Kinetics of interaction of disopyramide with the cardiac sodium channel: Fast dissociation from open channels at normal rest potentials

Block of cardiac sodium channels is enhanced by repetitive depolarization. It is not clear whether the changes in drug binding result from a change in affinity that is dependent on voltage or on the actual state of the channel. This question was examined in rabbit ventricular myocytes by analyzing the kinetics of block of single sodium channel currents with normal gating kinetics or channels with inactivation and deactivation slowed by pyrethrin toxins. At −20 and −40 mV, disopyramide 100 μm blocked the unmodified channel. Mean open time decreased45 and34% at −20 and −40 mV during exposure to disopyramide. Exposure of cells to the pyrethrin toxins deltamethrin or fenvalrate caused at least a tenfold increase in mean open time, and prominent tail currents could be recorded at the normal resting potential. The association rate constant of disopyramide for the normal and modified channel at −20 mV was similar, ∼10×10⁶/m/sec. During exposure to disopyramide, changes in open and closed times and in open channel noise at −80 and −100 mV are consistent with fast block and unblocking events at these potentials. This contrasts with the slow unbinding of drug from resting channels at similar potentials. We conclude that the sodium channel state is a critical determinant of drug binding and unbinding kinetics.     

The reaction force and the transition region of a reaction

The reaction force F(R) and the position-dependent reaction force constant κF(R) are defined by F(R)=-∂V(R)/∂R and κ(R)=∂²V(R)/∂R², where V(R) is the potential energy of a reacting system along a coordinate R. The minima and maxima of F(R) provide a natural division of the process into several regions. Those in which F(R) is increasing are where the most dramatic changes in electronic properties take place, and where the system goes from activated reactants (at the force minimum) to activated products (at the force maximum). κ(R) is negative throughout such a region. We summarize evidence supporting the idea that a reaction should be viewed as going through a transition region rather than through a single point transition state. A similar conclusion has come out of transition state spectroscopy. We describe this region as a chemically-active, or electronically-intensive, stage of the reaction, while the ones that precede and follow it are structurally-intensive. Finally, we briefly address the time dependence of the reaction force and the reaction force constant.     

Effects of unstructured video exposure on EEG power in situations of forced attention and rest

Group 1 (N = 30) and Group 2 (N = 22) of healthy volunteers participated in the experiment. In both groups, the EEG was recorded in the state of rest with the eyes closed (REC); at rest with eyes open (REO); and during passively watching TV channel noises (white noise). EEG was also recorded in the state of forced visual attention: when counting colored symbols appearing on the screen (group 1) and when searching for an image of a real object in the noise (group 2). The averaged values of EEG power in each derivation were calculated for every subject and for each state in the Δ, θ, α₁, α₂, β₁, β₂, and γ frequency bands. The results demonstrate that the exposure to unstructured noninformative video noise may lead to significant changes in the EEG power in various frequency bands, with the most prominent changes taking place in the α₂ band. These changes are topically wide, reflecting systemic changes in the corresponding brain mechanisms, but are much less intense compared to the difference between the states of rest with the eyes open and closed.     

Relationship between sleep stages and short‐term changes in rectal temperature in humans

Abstract

Sixteen volunteers have been studied during 3–4 control nights and eight of these subjects again during four successive sleeps on 30‐h “days”;. The experiments took place in a comfortable environment provided by an isolation chamber. Rectal temperature and the sleep EEG were measured throughout. The relationship between sleep stages, particularly SWS and REM sleep, and short‐term changes in rectal temperature has been investigated during both protocols. Care was taken to correct for or remove those temperature changes that could be attributed to circadian rhythmicity or the effects of loss of masking due to being awake. Results showed that there was a small but significant effect of sleep stages, with SWS producing a fall and REM sleep a rise in rectal temperature after a delay of about 30–48 minutes. It is concluded that such spontaneous changes in sleeping subjects accord with the results of other studies which indicate that thermoregulatory reflexes to hot or cold stimuli alter in different sleep stages.     

