Phase Of Inactivation Research Articles

Tryptic and chymotryptic cleavage sites in sequence of α-subunit of (Na + + K +)-ATPase from outer medulla of mammalian kidney

Localization of selective proteolytic splits in α-subunit of (Na + + K +)-ATPase is important for understanding the mechanism of active Na +,K +-transport. Proteolytic fragments of α-subunit from pig kidney were purified by chromatography in NaDodSO 4 on TSK 3000 SW columns. NH 2-terminal amino acid sequences of fragments as determined in a gas phase sequenator were unambiguously located within the total sequence of α-subunit from sheep kidney (Shull, C.E., et al. (1985) Nature 316, 691–695) and pig kidney (Ovchinnikov, Y.A., et al. (1985) Proc. Acad. Sci. USSR 285, 1490–1495). The primary chymotryptic split in the E 1-form is located between Leu-266 and Ala-267 while the tryptic cleavage site appears to be between Arg-262 and Ile-263 (Bond 3). Tryptic cleavage in the initial fast phase of inactivation of the E 1-form is located between Lys-30 and Glu-31 (Bond 2). In the E 2-form, primary tryptic cleavage is between Arg-438 and Ala-439 (Bond 1). Chymotryptic cleavage between Leu-266 and Ala-267 stabilizes the E 1-form of the protein without affecting the sites for binding of cations or nucleotides. Titration of fluorescence responses demonstrates the importance of the NH 2-terminal for E 1-E 2 transition. Protonation of His-13 facilitates transition from E 1- to E 2-forms of the protein. Removal of His-13 after cleavage of bond 2 can explain the increase in apparent affinity of the cleaved enzyme for Na + and the shift in poise of E 1-E 2 equilibrium in direction of E 1-forms. The NH 2-terminal sequence in renal α-subunit is not conserved in α + from rat neurolemma or in α-subunit from Torpedo or brine shirmp. A regulatory function of the NH 2-terminal part of the α-subunit may thus be a unique feature of the α-subunit in (Na + + K +)-ATPase from mammalian kidney.

Transient and delayed potassium currents in the Retzius cell of the leech, Macrobdella decora

The properties of a quickly inactivating transient K current (IA) and a slowly inactivating delayed K current (IK) were investigated with two-electrode voltage-clamp techniques in the isolated soma of the Retzius cell of the leech, Macrobdella decora. The two currents could be pharmacologically separated according to their different sensitivities to tetraethylammonium ions (TEA) and 4-aminopyridine (4-AP). IA was totally blocked by 3 mM 4-AP but not affected by 25 mM TEA. IK was suppressed almost completely by 25 mM TEA, whereas its peak amplitude only decreased by 10-15% in 3 mM 4-AP. IA was activated at membrane potentials more positive than -35 to -30 mV, whereas the threshold for IK was at more positive potentials of approximately -20 to -15 mV. The activation of IA was rapid with a voltage-dependent time constant [tau m(A)] that varied from 6 to 2 ms for command potentials between -20 and 10 mV (at 22-24 degrees C). The inactivation, which was independent of voltage, was somewhat slower with a time constant (tau A) of approximately 90-110 ms. The time constants for activation [tau m(K)] and the early inactivation phase (tau K) of IK were both voltage dependent. In the range of potential steps from 0 to 30 mV, tau m(K) varied from 12 to 4.5 ms and tau K from 1,500 to 700 ms. The steady-state inactivation of IA varied with holding potential and was complete at potentials more positive than -30 mV. IA was fully available from potentials more negative than -70 mV. IK did not show steady-state inactivation below its threshold of activation. The time course of IA during a maintained depolarization could be reasonably described by the expression IA(t) = IA(infinity) [1-exp(-t/tau m(A))]2 exp(-t/tau A). The time course of activation of IK without allowance for inactivation was approximated by the expression IK(t) = IK(infinity) [1-exp(-t/tau m(K))]2. The reversal potentials and magnitude of both IA and IK were dependent on extra-cellular K concentration, which suggest that a substantial part of the two currents was carried by K ions.

