Deactivation Time Research Articles

Direct activation of GABAA receptors by barbiturates in cultured rat hippocampal neurons.

1. The direct activation of the GABAA receptor by pentobarbitone (PB) and phenobarbitone (PHB) was characterized in cultured rat hippocampal neurons using whole-cell voltage clamp and single channel recording techniques. 2. In whole-cell recordings, PB and PHB produced a concentration-dependent activation of Cl- current (EC50 values, 0.33 and 3.0 mM, respectively). The response to the barbiturates was similar to that produced by GABA, although GABA was more potent (EC50, 5.5 microM). PB and PHB were substantially more potent in enhancing the response to 1 microM GABA (EC50 values, 94 microM and 0.89 mM, respectively). The maximal magnitude of the responses to PB was similar to that of the maximal response to GABA or GABA + PB. PHB appeared to be modestly less efficacious. 3. The mean deactivation time constant for whole-cell Cl- currents evoked by 1 mM PB + 1 microM GABA was significantly longer (480 +/- 34 ms) than for 1 mM PB (170 +/- 9 ms) or 1 microM GABA (180 +/- 14 ms) alone. 4. Whole-cell currents directly activated by 300 microM PB and 1 microM GABA were blocked by the GABA receptor antagonists bicuculline and picrotoxin. 5. Unitary GABAA receptor channel currents evoked by 300 microM PB had similar main conductance, mean open time and mean burst duration as those activated by 2 microM GABA alone. Single channel openings and bursts were of shorter mean duration when 100 and 300 microM PHB were used. 6. High concentrations of PB (1-3 mM) and PHB (3-10 mM) produced a rapid block of currents activated by the barbiturate alone or by the barbiturate in the presence of 1 microM GABA. The estimated IC50 values for block of PB- and PHB-potentiated GABA currents were 2.8 and 12.9 mM, respectively. 7. Single channel currents activated by high concentrations of PB and PHB alone or in the presence of GABA demonstrated flickering, probably reflecting fast channel block. 8. We conclude that the gating of the GABAA receptor channel by PHB and PB is functionally similar to that produced by the natural agonist GABA alone, but distinct from that obtained when barbiturates modulate the response to GABA. At high concentrations, the barbiturates produce a channel blocking action that limits the maximum total current conducted by the channel.

Calcium permeability increase of endplate channels in rat muscle during postnatal development.

1. Patches of endplate membrane were isolated from rat flexor digitorum brevis muscle at different postnatal stages to measure the time course of development changes in conductance, deactivation time constant and relative Ca2+ permeability of endplate channels. 2. The predominant channel conductance was 40 +/- 1 pS (n = 9) at postnatal day 9 (P9) or younger whereas it was 59 +/- 3 pS (n = 5) at P21 or in older muscle. The deactivation time constant of ensemble patch currents evoked by brief ACh application, decreased from 8 +/- 3 ms (n = 45) at P5-9 to 2.3 +/- 0.3 ms (n = 5) in P21-28 muscle. 3. The relative Ca2+ permeability, measured by the shift of biionic (Ca2+/Cs+) reversal potential of ensemble patch currents upon the replacement of high [Cs+] by high [Ca2+] extracellular solution and with Cs+ as internal reference ion, increased during postnatal development. THe biionic reversal potential shift changed from -21 +/- 1 mV (n = 8) at P5 to -8 +/- 1 mV (n = 10) in P15 or older muscle. 4. Recombinant gamma-AChR channels expressed in Xenopus laevis oocytes had a biionic (Ca2+/Cs+) reversal potential shift of -24.9 +/- 2 mV (n = 14) comparable to that of neonatal endplate channels whereas the reversal potential shift for recombinant epsilon-AChR channels was -7.6 +/- 0.9 mV (n = 13), comparable to that of endplate channels in adult muscle. 5. It is concluded that an approximately 3-fold increase in Ca2+ current through endplate channels during postnatal development is caused by replacement of the fetal gamma-subunit by the epsilon-subunit in juvenile and adult muscle.