Electroencephalogram bands modulated by vigilance states in an anuran species: a factor analytic approach

Dramatic changes in neocortical electroencephalogram (EEG) rhythms are associated with the sleep–waking cycle in mammals. Although amphibians are thought to lack a neocortical homologue, changes in rest–activity states occur in these species. In the present study, EEG signals were recorded from the surface of the cerebral hemispheres and midbrain on both sides of the brain in an anuran species, Babina daunchina, using electrodes contacting the meninges in order to measure changes in mean EEG power across behavioral states. Functionally relevant frequency bands were identified using factor analysis. The results indicate that: (1) EEG power was concentrated in four frequency bands during the awake or active state and in three frequency bands during rest; (2) EEG bands in frogs differed substantially from humans, especially in the fast frequency band; (3) bursts similar to mammalian sleep spindles, which occur in non-rapid eye movement mammalian sleep, were observed when frogs were at rest suggesting sleep spindle-like EEG activity appeared prior to the evolution of mammals.     

Topographic analysis of dimension estimates of EEG and filtered rhythms in epileptic patients with complex partial seizures

Nonlinear dynamic properties were analyzed on the EEG and filtered rhythms recorded from healthy subjects and epileptic patients with complex partial seizures. Estimates of correlation dimensions of control EEG, interictal EEG and ictal EEG were calculated. The values were demonstrated on topograms. The delta (0.5–4 Hz), theta (4–8 Hz), alpha (8–13 Hz), beta (13–30 Hz) and gamma (30–40 Hz) components were obtained and considered as signals from the cortex. Corresponding surrogate data was produced. Firstly, the influence of sampling parameters on the calculation was tested. The dimension estimates of the signals from the frontal, temporal, parietal and occipital regions were computed and compared with the results of surrogate data. In the control subjects, the estimates between the EEG and surrogate data did not differ (P > 0.05). The interictal EEG from the frontal region and occipital region, as well as its theta component from the frontal region, and temporal region, showed obviously low dimensions (P < 0.01). The ictal EEG exhibited significantly low-dimension estimates across the scalp. All filtered rhythms from the temporal region yielded lower results than those of the surrogate data (P < 0.01). The dimension estimates of the EEG and filtered components markedly changed when the neurological state varied. For each neurological state, the dimension estimates were not uniform among the EEG and frequency components. The signal with a different frequency range and in a different neurological state showed a different dimension estimate. Furthermore, the theta and alpha components demonstrated the same estimates not only within each neurological state, but also among the different states. These results indicate that the theta and alpha components may be caused by similar dynamic processes. We conclude that the brain function underlying the ictal EEG has a simple mechanism. Several heterogeneous dynamic systems play important roles in the generation of EEG. Received: 10 December 1999 / Accepted in revised form: 8 May 2000     

Sleep and EEG spectra in the Syrian hamster (Mesocricetus auratus) under baseline conditions and following sleep deprivation

Summary Sleep was studied by continuous 24-h recordings in adult male Syrian hamsters, chronically implanted with EEG and EMG electrodes. Three vigilance states were determined using visual scoring and EEG power spectra (0.25–25 Hz) computed for 4-s episodes.The effects of two methods of total sleep deprivation (SD) were examined on vigilance states and the EEG power spectrum. The animals were subjected to 24 h SD by: (1) forced locomotion in a slowly rotating drum, (2) gentle handling whenever the hamsters attempted a sleeping posture. In addition, the hamsters were subjected to SD by handling during the first 3 h of the L period.Sleep predominated in the L period (78.2% of 12 h) and the D period (51.2%). The power spectra of the 3 vigilance states were similar during the L and D period. In NREM sleep, power density values in the low frequency range (0.25–6.0 Hz) exceeded those of REM sleep and W by a maximum factor of 8.3 and 2.8, respectively. At frequencies above 16 Hz, NREM and REM sleep power density values were significantly lower than during W. A progressive decrease in power density for low EEG frequencies (0.25–7 Hz) during NREM sleep was seen in the course of the L period. Power density values of higher frequencies (8–25 Hz) increased at the end of the L period and remained high during the first hours of the D period.The effect of prolonged SD on vigilance states and EEG spectra was similar by both methods and strikingly small compared to similar results in rats. In contrast, 3 h SD induced a large and more prolonged effect. The similarities and differences of sleep and sleep regulation are summarized for the hamster, rat and man.Abbreviations EEG electroencephalogram - LD light dark - REM rapid eye movements - NREM sleep non REM sleep - W waking - SD sleep deprivation - TST total sleep time - L light - D dark     