Calcium channel current inactivation is selectively modulated by menthol

The effect of menthol on Ca channel current inactivation was studied in identified Helix neurons. External application of menthol accelerated the Ca-dependent rapid phase of inactivation. Menthol restored a fast inactivation phase after the Ca-dependent inactivation had been removed by strongly buffering changes in intracellular free Ca or by using Ba ions as current carriers. The menthol-induced inactivation was unchanged by variations in intracellular free Ca. A sensitizing effect of menthol on Ca-dependent inactivation appeared unlikely. Instead the results indicate a modulating action of menthol on Ca inactivation.

Three copies of the beta subunit must be modified to achieve complete inactivation of the bovine mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoyladenosine.

The modification of both beta-Tyr-368 and beta-His-427 can be correlated with the loss of activity observed when the bovine mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoyl[3H]adenosine ([3H]FSBA). At pH 8.0, where the rate of inactivation is fast, beta-Tyr-368 is modified predominantly, while at pH 6.0, where the rate of inactivation is slow, beta-His-427 is modified predominantly. At pH 7.0, the 2 residues are modified with about equal efficiency. When the F1-ATPase was inactivated by 80% at pH 6.5, 7.0, and 7.5, the sum of radioactivity incorporated into beta-Tyr-368 and beta-His-427 was 1.99, 1.87, and 1.82 mol of label incorporated per mol of enzyme, respectively. Examination of the rate of inactivation of the enzyme by FSBA as a function of pH revealed two pKa values, one of about 7.6 associated with the modification of beta-Tyr-368 and the other of about 5.8 associated with the modification of beta-His-427. The inactivation of the F1-ATPase by FSBA exhibited an initial fast rate followed by a slower rate in triethanolamine-HCl, pH 7.0. In contrast, only a single rate, equivalent to the fast phase of inactivation in the absence of phosphate, was observed in 0.2 M phosphate, pH 7.0. The dependence of this stimulation on phosphate concentration is sigmoidal with half-maximal stimulation occurring at approximately 160 mM. The ratio of 3H incorporated into beta-Tyr-368 to that incorporated into beta-His-427 was approximately the same during the fast and slow phases of inactivation in triethanolamine-HCl, pH 7.0. Approximately the same ratio was observed when the enzyme was modified during the single phase of inactivation exhibited in the presence of 0.2 M phosphate, pH 7.0. The sum of the 3H incorporated into beta-Tyr-368 and beta-His-427 during inactivation of the F1-ATPase from bovine heart mitochondria by [3H]FSBA in the presence and absence of phosphate was linear and extrapolated to a value of about 2.6 residues modified on complete inactivation of the enzyme. From these data, it is concluded that FSBA binds to a single binding site on the beta subunits of the enzyme where it reacts with either beta-Tyr-368 or beta-His-427 in mutually exclusive reactions. All three beta subunits must be modified in this manner for complete inactivation to be observed.

Nucleotide specificity of the [formula omitted] transition in [formula omitted] as probed with tryptic inactivation and fragmentation

The nucleotide specificity for the E 2K → E 1K conformational transition in ( Na + + K +)- ATPase as the key step for overall hydrolytic activity and coupled cation transport has been investigated. Use has been made of tryptic inactivation, which is biexponential in time for the enzyme in the presence of Na + with or without nucleotides (E 1 conformation) and monoexponential in the presence of K + (E 2 conformation). ATP, AdoPP[NH]P and CTP in order of decreasing effectivity induce the biphasic tryptic inactivation pattern in the presence of K +. Their order of effectivity is inversely related to the rate constant of the second (slow) phase of inactivation. In the presence of K + and either ITP or GTP tryptic inactivation remains monoexponential, indicating that these nucleotides cannot drive the E 2K → E 1K transition. Tryptic inactivation has been compared with tryptic fragmentation of the α-subunit (apparent mol. wt. 94 kDa) of ( Na + + K +)- ATPase . In the E 1 conformation (Na + present) a 71 kDa fragment is formed during the second phase of inactivation. In the E 2 conformation (K + present) the α-subunit is split to fragments of 41 and 52 kDa. In the presence of K + and ATP, ADP, AdoPP[NH]P or CTP the 71 kDa fragment is formed in amounts which decrease in the order ATP ≈ ADP > AdoPP[NH]P > CTP . In the presence of K + and AMP, ITP or GTP the 71 kDa fragment is absent and only the E 2 fragments are formed. From these and literature data we arrive at a specificity order for the E 2K → E 1K transition of ATP > ADP > AdoPP[NH]P > CTP > ITP = GTP = AMP . The same order holds for K + transport in the K +− K + exchange and for overall hydrolytic activity ( Na + + K + present) with the natural nucleoside triphosphates as substrates. This marks the E 2K → E 1K transition as the step in the reaction mechanism that determines nucleotide specificity for ( Na + + K +)- activated hydrolysis and coupled cation transport.