Interaction of Extracellular Potassium and Cesium with the Kinetics of Inward Rectifying K+ Channels in the Plasma Membrane of Mesophyll Protoplasts of Avena sativa

Using the patch-clamp technique the kinetics of whole-cell and single channel inwardly rectifying K+ currents were measured in enzymatically -isolated protoplasts from Avena sativa mesophyll leaf cells. The hyperpolarization-activated whole-cell current had an initial K+ component (I|d) and a time-dependent K+ component which reaches steady state (IKss) within 500 ms. After an initial delay, the activation of I Kss and the deactivation of the tail K+ current (IKT) followed an exponential time course. The time-constants of activation (ra), at 10 mM and 50 mM [K+]o, and of deactivation (rd) could be described by exponential and sigmoidal dependence on membrane voltage (Vm), respectively. The relative number of the activated K+ channel population increased sigmoidally as a function of hyperpolarized Vm. On the other hand, the relative number of the deactivated K+ channel population decreased exponentially as a function of hyperpolarize d V m. The kinetics of the inward rectifying K+ current were dependent on V m but not on extracellular K+ concentration, [K+]o. The presence of Cs+ in the bathing medium reduced r, at voltage steps less negative than —125 mV and increased r, for voltage steps between —150 mV and — 200 mV. By comparison, the dependence of r d on V m was not altered significantly by changing [Cs+]o. Cs+ reversibly blocked the voltage-dependent sigmoidal rise of K+ current activation and the voltage-dependent exponential decrease of current deactivation. The Cs+-induced block of K+ current was both voltage and concentration dependent. Single channel current (IK), the probability of the channel being open (Po) and the mean open-time (ro) increased as a function of hyperpolarize d potentials. The mean closed-time (rc) and the mean lag time before the channel opened were exponentially dependent on voltage and they became shorter as the membrane was hyperpolarized. The kinetics of single K+ channels i.e. activation, deactivation and lag time, and the absence of outward single K+ channel currents were consistent with whole-cell current measurements. The inward rectification of single channel currents and simulated cell currents suggest that this current rectification is an intrinsic nature of the inward rectifying K+

Class III antiarrhythmic effects of zatebradine. Time-, state-, use-, and voltage-dependent block of hKv1.5 channels.

Zatebradine is a bradycardic agent that inhibits the hyperpolarization-activated current (I(f)) in the rabbit sinoatrial node. It also prolongs action potential duration in papillary muscles in guinea pigs and in Purkinje fibers in rabbits. The underlying mechanism by which zatebradine induces this effect has not been explored, but it is likely to involve K+ channel block. Cloned human cardiac K+ delayed rectifer currents (hKv1.5) were recorded in Ltk- cells transfected with their coding sequence. Zatebradine 10 mumol/L did not modify the initial activation time course of the current but induced a subsequent decline to a lower steady-state current level with a time constant of 109 +/- 16 ms. Zatebradine inhibited hKv1.5 with an apparent KD of 1.86 +/- 0.14 mumol/L. Block was voltage dependent (electrical distance delta = 0.177 +/- 0.003) and accumulated in a use-dependent manner during 0.5- and 1-Hz pulse trains because of slower recovery kinetics in the presence of the drug. Zatebradine reduced the tail current amplitude, recorded at -30 mV, and slowed the deactivation time course, which resulted in a "crossover" phenomenon when control and zatebradine tail currents were superimposed. These results indicate that (1) zatebradine is an open-channel blocker of hKv 1.5, (2) binding occurs in the internal mouth of the ion pore, (3) unbinding is required before the channel can close, and (4) zatebradine-induced block is use dependent because of slower recovery kinetics in the presence of the drug. These effects may explain the prolongation of the cardiac action potential and could be clinically relevant.

Two distinct low-voltage-activated Ca2+ currents contribute to the pacemaker mechanism in cockroach dorsal unpaired median neurons.