Alpha blocking and 1/fβ spectral scaling in resting EEG can be accounted for by a sum of damped alpha band oscillatory processes

The dynamical and physiological basis of alpha band activity and 1/f^β noise in the EEG are the subject of continued speculation. Here we conjecture, on the basis of empirical data analysis, that both of these features may be economically accounted for through a single process if the resting EEG is conceived of being the sum of multiple stochastically perturbed alpha band damped linear oscillators with a distribution of dampings (relaxation rates). The modulation of alpha-band and 1/f^β noise activity by changes in damping is explored in eyes closed (EC) and eyes open (EO) resting state EEG. We aim to estimate the distribution of dampings by solving an inverse problem applied to EEG power spectra. The characteristics of the damping distribution are examined across subjects, sensors and recording condition (EC/EO). We find that there are robust changes in the damping distribution between EC and EO recording conditions across participants. The estimated damping distributions are found to be predominantly bimodal, with the number and position of the modes related to the sharpness of the alpha resonance and the scaling (β) of the power spectrum (1/f^β). The results suggest that there exists an intimate relationship between resting state alpha activity and 1/f^β noise with changes in both governed by changes to the damping of the underlying alpha oscillatory processes. In particular, alpha-blocking is observed to be the result of the most weakly damped distribution mode becoming more heavily damped. The results suggest a novel way of characterizing resting EEG power spectra and provides new insight into the central role that damped alpha-band activity may play in characterising the spatio-temporal features of resting state EEG.     

Modeling the effect of sleep regulation on a neural mass model

In mammals, sleep is categorized by two main sleep stages, rapid eye movement (REM) and non-REM (NREM) sleep that are known to fulfill different functional roles, the most notable being the consolidation of memory. While REM sleep is characterized by brain activity similar to wakefulness, the EEG activity changes drastically with the emergence of K-complexes, sleep spindles and slow oscillations during NREM sleep. These changes are regulated by circadian and ultradian rhythms, which emerge from an intricate interplay between multiple neuronal populations in the brainstem, forebrain and hypothalamus and the resulting varying levels of neuromodulators. Recently, there has been progress in the understanding of those rhythms both from a physiological as well as theoretical perspective. However, how these neuromodulators affect the generation of the different EEG patterns and their temporal dynamics is poorly understood. Here, we build upon previous work on a neural mass model of the sleeping cortex and investigate the effect of those neuromodulators on the dynamics of the cortex and the corresponding transition between wakefulness and the different sleep stages. We show that our simplified model is sufficient to generate the essential features of human EEG over a full day. This approach builds a bridge between sleep regulatory networks and EEG generating neural mass models and provides a valuable tool for model validation.     

Decision Tree in Working Memory Task Effectively Characterizes EEG Signals in Healthy Aging Adults