Affinity labeling of NADP+-specific isocitrate dehydrogenase by a new fluorescent nucleotide analogue, 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate.

A new reactive fluorescent adenine nucleotide analogue has been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (BDB-T epsilon ADP). This compound reacts irreversibly with NADP+-specific isocitrate dehydrogenase. Biphasic kinetics of inactivation are observed that can be described in terms of a fast initial phase of inactivation resulting in partially active enzyme of 8-10% residual activity, followed by a slower phase leading to total inactivation. NADPH protects completely against the fast phase of the reaction, indicating that modification occurs at the coenzyme binding site, whereas isocitrate with MnSO4 protects totally against the slow phase of reaction. The inactivation rate constants for both phases exhibit nonlinear dependence on BDB-T epsilon ADP concentration, consistent with the formation of a reversible complex with the enzyme prior to irreversible modification. Covalent incorporation of BDB-T epsilon ADP is limited and specific; only 0.99 mol of reagent/mol of subunit is incorporated when the enzyme is 98% inactivated in the absence of ligands. A maximum incorporation of 0.5 mol of reagent/mol of subunit is obtained in the presence of isocitrate and MnSO4, while incorporation in the presence of NADPH equals the difference between the incorporation in the absence of ligands and that measured in the presence of isocitrate and MnSO4. It appears that 0.5 mol of reagent/mol of subunit is responsible for the fast phase of inactivation and the remaining incorporation causes the slow phase. Under all conditions used in this study, isocitrate dehydrogenase has been shown to exist as a dimer by analytical ultracentrifugation and by cross-linking with dimethyl suberimidate followed by analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate. It is proposed that, in the fast phase of inactivation, 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate reacts at the coenzyme binding site of one subunit of dimeric isocitrate dehydrogenase, causing complete inactivation of the modified subunit and substantial inactivation of the other subunit. This new reagent is likely to have general applicability as an affinity label for other NADP+ binding enzymes.

Chemical modification of the bifunctional human serum pseudocholinesterase. Effect on the pseudocholinesterase and aryl acylamidase activities.

The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenylglyoxal and 2,4-pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N-Ethylmaleimide and p-hydroxy-mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase. The use of N-acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation. Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imipramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N-acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non-active-site tyrosine residues.

Removal of Ca current inactivation in dialysed guinea-pig atrial cardioballs by Ca chelators

Ca currents flowing during voltage clamp depolarizations were studied in cultured guinea-pig atrial cardioballs by means of single low resistance patch clamp pipettes. The pipettes were filled with solutions containing Cs+ as major cation in order to block K+ currents and high concentrations of various Ca chelating agents (EGTA, nitrilotriacetic acid, citrate, dipicolinic acid) to prevent rises of the intracellular Ca-activity by Ca-entry. Ca currents of myocytes loaded with 20 mM of either EGTA [(ethylenedioxy)-diethylenedinitrilo)tetra-acetic acid] or NTA (nitrilotriacetic acid) display a biphasic time course of inactivation at membrane potentials between -25 and +45 mV. The fast phase is reduced with increasingly positive membrane potentials. In cells loaded with either citrate or DPA (dipicolinic acid, pyridine-2,6-dicarboxylic acid) inactivation is negligible or absent for small depolarizations. In the range of membrane potentials where maximum current flows (0-+10 mV) a monophasic slow time course of inactivation is observed. At more positive membrane potentials inactivation is slowed. The amount of inactivation under this condition is related to the current density of the cell. Conditions, which for a given membrane potential reduce the amplitude of ICa such as extracellular application of blocking ions (Co2+, Cd2+), a conditioning depolarization, or 'rundown' of Ca-channels lead to a slowing or a complete removal of inactivation in cells dialysed with citrate or DPA respectively. Cells loaded with these Ca chelators did not show any symptom of voltage dependent inactivation of ICa. Under the conditions described action potentials were recorded in the current clamp mode. Upon dialysis with EGTA the typical 'triangular shaped' atrial action potential develops a plateau of 500 to 800 ms in duration. With citrate-containing pipette solutions the action potential duration usually is several seconds. The results for the first time demonstrate that inactivation of cardiac ICa can be considerably slowed or even removed. They provide further strong support for the hypothesis that inactivation of this current depends on Ca entry rather than membrane potential. The fast phase of inactivation observed with EGTA (NTA) possibly reflects the slow kinetics of the binding reaction of this type of Ca chelators.