1. The contribution of Ca2+ currents to the endogenous firing properties of cockroach isolated adult dorsal unpaired median neurons was investigated using whole cell patch-clamp technique with 5 mM Ca2+ as the charge carrier. At least three types of Ca2+ currents, a high-voltage-activated Ca2+ current and two low-voltage-activated (LVA) Ca2+ currents, have been found in these neurons. This study focused on the LVA Ca2+ currents, which are suitable candidates in the generation of the slow predepolarization because of their low threshold of activation. 2. The global LVA Ca2+ current could be dissociated by means of nickel sensitivity, deactivation time constant and voltage dependence of time to peak, tail current amplitude and inactivation, as transient and maintained LVA Ca2+ currents. 3. The transient LVA Ca2+ current, sensitive to 100 microM Ni2+, was isolated by using a subtraction procedure. It was activated at -70 mV and half-inactivated at -59.5 mV. The inactivation was purely voltage dependent. Current-clamp experiments performed with 150 microM Ni2+ indicated that this current was involved in the initial part of the predepolarization. 4. The maintained LVA Ca2+ current, resistant to 100 microM Ni2+, was activated in a range of potential 10 mV more positive than the transient LVA Ca2+ current, and its voltage dependence of inactivation displayed a U-shaped-curve. 5. Replacing Ca2+ with Ba2+ in equimolar amount or low internal Ca2+ concentration [5 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) in the pipette] induced a monotonic voltage dependence of inactivation and increased the rate of relaxation of this current. These effects were mimicked by high internal Ca2+ concentration [0.1 mM Ca2+ and no ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the pipette]. This demonstrated an unusual Ca2+-sensitive inactivation process that varied over a narrow range of Ca2+ concentrations. 6. Current-clamp experiments performed under 150 microM Ni2+, with 15 mM external Ca2+ concentration (which potentiated the maintained LVA current within 30 s of superfusion) or with 5 mM BAPTA in the pipette demonstrated the participation of this current in the last two-thirds of the slow predepolarizing phase. 7. Our findings demonstrated, for the first time in neurosecretory cells, the coexistence of two distinct LVA Ca2+ currents, which have specialized function in the generation of the pacemaker activity.

Different properties of the inward rectifying potassium conductance of aleurone protoplasts from dormant and non-dormant barley grains

Isogenic dormant and non-dormant barley grains provide a useful system to study the molecular mechanisms of grain dormancy and the role of plant hormones in this process. As ion fluxes are associated with dormancy-related plant hormone responses, we compared the properties of the inward rectifying potassium conductance in aleurone protoplasts isolated from dormant and non-dormant Triumph grains and in germinating Himalaya grains. Maximal conductance, voltage dependency of steady-state activation, activation and deactivation kinetics were studied in the whole-cell patch-clamp configuration. Activation and deactivation time courses were single exponential. No differences in the above described properties were found between the protoplasts isolated from non-dormant Triumph and Himalaya grains. However, the maximal conductance (corrected for cell size) in protoplasts from dormant Triumph grains was much smaller (65%), and activation time constants were much larger as compared to protoplasts from non-dormant grains. No differences were found in the deactivation kinetics in the three different types of protoplasts. The half-maximal activation potential was slightly more negative in protoplasts from dormant grains than from non-dormant grains.

Voltage-Dependence of Extracellular Ca2+-Induced Modification in Properties of the Inward Rectifying K+ Channels in the Plasma Membrane of Mesophyll Protoplasts of Avena sativa

Data obtained using the whole-celi configuration of the patch-clamp technique reveal that characteristics of the inward rectifying K+ current across the plasma membrane of protoplasts isolated from mesophyll cells of leaves of oat (Avena sativa) are modified by increasing concentrations or removing the extracellular Ca2+. The whole-cell membrane current reveals two components. The first component an initial current II* which is the sum of two currents: (a) a linear ohmic leak current passing through non-gated channels, liNGC, and (b) a rectifying inward K+ current passing through inward rectifying gated K+ channels, IKi, that are instantaneously open. The second component of the membrane current at the steady state Iss is a time-dependent K+ current IKss defined as Iss-IiNGC and passes through inward rectifying gated K+ channels. The tail K+ current, IKT, is also defined as IT-IiNGC. Raising external calcium concentration, [Ca2+]o, from 0.1 mM to 10 mM blocked the inward rectifying currents IKi, IKss and IKT. The voltage-dependence of the activation time constant (τa) for time-dependent KC current IKss was not altered significantly by increasing [Ca2+]o whereas the deactivation time constant (τd) of the IKT increased from 16 ms to 30 ms at a Vm of -100 mV. Removal of [Ca2+]o increased the amplitude and altered the characteristics of the inward rectifying K+ current. Ten minutes after the removal of [Ca2+]o the increase in IKi was 3.5-fold larger than the increase in IKss. Furthermore, removing [Ca2+]o hastened the activation of IKss and the deactivation of IKT. However, the deactivation time constant (Td) remained dependent on membrane voltage (Vm). Extracellular Ca2+ may modulate the function of mesophyll cells by regulating K+ transport through the inward rectifying K+ channels and this may have significant implications for photosynthesis and cell expansion.