BackgroundThe changes in electroencephalogram (EEG) signals that reflect the changes in physiological structure, cognitive functions, and activities have been observed in healthy aging adults. It is unknown that when the brain aging initiates and whether these age-related alterations can be associated with incipient neurodegenerative diseases in healthy elderly individuals.Materials and methodsWe employed feature extraction and classification methods to classify and compare the EEG signals of middle-aged and elderly age groups. This study included 20 healthy middle-aged and 20 healthy elderly subjects. The EEG signals were recorded during a resting state (eyes-open and eyes-closed) and during a working memory (WM) task using eight electrodes. The minimum redundancy maximum relevance technique was employed in the selection of the optimal feature. Four classification methods, including decision tree, support vector machine, Naïve Bayes, and K-nearest neighbor, were used to distinguish the elderly age group from the middle-aged group based on their EEG signals.ResultsIn the resting state, a good correlation was observed among absolute power delta and theta bands and aging, whereas between beta absolute power and aging, a WM task correlation was observed. The results also indicated that the mean frequency and absolute power might be useful for the prediction and classification of EEG signals in aging individuals. Furthermore, the use of the decision tree method in a WM task state distinguished the elderly group from the middle-aged group with an accuracy of 87.5%.ConclusionsWorking memory could play a vital role in the optimization of classification of EEG signals in aging and discrimination of age-related issues associated with neurodegeneration.     

Modulation of attention in healthy children using a course of EEG-feedback sessions

We studied changes in the power spectra of EEG in the course of sessions of feedback by EEG characteristics (neurofeedback sessions) and estimated the effects of neurofeedback on psychological and EEG correlates of voluntary attention. Indices of the latter were estimated using Bourdon’s test (a correcture test) and Schulte’s tables. Twenty-nine reasonably healthy 10-to 13-year-old children took part in the study; they were divided into two groups, an experimental group (n = 12) and a control group (n = 15). The results obtained support the statement on noticeable changes in the functional state of the brain both immediately in the course of a neurofeedback session and after a course of such trainings. Changes in the ratios of the spectral powers of the beta₁ vs theta rhythms and the low-frequency beta vs theta rhythms were found in EEG recorded from the sensorimotor zone of the right hemisphere (C4). The observed changes in the spectral characteristics of EEG induced by the course of neurofeedback sessions were accompanied by the improvement of a few indices of voluntary attention. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 458–465, September–December, 2006.     

Sleep and cortical temperature in the Djungarian hamster under baseline conditions and after sleep deprivation

The Djungarian hamster (Phodopus sungorus) is a markedly photoperiodic rodent which exhibits daily torpor under short photoperiod. Normative data were obtained on vigilance states, electroencephalogram (EEG) power spectra (0.25–25.0 Hz), and cortical temperature (T_CRT) under a 168 h light-dark schedule, in 7 Djungarian hamsters for 2 baseline days, 4 h sleep deprivation (SD) and 20 h recovery.During the baseline days total sleep time amounted to 59% of recording time, 67% in the light period and 43% in the dark period. The 4 h SD induced a small increase in the amount of non-rapid eye movement (NREM) sleep and a marked increase in EEG slow-wave activity (SWA; mean power density 0.75–4.0 Hz) within NREM sleep in the first hours of recovery. T_CRT was lower in the light period than in the dark period. It decreased at transitions from either waking or rapid eye movement (REM) sleep to NREM sleep, and increased at the transition from NREM sleep to waking or REM sleep. After SD, T_CRT was lower in all vigilance states.In conclusion, the sleep-wake pattern, EEG spectrum, and time course of T_CRT in the Djungarian hamster are similar to other nocturnal rodents. Also in the Djungarian hamster the time course of SWA seems to reflect a homeostatically regulated process as was formulated in the two-process model of sleep regulation.Abbreviations EEG electroencephalogram - EMG electromyogram - N NREM sleep - NREM non-rapid eye movement - R REM sleep - REM rapid eye movement - SD sleep deprivation - SWA slow-wave activity - T_CRT cortical temperature - TST total sleep time - VS vigilance state - W waking     

Electrocoupling of Ion Transporters in Plants: Interaction with Internal Ion Concentrations