Evidence for two transient sodium currents in the frog node of Ranvier.

Na current (INa) was monitored in isolated voltage-clamped frog nodes of Ranvier in order to analyse the pharmacological and kinetic properties of fast and slow phases of inactivation. Niflumic acid (0.1-10 mM) and tetrodotoxin (0.3-30 nM) did not alter fast and slow inactivation time courses but preferentially reduced the amplitude of the fast phase of inactivation. The block of both phases of inactivation by niflumic acid and tetrodotoxin was well described if one assumed that more than one molecule of drug reacted with one channel. Fast and slow currents, corresponding respectively to fast and slow phases of inactivation, reversed at different potentials, had different threshold voltages of activation and the slopes of their steady-state inactivation curves were different. The recovery from inactivation of the compound INa could be described by the sum of two exponentials (plus a delay) corresponding respectively to fast and slow currents. When calculated from INa recorded without and with niflumic acid or tetrodotoxin, the slow current activated about three times more slowly than the fast current. Large prehyperpolarizations delayed both the activation and the inactivation of the fast current but only the activation of the slow current. Lowering the temperature decreased the fast current but increased the slow current. We conclude that the inactivatable Na current of the nodal membrane is made up of two components (INa,f and INa,s) corresponding to two different and interconvertible forms of the Na channel.

The calcium current in a myenteric neurone of the guinea-pig ileum.

The active and passive electrical properties of the after-hyperpolarizing (AH) cell of the guinea-pig myenteric plexus were analysed using a single-electrode voltage or current clamp. Action potentials were compared under normal conditions, in the presence of tetrodotoxin (TTX) and in the presence of both TTX and tetraethylammonium chloride (TEA). Calcium action potentials were studied by examining their calcium dependence, the actions of manganese and the effect of substituting barium for calcium. The maximum rate of rise of the action potential did not increase in calcium concentrations above 10 mM. The half-saturation concentration was 2 mM-calcium. AH cells exhibited five predominant currents consisting of an inward sodium current, an inward calcium current and three outward currents. There was a transient outward current which was inactivated at holding potentials more positive than -65 mV and was suppressed by 4-aminopyridine and barium but not by external TEA. A second outward current observed in the presence of 10 mM-external TEA had properties consistent with that of the delayed rectifier (Hodgkin & Huxley, 1952). A third outward current was the calcium-dependent slow after-hyperpolarizing current (Hirst, Johnson & van Helden, 1985). The voltage dependence, the action of calcium antagonists, the effect of barium substitution and the temporal characteristics of calcium currents were studied. The peak calcium current density was in excess of 100 microA/cm2 in 2.5 mM-calcium solution at 35 degrees C for depolarizations to -10 mV. Calcium currents showed at least two phases of inactivation. Both calcium and barium currents showed early inactivation with decay occurring over the first 10-40 ms. The calcium-activated current precluded direct measurement of slow inactivation of the calcium current. Barium currents studied over the first 100-150 ms had a very slow inactivating component.

Reaction of 5′- p-fluorosulfonylbenzoyl-1, N6-ethenoadenosine with histidine and cysteine residues in the active site of rabbit muscle pyruvate kinase