Ca2+ activation and Ca2+ inactivation of canine reconstituted cardiac sarcoplasmic reticulum Ca(2+)-release channels.

1. Calcium-release channels (ryanodine receptors) of canine cardiac sarcoplasmic reticulum (SR) were incorporated into lipid bilayer membranes at the tip of a patch pipette. Using symmetrical 150 mM KCl solutions, [Ca2+] > 0.3 microM activated single channels of 627 pS conductance. The kinetics of Ca(2+)-mediated channel activation, deactivation and inactivation were studied by stepwise changes in pCa (-log[Ca2+]) and analysis of current means. 2. Steps of [Ca2+] activated the channel open probability (Po) along a time course which could be fitted by a single exponential. The activation time constant was dependent on [Ca2+], which decreased from 4.9 ms at pCa 6.5 to 0.2 ms at pCa 3. Subsequent rapid reduction in [Ca2+] decreased Po along a mono-exponential deactivation time course, the time constant of which was independent of the [Ca2+] during the preceding activation period. Further analysis yielded the rate constants kon of 2 x 10(8) (M s)-1 and koff of 2 x 10(2) s-1, an apparent dissociation constant (KD) of 1 microM, and a Hill coefficient of 1.05. 3. The open probability increased with [Ca2+], reaching a peak at about pCa 5.5. At pCa < or = 5.5, Po decreased time dependently, the time constants decreasing along with [Ca2+] from 1 s at 3 microM to 0.2 s at 1 mM. During the 0.5 s period at 3 microM Ca2+, Po fell by 13% due to an extension of the closed times. At 1 mM Ca2+, Po 'inactivated' by 72%, which was due mostly to long closures. These differences suggest that the Ca(2+)-mediated decay of Po was dependent on Ca2+ binding to an intermediate (KD, 3 microM) and a low affinity site (KD, 360 microM). On the return of pCa from 3 to > 8, the channels briefly re-opened. 4. A 'refractory' behaviour of the channel was not observed for 20 ms steps between < 10 nM and < 10 microM [Ca2+] (25 Hz). For steps between 10 nM and 1 mM, however, such behaviour was marked by infrequent and irregular channel openings. 5. The results are described by a three Ca2+ binding site model and compared with the literature.

Interaction of Ca2+ agonist and depolarization on Ca2+ channel current in guinea pig detrusor cells.

The interaction of large depolarization and dihydropyridine Ca2+ agonists, both of which are known to enhance L-type Ca2+ channel current, was examined using a conventional whole-cell clamp technique. In guinea pig detrusor cells, only L-type Ca2+ channels occur. A second open state (long open state: O2) of the Ca2+ channels develops during large depolarization (at +80 mV, without Ca2+ agonists). This was judged from lack of inactivation of the Ca2+ channel current during the large depolarizing steps (5 s) and slowly deactivating inward tail currents (= 10-15 ms) upon repolarization of the cell membrane to the holding potential (-60 mV). Application of Bay K 8644 (in 2.4 mM Ca(2+)-containing solutions) increased the amplitude of the Ca2+ currents evoked by simple depolarizations, and made it possible to observe inward tail currents (= 2.5-5 ms at -60 mV). The open state induced by large depolarization (O2*) in the Bay K 8644 also seemed hardly to inactivate. After preconditioning with large depolarizing steps, the decay time course of the inward tail currents upon repolarization to the holding potential (-60 mV) was significantly slowed, and could be fitted reasonably with two exponentials. The fast and slow time constants were 10 and 45 ms, respectively, after 2 s preconditioning depolarizations. Qualitatively the same results were obtained using Ba2+ as a charge carrier. Although the amplitudes of the inward currents observed in the test step and the subsequent repolarization to the holding potential were decreased in the same manner by additional application of nifedipine (in the presence of Bay K 8644), the very slow deactivation time course of the tail current was little changed. The additive enhancement by large depolarization and Ca2+ agonists of the inward tail current implies that two mechanisms separately induce long opening of the Ca2+ channels: i.e., that there are four open states.

