagent N-bromoacetamide (NBA) (0.3-1 mM) and studied in excised patches using patch-clamp techniques. After NBA treatment, channels opened only occasionally, and, in contrast to untreated channels, the open probability was no longer sensitive to intracellular surface calcium ions (1 nM to 100 microM). Channel activity did, however, exhibit a voltage dependence similar in direction and magnitude to that shown before NBA treatment (increasing e-fold with 19 mV depolarization). Distributions of open channel lifetimes revealed that NBA treatment virtually abolished openings of long duration, which suggests that this class of openings requires calcium sensitivity. These effects were not reversed by subsequent washing. Quantitatively similar open probability, voltage dependence, and open-interval distributions were observed in untreated channels in calcium-free medium. These results suggest that NBA removed a calcium-dependent component of channel opening, and that normal channels are able to open in the absence of significant intracellular calcium concentrations.