The inactivation of rabbit muscle pyruvate kinase by 0.3 mM 5′- p-fluorosulfonylbenzoyl-1, N 6-ethenoadenosine at pH 7.8 is biphasic. The first phase proceeds rapidly to yield a partially active enzyme (46% residual activity) followed by a slower rate which leads to total inactivation. The inactivation of the first phase can be reversed by addition of 20 mM dithiothreitol, whereas the second phase is unaffected. These two phases have second-order rate constants of 250 M −1·min −1 (dithiothreitol-sensitive reaction) and 52 M −1·min −1 (dithiothreitol-insensitive reaction), respectively. Marked protection against inactivation is afforded by phospho enolpyruvate and by metal-nucleotide complexes in the presence of free metal, indicating that reaction occurs in the region of the active site. Loss of approximately two sulfhydryls per enzyme subunit correlates well with the dithiothreitol-sensitive inactivation, suggesting that this phase of the inactivation may be attributable to disulfide formation. Incorporation of about one mole of fluorescent reagent per enzyme subunit correlates closely with the dithiothreitol-insensitive phase of inactivation, yielding a modified histidine residue. The quantum yield of the fluorescent sulfonylbenzoyl-1, N 6-ethenoadenosine-pyruvate kinase is only 0.007, as compared to 0.54 for the parent nucleoside 1, N 6-ethenoadenosine. The quenched fluorescence is consistent with stacking of the sulfonylbenzoyl moiety on the purine ring in the modified enzyme, which suggests that the altered histidine may be located in the adenine region of the metal-nucleotide binding site.

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)

Slow inactivation of Vmax in guinea pig ventricular myocardium.

Measurements of maximum upstroke velocity (Vmax) of guinea pig ventricular action potentials were used to investigate the effect of prolonged depolarization on the inactivation and recovery kinetics of cardiac sodium channels. Membrane potential before stimulated upstrokes was controlled by passing current across a sucrose gap. Two phases of inactivation ("slow" and "ultra-slow") having kinetics and voltage dependence different from the commonly observed fast inactivation process were observed. Ultra-slow inactivation developed exponentially with a time constant of several minutes between -60 and -20 mV. In contrast, slow inactivation developed with a time constant of 1-6 s between -60 and 40 mV. Under steady-state conditions slow and ultra-slow inactivations were virtually absent at -85 mV, while 50% of Vmax underwent slow inactivation at approximately 10 mV and 50% underwent ultra-slow inactivation at approximately -40 mV. Recovery from slow inactivation occurred exponentially with a time constant of about 2 s at -70 to -85 mV and 0.7 s at -100 mV. Recovery from ultra-slow inactivation was not completely characterized but was complete within 20 s at -85 mV. No significant effect of external [K+] (1-10 mM) on slow inactivation was found. The results suggest the existence of two additional inactivated states of the cardiac sodium channel distinctly different from the fast inactivated state.

Kinetics of calcium-dependent inactivation of calcium current in voltage-clamped neurones of Aplysia californica.

Ca currents flowing during voltage-clamp depolarizations were examined in axotomized Aplysia neurones under conditions that virtually eliminated other currents. Moderate to large currents exhibited a two-component time course of relaxation that can be approximated reasonably well by the sum of two exponentials. The rapid phase (tau 1 approximately equal to 70 ms at 0 mV) plus the slower phase (tau 2 approximately equal to 300 ms at 0 mV) ride upon a steady, non-inactivating current, I infinity. Conditions that diminish the peak current amplitude, such as reduced stimulus depolarization, inactivation remaining from a prior depolarization, or partial blockade of the Ca conductance by Cd, slowed both phases of inactivation, and all selectively eliminated the tau 1 phase, such that weak currents exhibited only the slower phase of decline. Injection of EGTA slowed both phases of inactivation, decreased the extent of the tau 1 phase, and increased the intensity of I infinity and of the current during the tau 2 phase. For a given voltage, the rate of inactivation increased as the peak current strength was increased, and decreased as the peak current strength was decreased. For a given peak current the rate of inactivation decreased as depolarization was increased. The relation of inactivation to prior Ca2+ entry was essentially linear for small currents, but decreased in slope with time during strong currents. The relation also became shallower with increasing depolarization, suggesting an apparent decrease in the efficacy of Ca in causing inactivation at more positive potentials. The basic kinetics of Ca current inactivation along with experimentally induced changes in those kinetics were simulated with a binding-site model in which inactivation develops during current flow as a function of the entry and accumulation of free Ca2+. This demonstrated that a single Ca-mediated process can account for the two-component time course of inactivation, and that the nearly bi-exponential shape need not arise from two separate processes. The two-component time course emerges as a consequence of a postulated hyperbolic reaction between diminishing probability of channels remaining open and the accumulation of intracellular free Ca2+. The occurrence of a single- or a two-component time course of inactivation thus appears to depend on the levels of internal free Ca2+ traversed during current flow.