Different effects on gating of three myotonia-causing mutations in the inactivation gate of the human muscle sodium channel.

1. Three mutations at the same site in the inactivation gate of the alpha-subunit of the human muscle Na+ channel, G1306E, G1306V and G1306A, cause three phenotypes of K(+)-aggravated myotonia: G1306E as the most severe and G1306A as the most benign form. 2. Recombinant wildtype (WT) and mutant (G1306E, G1306V and G1306A) human Na+ channels were expressed in human embryonic kidney cells (HEK293). G1306E and G1306V channels showed a distinct increase in the time constants of inactivation (tau h1 and tau h2) and in the ratios of steady-state to peak currents (Iss/Ipeak) (e.g. at 0 mV, G1306E vs. WT; tau h1, 1.29 +/- 0.10 vs. 0.52 +/- 0.01 ms; Iss/Ipeak, 2.90 +/- 0.40 vs. 0.93 +/- 0.19%). G1306A channels showed only an increase in tau h1 (0.74 +/- 0.07 ms). For G1306E and G1306V channels, the steady-state inactivation curves, as well as the voltage dependence of the rate of recovery from inactivation, were shifted by +15 mV. For G1306A the h infinity curve was shifted by only +5 mV. 3. G1306E and G1306V channels showed prolonged current rise times and later first openings suggesting slowing of activation. For G1306E channels only, the steady-state activation curve was shifted by -7 mV. For all mutants the deactivation time constants were increased. 4. We conclude that (i) the combination of alterations in inactivation and activation produces the slowing of the current decay, (ii) the slowed inactivation is most responsible for myotonia, and (iii) the shift of the steady-state activation curve, seen only with G1306E channels, may explain the severity of this phenotype. 5. The results suggest that two of the mutations in the Na+ channel inactivation gate also alter channel activation and deactivation.

A rapidly activating delayed rectifier K+ channel in rabbit sinoatrial node cells

The single-channel current of the delayed rectifier K+ current (IK) was recorded in rabbit sinoatrial node cells. In the cell-attached patch, depolarization from -70 mV to potentials more positive than -50 mV activated the IK channel while repolarization deactivated it. The single-channel conductance was 7.8 pS for the outward current and 10.8 pS for the inward current (n = 6). The steady-state open probability (NPo) was maximum at around -30 mV and markedly decreased at more positive potentials. On repolarization from positive potentials, the channel was initially closed and then rapidly opened. The ensemble average showed an initial rise to a peak followed by the deactivation time course. Because the channel events were completely blocked by E-4031, the drug-sensitive component was examined in the whole cell current. The steady-state current-voltage relation of the drug-sensitive current showed a marked negative slope at potentials more positive than -10 mV. Upon repolarization, the drug-sensitive current initially increased (removal of inactivation) to the peak of the outward tail current, which was in agreement with the ensemble average of the single-channel current. We conclude that IK in the sinoatrial node cells is largely composed of the rapidly activating IK (IK,r) channels and that the inward rectification of IK,r, which is more marked than had been assumed in previous studies, is due to the decrease in NPo.

Cd2+ regulation of the hyperpolarization-activated current IAB in crayfish muscle.

The effects of Cd2+ on the hyperpolarization-activated K(+)-mediated current called IAB (Araque, A., and W. Buño. 1994. Journal of Neuroscience. 14:399-408.) were studied under two-electrode voltage-clamp in opener muscle fibers of the crayfish Procambarus clarkii. IAB was reversibly reduced by extracellular Cd2+ in a concentration-dependent manner, obeying the Hill equation with IC50 = 0.452 +/- 0.045 mM and a Hill coefficient of 1 (determined from the maximal chord conductance of IAB). Cd2+ decreased the IAB conductance (GAB) and shifted its voltage dependence towards hyperpolarized potentials in a similar degree, without affecting the slope of the voltage dependence. The IAB activation time constant increased, whereas the IAB deactivation time constant was not modified by Cd2+. The IAB equilibrium potential (EAB) was unmodified by Cd2+, indicating that the selective permeability of IAB channels was not altered. IAB was unaffected by intracellular Cd2+. The Cd(2+)-regulation of IAB did not depend on [K+]o, and the effects of [K+]o on IAB were unchanged by Cd2+, indicating that Cd2+ did not compete with K+. Therefore, Cd2+ probably bound to a different site to that involved in the K+ permeability pathway. We conclude that Cd2+ affected the gating of IAB channels, interfering with their opening but not with their closing mechanism. The results can be explained by a kinetic model in which the binding of Cd2+ to the IAB channels would stabilize the gating apparatus at its resting position, increasing the energy barrier for the transition from the closed to the open channel states.