There are five major electroenzymes in the plasmalemma of plant cells: a driving electrogenic pump, inward and outward rectifying K⁺ channels, a Cl⁻-2H⁺ symporter, and Cl⁻-channels. It has been demonstrated previously (Gradmann, Blatt & Thiel 1993, J. Membrane Biol. 136:327–332) how voltage-gating of these electroenzymes causes oscillations of the transmembrane voltage (V) at constant substrate concentrations. The purpose of this study is to examine the interaction of the same transporter ensemble with cytoplasmic concentrations of K⁺ and Cl⁻. The former model system has been extended to account for changing internal concentrations. Constant-field theory has been applied to describe the influence of ion concentrations on current-voltage relationships of the active channels. The extended model is investigated using a reference set of model parameters. In this configuration, the system converges to stable slow oscillations with intrinsic changes in cytoplasmic K⁺ and Cl⁻ concentrations. These slow oscillations reflect alternation between a state of salt uptake at steady negative values of V and a state of net salt loss at rapidly oscillating V, the latter being analogous to the previously reported oscillations. By switching off either concentration changes or gating, it is demonstrated that the fast oscillations are mostly due to the gating properties of the Cl⁻ channel, whereas the slow oscillations are controlled by the effect of the Cl⁻ concentration on the current. The sensitivity of output results y (e.g., frequency of oscillations) to changes of the model parameters x (e.g., maximum Cl⁻ conductance) has been investigated for the reference system. Further examples are presented where some larger changes of specific model parameters cause fundamentally different behavior, e.g., convergence towards a stable state of only the fast oscillations without intrinsic concentration changes, or to a steady-state without any oscillations. The main and general result of this study is that the osmotic status of a plant cell is stabilized by the ensemble of familiar electroenzymes through oscillatory interactions with the internal concentrations of the most abundant ions. This convergent behavior of the stand-alone system is an important prerequisite for osmotic regulation by means of other physiological mechanisms, like second messengers and gating modifiers. Received: 23 February/Revised: 16 July 1998     

Applications of the ETS-NOCV method in descriptions of chemical reactions

The present study characterizes changes in the electronic structure of reactants during chemical reactions based on the combined charge and energy decomposition scheme, ETS-NOCV (extended transition state–natural orbitals for chemical valence). Decomposition of the activation barrier, ΔE ^#, into stabilizing (orbital interaction, ΔE _orb, and electrostatic, ΔE _elstat) and destabilizing (Pauli repulsion, ΔE _Pauli, and geometry distortion energy, ΔE _dist) factors is discussed in detail for the following reactions: (I) hydrogen cyanide to hydrogen isocyanide, HCN → CNH isomerization; (II) Diels-Alder cycloaddition of ethene to 1,3-butadiene; and two catalytic processes, i.e., (III) insertion of ethylene into the metal-alkyl bond using half-titanocene with phenyl-phenoxy ligand catalyst; and (IV) B–H bond activation catalyzed by an Ir-containing catalyst. Various reference states for fragments were applied in ETS-NOCV analysis. We found that NOCV-based deformation densities (Δρ _i) and the corresponding energies ΔE _orb(i) obtained from the ETS-NOCV scheme provide a very useful picture, both qualitatively and quantitatively, of electronic density reorganization along the considered reaction pathways. Decomposition of the barrier ΔE^# into stabilizing and destabilizing contributions allowed us to conclude that the main factor responsible for the existence of positive values of ΔE ^# for all processes (I, II, III and IV) is Pauli interaction, which is the origin of steric repulsion. In addition, in the case of reactions II, III and IV, a significant degree of structural deformation of the reactants, as measured by the geometry distortion energy, plays an important role. Depending on the reaction type, stabilization of the transition state (relatively to the reactants) originating either from the orbital interaction term or from electrostatic attraction can be of vital importance. Finally, use of the ETS-NOCV method to describe catalytic reactions allows extraction of information on the role of catalysts in determination of ΔE ^#.     