Inactivation of calcium conductance characterized by tail current measurements in neurones of Aplysia californica.

Calcium tail currents, recorded at -40 mV after repolarization from 7 or 10 ms voltage-clamp depolarizations in axotomized Aplysia neurones in the presence of tetrodotoxin and tetraethylammonium, were used to investigate the inactivation of the calcium conductance without interference from contaminating potassium currents. Prior depolarization with a prepulse (V1) resulted in a reduction in size of the tail currents recorded following the test pulse (V2). The reduction occurred in both the fast (tau 1 less than 0.4 ms) and slow (tau 2 approximately equal to 2.0 ms) components of the tail current. The degree of inactivation remained constant when tail currents were measured at potentials ranging up to 30 mV on either side of the potassium equilibrium potential. Thus, any changes in potassium current must have contributed virtually nothing to the changes in tail current amplitude seen following presentation of the prepulse. Inactivation was greatest following prepulses to potentials (+10 to +40 mV) that produce maximal entry of calcium ions, and declined to about zero as the prepulse approached the calcium equilibrium potential. For V1 potentials above +50 mV, the prepulse caused an apparent short-term facilitation of V2 tail currents in EGTA-injected neurones. This effect, detected up to 50 ms following the pulse, is of uncertain origin. Pressure injection of calcium ions caused reduction in the size of the tail current, which was restored by subsequent injection of EGTA. Tail current amplitude was reduced by presentation of the prepulse for all test pulse voltages, but the measured inactivation declined exponentially towards a minimum with test pulses of increasingly positive potential. Removal of inactivation following a 200 ms prepulse was greatly accelerated by injection of EGTA. The EGTA-resistant inactivation remaining at short times decayed with a time constant of about 0.12 s. The relation of tail current reduction to prepulse voltage is consistent with the interpretation that the EGTA-resistant inactivation remaining at short times depends on entry of calcium ions during the prepulse, as does the EGTA-sensitive inactivation remaining at later times. It is proposed that the 'EGTA-resistant' phase of inactivation results from loading of EGTA with calcium ions near the inner surface of the membrane during sustained calcium entry, allowing the intracellular calcium concentration to rise. The results provide further evidence for a calcium-mediated inactivation of the calcium conductance.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in nucleolar structure, number and size in cellular activation and inactivation. Observations in human phytohaemagglutinin-treated lymphocytes.

Lymphocytes in human peripheral blood are small, relatively inactive cells. The addition of phytohaemagglutinin (PHA) to cultures of these cells causes a marked increase in cellular and nuclear size, DNA-synthesis and metabolic activity, which reaches a maximum three days after the onset of culturing. The cells then undergo an inactivation process over a period of approximately ten days by which time they have reverted to cells resembling small, inactive lymphocytes. Within the first three days, nucleoli increase in size and number, changing from ring-shaped to nucleolonema-exhibiting to compact nucleoli. In the course of the inactivation process the nucleoli decrease in size and change from compact nucleoli directly into ring-shaped nucleoli. Thus activation and inactivation pathways are different. There is an increase in the number of nucleoli during the inactivation phase up to the seventh day in culture, followed by a slight decrease until day 14. This suggests that nucleoli in metabolically active cells have a tendency to fuse, whereas those in inactive cells tend to fragment.

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.

An exploration of the primary specificity site of cathepsin B

Peptidyl diazomethyl ketones inactivate cathepsin B apparently by alkylation of the active center thiol following complex formation as in the case of benzyloxycarbonyl (Cbz)PheAlaCHN 2. The phenylalanine contributes considerably to binding in the secondary specificity site. In order to define the topography of the active center region comprising the primary specificity site of beef spleen cathepsin B, a series of peptidyl diazomethyl ketones having the general structure CbzPheXCHN 2 has now been synthesized. The amino acid, X, has been varied in size to include rather large side chains which might reveal available binding potential or limitations. Some of the reagents, in fact, were not inhibitory even at 10 −4 m. Others, however, that did measurably inactivate cathepsin B provided a range of reactivities that extended over 5 orders of magnitude and correlated with affinity in the reversible phase of inactivation. Some large side chains, for example, that of tryptophan, were very poorly tolerated in this region of the active center, whereas others, such as O-benzyl threonine, provided remarkably active inhibitors. A topographical rationalization of the results is offered.