Chloride currents across the membrane of mammalian skeletal muscle fibres.

1. Chloride currents through the membrane of rat psoas muscle fibre segments were investigated with a double Vaseline gap under conditions minimizing the currents of other ion species. 2. In Cl(-)-free solutions a time- and voltage-independent conductance of 1.1 +/- 0.4 microS was observed. 3. As with intact fibres, the steady-state Cl- conductance was 2.5 +/- 0.9 mS cm-2; the halide selectivity was Cl- > Br- > I-, and Cl- currents were completely blocked by 0.1 mM 9-anthracene carboxylic acid (9-AC). 4. Voltage steps from -85 mV to between -125 and +55 mV elicited currents with deactivation upon hyperpolarization and activation upon depolarization. Activation was fitted with two exponentials, the smaller time constant increasing from 37.5 ms at +55 mV to 67.0 ms at -5 mV, the larger time constant (450 ms) being independent of potentials more positive than -5 mV. The two deactivation time constants ranged between 30.6 (-105 mV) and 99.3 ms (-35 mV), and 139.4 (-105 mV) and 738.5 ms (-35 mV). 5. The activation curve was fitted with a Boltzmann distribution (half-maximum, -39 mV; slope at inflexion point, 1/17.2 mV). Deactivation was incomplete. At very negative potentials about one-quarter of the maximum number of channels were open. 6. When tested with 5 and 61 mM intracellular Cl- concentration ([Cl-]i) the kinetic parameters were not different. 7. During depolarizations lasting > 5 s, activation was followed by a decline. With progressively longer prepulses going positive to the reversal potential and test pulses going negative, the responses to test and prepulses decreased with similar time constants, suggesting a real inactivation process.

Effects of ajmaline on non-sodium ionic currents in guinea pig ventricular myocytes.

The lack of currently available data stimulated us to investigate the electrophysiological effects of ajmaline, a classical class Ia antiarrhythmic agent, on various currents responsible for the action potential plateau and repolarization phases. The whole cell patch clamp recording technique was applied to guinea pig ventricular myocytes. Ajmaline suppressed the Ca2+ current (Ica) in a dose-dependent manner (Kd = 1.2 x 10(-5) M) without affecting the steady-state inactivation kinetics and the voltage dependency of the current-voltage relationship. Ajmaline inhibited the inward portion of the inward rectifying K+ current (IKl). Ajmaline decreased the delayed rectifier K+ current (IK) without altering the activation or deactivation time courses. All these inhibitory effects of ajmaline prolonged the action potential duration in a dose dependent manner. The inhibitory actions of ajmaline on the action potential upstroke and various currents responsible for the plateau and repolarization may contribute to the observed suppression of depolarization-induced abnormal automaticities by this agent.

Modulation of the GTPase activity of transducin. Kinetic studies of reconstituted systems.

We seek to define the influence of retinal cGMP phosphodiesterase (PDE) on the GTPase activity of transducin (T). A novel stopped-flow/fast filtration apparatus [Antonny, B., et al. (1993) Biochemistry 32, 8646-8653] is used to deliver T alpha GTP free of rod outer segment (ROS) membranes to a suspension of phospholipid vesicles bearing holoPDE. As measured by a pH electrode, the decay of cGMP hydrolysis from these samples, which contain no other proteins but T alpha and holoPDE, requires GTP hydrolysis and occurs in 40 s. The addition of T beta gamma to the vesicles does not accelerate this deactivation. When ROS membranes are urea-stripped, reconstituted with transducin + holoPDE, and illuminated, the injection of an amount of GTP that is substoichiometric to holoPDE gives a cGMP hydrolysis pulse that lasts for 30 s. However, the same reconstitution performed with ROS stripped by extensive dilution in isotonic buffer results in a deactivation time of only 8 s, which resembles the 7 s observed with native ROSs. With these isotonically stripped ROSs, when GTP injection comes after a first injection with GTP gamma S, the cGMP hydrolysis pulse is lengthened and lasts for 17 s; with urea-washed ROS, no such lengthening is observed. These results clearly demonstrate that holoPDE by itself cannot enhance the GTPase activity of transducin, even when the two proteins are localized on a membrane surface. Instead, they point to the existence of a membrane-bound, urea-sensitive protein factor that activates the GTPase of T alpha in the transducin-holoPDE complex.