Contrasting patterns of wetland use by sympatric larids in winter

The population dynamics and behaviour of the larid assemblage of a Mediterranean coastal wetland, the Vourkari inlet in Greece, were studied during the winter of 2008–2009. More black-headed gulls (Larus ridibundus) were seen in the inlet in December, more Mediterranean gulls (Larus melanocephalus) were present from mid-January to mid-February, while little variation was observed in yellow-legged gull (Larus michahellis) numbers throughout winter. Bird numbers remained stable through the day for the yellow-legged gull, but fewer black-headed and Mediterranean gulls were present in the late morning than the other periods. Diurnal activity patterns showed that feeding was the primary behaviour for yellow-legged gulls and resting and sleeping for black-headed and Mediterranean gulls, with locomotion being equally important for all species. All larids were primarily feeding in the late morning period, but yellow-legged gulls were doing so in much higher proportions. These contrasting patterns suggested that the Vourkari inlet was more important as a day roost for black-headed and Mediterranean gulls and a preferred feeding ground for yellow-legged gulls. Furthermore, results suggested that resting and sleeping were interchangeable activities and all other activities had more or less the same time demands on a daily basis, and also a consistent pattern across species in sleeping proportions that might indicate cross-species synchrony in sleep patterns; however future research is needed to resolve these issues. This study provided important new information on the winter ecology of three larids and revealed patterns of wetland use by these species that could help assess the importance of certain areas and improve coastal habitat management strategies to benefit birds.     

Diurnal sleep depth changes in the king penguin (<Emphasis Type="Italic">Aptenodytes patagonicus</Emphasis>)

Avian sleep quality depends on its depth (deeper sleep being of better quality). In king penguins (Aptenodytes patagonica), sleep may be disturbed by congeners passing in the sleeper's vicinity. As king penguin activity is increased in the morning, sleep disturbances are more likely to occur during this time period. One might therefore assume that afternoon sleepers (AS) sleep more profoundly than morning sleepers (MS). To test this hypothesis, we examined the diurnal variations in sleep depth of king penguins sleeping in resting sites adjacent to the colony of 'La Baie du Marin' (Crozet Archipelago). We measured the bodily tactile arousal threshold at the upper back level. The arousal threshold in AS was twice as high as in MS. This study demonstrates for the first time that sleep depth changes according to time of day in a diurnal wild bird. We postulate that diurnal sleep depth is increased due to decreased congener movements close to the sleeping penguin.     

Kososan,a standardized traditional Japanese herbal medicine,reverses sleep disturbance in socially isolated mice via GABAA-benzodiazepine receptor complex activation

PurposeKososan (KSS), a traditional Japanese medicine with a distinct aroma, is clinically used to treat affective disorders but its antidepressant-like effect has not been thoroughly investigated. In this study, we investigated the effects of inhaled and orally administered KSS on sleep disturbances in socially isolated mice.MethodsFour-weeks-old male ddy mice were housed either in social isolation or in groups for 4–6 weeks before the experiment. KSS was orally administered (0.5 or 1.0 g/kg) or inhaled (0.5, 1.0, or 2.5 g/0.125 m³) 60 min before pentobarbital administration. Stress levels in mice were evaluated by the duration of pentobarbital-induced sleeping time.ResultsSleeping time was shorter in socially-isolated mice than in group-housed mice. Oral and inhaled KSS prolonged sleeping time in stressed mice, but had no effect on sleeping time of group-housed mice. Prolonged sleeping time after oral KSS was significantly inhibited (p < 0.05) by bicuculline (3 mg/kg, i.p.), a GABA_A antagonist, but not by flumazenil (3 mg/kg, i.p.), a selective benzodiazepine antagonist. Prolonged sleeping time after KSS inhalation was significantly inhibited (p < 0.05) by flumazenil but not by bicuculline.ConclusionsOur findings suggest that KSS activates GABA_A-benzodiazepine receptor complex and reverses shortened pentobarbital-induced sleep caused by social isolation.     