NMDA receptor channels: Subunit-specific potentiation by reducing agents

Sulfhydryl redox agents affect NMDA receptor activity. We investigated a putative redox site in four recombinant NMDA receptors. In 293 cells expressing NR1-NR2A channels dithiothreitol (DTT) rapidly potentiated l-glutamate-activated whole-cell currents and decreased the time course of desensitization and deactivation. Part of the current potentiation (reversible component) and all kinetic changes reversed upon washout. The remaining potentiation (persistent component) was abolished by an oxidizing agent. The N-terminal 370 residues of NR2A mediate the reversible component in chimeric N R2 subunits. I n cells expressing the NR1-NR2B, -NR2C, and -NR2D channels DTT elicited only a slowly developing, persistent potentiation and increased the deactivation time course. In these, but not in NR1-NR2A, the DTT effect was rendered insensitive to reoxidation by alkylation. Reduced glutathione mimicked the DTT effects only in the NR1-NR2A receptor. Hence, molecularly distinct NMDA receptors differ profoundly in their responses to sulfhydryl redox agents.

Selective block of delayed rectifying potassium current in the rabbit sinoatrial node by a novel class III antiarrhythmic agent MS-551

Electrophysiological actions of MS-551, a novel class III antiarrhythmic agent, were studied using small preparations (0.2 × 0.2 × 0.1mm) of the rabbit sinoatrial (SA) node. MS-551 (0.1–3 µM) exerted a negative chronotropic action by prolonging the action potential duration and diastolic interval. Automaticity was completely suppressed in 5 of 6 preparations at 1–3 µM. Voltage clamp experiments using double microelectrode techniques revealed that MS-551 (0.1–10µM) blocked the delayed rectifying K+ current (IK) in a concentration-dependent manner, and the block was almost saturated at >1 µM, attaining 60% ± 10% at 10 µM (n = 5). The MS-551-sensitive IK tail (Kd = 0.4µM, Hill r = 1.4,n = 5) had fast and slow components of deactivation. MS-551 (1 µM) reduced the amplitudes of control IK fast and IK slow from 20 ± 4 and 11 ± 4 nA to 8 ± 3 and 5 ± 3 nA, respectively (P < 0.01,n = 4). Although the fast deactivation time constant at −60mV remained unaltered (127 ± 12 vs 113 ± 13ms), the slow deactivation time constant was prolonged from 1,117 ± 130 to 1,555 ± 407ms by 1µM MS-551 (P < 0.05). This agent shifted the steady-state activation curve for IK from −21 ± 2 to −26 ± 4mV and increased the slope factor from 8 ± 1 to 9 ± 1mV (P < 0.05,n = 4). The fully-activated IK exhibited prominent inward rectification and was reduced by MS-551. These results suggest that (1) MS-551 prolongs the action potential duration and diastolic interval, and exerts a negative chronotropic action by blocking IK, (2) MS-551 has a higher affinity for the activated than the resting state K+ channel, and (3) this agent may either preferentially block one type of IK, or stabilize a single population of IK in a subconductance state in the rabbit SA node.

Electrochemical process recovers spent catalyst

A catalyst regeneration process designed to provide environmental and cost advantages over conventional spent catalyst reclamation processes has been developed by researchers at Battelle Pacific Northwest Laboratories, Richland, Wash. The process, based on electrochemical dissolution, recovers metal from the spent catalysts. Hydrocarbon deposits on the catalysts are also removed and are converted to carbon dioxide and water at moderate temperatures and atmospheric pressure. Development of the process was spurred by stricter environmental regulations and the tighter economics of catalyst regeneration. The trend in oil refining toward ever heavier feedstocks puts a correspondingly greater demand on catalyst performance. Heavier feedstocks typically have higher concentrations of impurities, which promote catalyst deactivation and decrease on-stream cycle time. Simultaneously, the economic encourage lower energy consumption and an increase in reaction selectivities to minimize production of waste.

Mechanism of Ba2+ block of M-like K channels of rod photoreceptors of tiger salamanders.

IKx is a voltage-dependent K+ current in the inner segment of rod photoreceptors that shows many similarities to M-current. The depression of IKx by external Ba2+ was studied with whole-cell voltage clamp. Ba2+ reduced the conductance and voltage sensitivity of IKx tail currents and shifted the voltage range over which they appeared to more positive potentials. These effects showed different sensitivities to Ba2+: conductance was the least sensitive (K0.5 = 7.6 mM), voltage dependence intermediate (K0.5 = 2.4 mM) and voltage sensitivity the most sensitive (K0.5 = 0.2 mM). Ca2+, Co2+, Mn2+, Sr2+, and Zn2+ did not have actions comparable to Ba2+ on the voltage dependence or the voltage sensitivity of IKx tail currents. In high K+ (100 mM), the voltage range of activation of IKx was shifted 20 mV negative, as was the tau-voltage relation. High K+ did not prevent the effect of Ba2+ on conductance, but abolished its ability to affect voltage dependence and voltage sensitivity. Ba2+ also altered the apparent time-course of activation and deactivation of IKx. Low Ba2+ (0.2 mM) slowed both deactivation and activation, with most effect on deactivation; at higher concentrations (1-25 mM), deactivation and activation time courses were equally affected, and at the highest concentrations, 5 and 25 mM Ba2+, the time course became faster than control. Rapid application of 5 mM Ba2+ suggested that the time dependent currents in Ba2+ reflect in part the slow voltage-dependent block and unblock of IKx channels by Ba2+. This blocking action of Ba2+ was steeply voltage-dependent with an apparent electrical distance of 1.07. Ba2+ appears to interact with IKx channels at multiple sites. A model which assumes that Ba2+ has a voltage-independent and a voltage-dependent blocking action on open or closed IKx channels reproduced many aspects of the data; the voltage-dependent component could account for both the Ba(2+)-induced shift in voltage dependence and reduction in voltage sensitivity of IKx tail currents.

Effects of metabolic blockers on Ca2+‐dependent currents in cultured sensory neurones from neonatal rats

1. The whole cell variant of the patch clamp technique was used to record high voltage-activated Ca2+ currents and Ca(2+)-activated Cl- tail currents from cultured neonatal rat dorsal root ganglion neurones. The aim of the project was to use these currents as physiological indices of intracellular Ca2+ regulation under control conditions and in the presence of metabolic inhibitors. 2. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (5 microM) and sodium cyanide (1 microM) inhibited Ca2+ currents within 20 s, even when ATP was present in the patch pipette solution, suggesting a direct action on Ca2+ channels. These metabolic inhibitors did not affect Ca2+ current 'run down' or inactivation kinetics. 3. Cultured neonatal dorsal root ganglion neurones of the rat were relatively insensitive to the removal of glucose and ATP from the recording solutions for up to 3 h. These data suggest that the Ca2+ homeostatic mechanisms in these cells are highly resistant to metabolic insult. 4. However 2-deoxy-D-glucose (5 mM) in the extracellular recording medium with no ATP or glucose present did prolong the deactivation time of Ca(2+)-activated Cl- tail currents and increase the total charge flow following activation of a 500 ms voltage-activated Ca2+ current. This effect was prevented by inclusion of D-fructose 1,6-diphosphate (500 microM) in the patch pipette solution. 5. We conclude that some agents used to induce chemical hypoxia, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone and sodium cyanide, may interact directly with voltage-activated Ca2+ channels and are therefore not appropriate for use in studying disturbed neuronal Ca2+ homeostasis. However, the use of 2-deoxy-D-glucose in the absence of glucose and ATP does represent a model of disturbed Ca2+ homeostasis in cultured dorsal root ganglion neurones. In this study we have combined the whole cell recording technique with cultured neurones under conditions which produce a degree of metabolic stress as reflected by prolonged Ca(2+)-activated Cl- tail currents. The reduced efficiency of handling of intracellular Ca2+ loads may be an important factor contributing to the onset of neuronal damage during hypoxia and ischaemia.