Modeling the Current-Voltage Characteristics of Chara Membranes: I. The Effect of ATP Removal and Zero Turgor

We have obtained and modeled the electrical characteristics of the plasma membrane of Chara internodal cells: intact, without turgor and perfused with and without ATP. The cells were voltage and space-clamped to obtain the I/V (current-voltage) and G/V (conductance-voltage) profiles of the cell membrane. The intact cells yielded similar I/V characteristics with resting p.d.s of −221 ± 12 mV (cytoplasmic clamp, 5 cells) and −217 ± 12 mV (vacuolar clamp, 5 cells). The cut unperfused cells were depolarized at −169 ± 12 mV (7 cells) compared to the vacuole-clamped intact cells. The cells perfused with ATP fell into three groups: hyperpolarized group with resting p.d. −175 ± 12 mV (4 cells) and I/V profile similar to the intact and cut unperfused cells; depolarized group with resting p.d. of −107 ± 12 mV (6 cells) and I/V profiles close to linear; and excited cells with profiles showing a negative conductance region and resting p.d. at −59 ± 12 mV (5 cells). The cells perfused with medium containing no ATP showed upwardly concave I/V characteristics and resting p.d. at −81 ± 12 mV (6 cells). The I/V curves were modeled employing the ``Two-state' model for the H<sup>+</sup> pump (Hansen et al., 1981). The inward and outward rectifiers were fitted to exponential functions and combined with a linear background current. The excitation state in perfused cells was modeled by including an inward current, i <sub>excit</sub>, with p.d.-dependence described by a combination of hyperbolic tangent functions. An inward current, i <sub>no-ATP</sub>, with a smaller amplitude, but very similar p.d.-dependence was also included in the simulation of the I/V curves from cells without ATP. This approach avoided I/V curve subtraction. The modeling of the total I/V and G/V characteristics provided more information about the parameters of the ``Two-state' pump model, as well as more quantitative understanding of the interaction of the major transport systems in the plasmalemma in generation of the resting potential under a range of circumstances. ATP had little effect on nonpump currents except the excitation current; depolarization profoundly affected the pump characteristics. Received: 23 January/Revised: 10 October 1995     

Characterization of the High-Affinity Verapamil Binding Site in a Plant Plasma Membrane Ca2+-selective Channel

Despite biochemical evidence for the existence of high-affinity phenylalkylamine receptors in higher plants, their effects on channel activity have only been demonstrated at relatively high concentrations. We have performed a quantitative single-channel analysis of the changes induced by extracellular verapamil in the rca channel [a wheat root plasma membrane Ca²⁺-selective channel (Pi?eros & Tester, 1995. Planta 195:478–488)]. Concentrations as low as 0.5 μm verapamil induced a blockade of the inward current, with no evident reduction of the single-channel current amplitude. Blockade by verapamil was concentration and voltage dependent. Preliminary analysis suggested the blockade was due to a reduction in the maximum open state probability rather than a change in V_0.5. Further analysis of the association and dissociation rate constants revealed a binding site located 56 to 59% down the voltage drop from the extracellular face of the channel, with a K _d (0) of 24 to 26 μm. This results in a K _d at −100 mV of 2 μm. Methoxyverapamil had qualitatively the same effects. This intra-pore binding site can be accessed directly from the extracellular side of the rca channel, but apparently not from the cytosolic side. Received: 15 August 1996/Revised: 23 December 1996     

Two-step concerted mechanism for alkane hydroxylation on the ferryl active site of methane monooxygenase

 A two-step concerted mechanism for the conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO) is discussed. We propose that the enzymatic reaction mechanism is essentially the same as that of the gas-phase methane-methanol conversion by the bare FeO⁺ complex. In the initial stage of our mechanism, the ferryl (Fe—O) "iron" active site of intermediate Q and substrate methane come into contact to form the initial Q (CH₄) complex with an OFe—CH₄ bond. The C—H bonds of methane are significantly weakened by the formation of a five-coordinate carbon species, through orbital interactions between a C _3v - or D _2d -distorted methane and the Fe—O active site. The important transition state for an H atom abstraction exhibits a four-centered structure. The generated intermediate involves an HO—Fe—CH₃ moiety, and it is then converted into the final product complex including methanol as a ligand through a methyl migration that occurs via a three-centered transition state. The two-step concerted mechanism is consistent with recent experiments on regioselectivity of enzyme-catalyzed alkane hydroxylations. Received: 15 September 1997 / Accepted: 20 December 1997