Can presynaptic depolarization release transmitter without calcium influx?

Recent experimental evidence suggesting that presynaptic depolarization can evoke transmitter release without calcium influx has been re-examined. The presynaptic terminal of the squid giant synapse can be depolarized by variable amounts while recording presynaptic calcium current under voltage clamp and postsynaptic responses. Small depolarizations open few calcium channels with large single channel currents. Large depolarizations approaching the calcium equilibrium potential open many channels with small single channel currents. When responses to small and large depolarizations eliciting similar total macroscopic calcium currents are compared, the large pulses evoke more transmitter release. This apparent voltage-dependence of transmitter release may be explained by the greater overlap of calcium concentration domains surrounding single open calcium channels when many closely apposed channels open at large depolarizations. This channel domain overlap leads to higher calcium concentrations at transmitter release sites and more release for large depolarizations than for small depolarizations which open few widely dispersed channels. At neuromuscular junctions, a subthreshold depolarizing pulse to motor nerve terminals may release over a thousand times as much transmitter if it follows a brief train of presynaptic action potentials than if it occurs in isolation. This huge synaptic facilitation has been taken as indicative of a direct effect of voltage which is manifest only when prior activity raises presynaptic resting calcium levels. This large facilitation is actually due to a post-tetanic supernormal excitability in motor nerve terminals, causing the previously subthreshold test pulse to become suprathreshold and elicit a presynaptic action potential. When motor nerve terminals are depolarized by two pulses, as the first pulse increases above a certain level it evokes more transmitter release but less facilitation of the response to the second pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Block of squid axon K channels by internally and externally applied barium ions

We have studied the interactions of Ba ion with K channels. Ba2+ blocks these channels when applied either internally or externally in millimolar concentrations. Periodic depolarizations enhance block with internal Ba2+, but diminish the block caused by external Ba2+. At rest, dissociation of Ba2+ from blocked channels is very slow, as ascertained by infrequent test pulses applied after washing Ba2+ form either inside or outside. The time constant for recovery from internal and external Ba2+ is the same. Frequent pulsing greatly shortens recovery time constant after washing away both Ba2+in and Ba2+out. Block by Ba2+ applied internally or externally is voltage dependent. Internal Ba2+ block behaves like a one-step reaction governed by a dissociation constant (Kd) that decreases e-fold/12 mV increase of pulse voltage: block deepens with more positive pulse voltage. For external Ba2+, Kd decreases e-fold/18 mV as holding potential is made more negative: block deepens with increasing negativity. Millimolar external concentrations of some cations can either lessen (K+) or enhance (NH+4, Cs+) block by external Ba2+. NH+4 apparently enhances block by slowing exist of Ba ions from the channels. Rb+ and Cs+ also slow clearing of Ba ions from channels. We think that (a) internally applied Ba2+ moves all the way through the channels, entering only when activation gates are open; (b) externally applied Ba2+ moves two-thirds of the way in, entering predominantly when activation gates are closed; (c) at a given voltage, Ba2+ occupies the same position in the channels whether it entered from inside or outside.     

Electrical activity in white adipose tissue of rat

Intracellular recording of white adipocytes was performed in an in vitro preparation. Resting potential, input resistance and membrane time constant averaged: -34 +/- 9 mV, 295 +/- 161 M omega, and 58 +/- 19 ms respectively (mean +/- SD, n = 32). Intracellular injection of positive and negative square current pulses elicited membrane voltage responses, characterized by a rectification of the voltage change evoked by positive pulses, and a slow return to baseline at the offset of hyperpolarizing pulses. The amplitude and duration of the slow return to resting potential was dependent on membrane potential, pulse duration, and extracellular K+ concentration. This response was depressed when external Ca2+ was replaced by Co2+, and by external application of 4-aminopyridine. These results indicate that white adipocytes can generate membrane voltage responses which may mostly be a consequence of the activity of ionic channels. The properties of the slow return to baseline suggest that it may be due to a transient K+ current.     

Initiation and abolition of the action potential of a single node of Ranvier

1. Using single node preparations of the bull frog or the toad, observations were made on the variation of the voltage across the nodal membrane under various experimental conditions. 2. The time constant of the variation in the membrane voltage caused by a long subthreshold rectangular pulse was of the order of 0.1 msec. 3. The action potential was initiated when the potential inside the node was raised stimulating pulses above a threshold level of approximately 15 mv. for a node in normal Ringer; it was greater in a relatively refractory node and in a partially narcotized node. 4. The variation of the membrane voltage caused by long stimulating pulses of subrheobasic strengths was in general proportional to the strength of the applied pulse. A non-linear behavior of the membrane voltage was observed with barely subthreshold stimulating pulses. 5. The early portion of the action potential of a node was not modified by a direct current which was strong enough to produce measurable potential changes (IR drops) across the resting membrane. 6. A strong pulse of inward current applied to the node during activity abolished the portion of the action potential following the pulse in all-or-none manner. 7. There was no refractory period after a response abolished in its early phase. Following a response abolished later, the recovery in the spike height started from the level of the action potential at the time of abolition. 8. Initiation and abolition of action potentials at a single node are interpreted as "transitions" between the two "equilibrium potential levels" at the node.     

Voltage-independent Adaptation of Mechanosensitive Channels in Escherichia coli Protoplasts

Mechanosensitive (MS) ion channels, with 560 pS conductance, opened transiently by rapid application of suction pulses to patches of E. coli protoplast membrane. The adaptation phase of the response was voltage-independent. Application of strong suction pulses, which were sufficient to cause saturation of the MS current, did not abolish the adaptation. Multiple-pulse experimental protocols revealed that once MS channels had fully adapted, they could be reactivated by a second suction pulse of similar amplitude, providing the time between pulses was long enough and suction had been released between pulses. Limited proteolysis (0.2 mg/ml pronase applied to the cytoplasmic side of the membrane patch) reduced the number of open channels without affecting the adaptation. Exposing patches to higher levels of pronase (1 mg/ml) removed responsiveness of the channel to suction and abolished adaptation consistent with disruption of the tension transmission mechanism responsible for activating the MS channel. Based on these data we discuss a mechanism for mechanosensitivity mediated by a cytoplasmic domain of the MS channel molecule or associated protein. Received: 29 January 1998/Revised: 16 April 1998     

Memory is a property of an ion channels pool: ion channels formed by Staphylococcus aureus alpha-toxin.

The short-time depolarization effects on the integral conductance induced by S. aureus alpha-toxin (ST) in planar lipid bilayer membranes has been studied. Ion channels formed by ST were found to have several potential-induced nonconductance (closed) states. The transitions of ion channels between the states are only through one conductance state. The transition of ST-channels from closed to open state is induced by membrane depolarization. The amplitude current after a series of voltage pulses is a function of pulse number, and is effectively independent of the time interval between the neighbouring pulses. Therefore, a membrane which contains a pool of ion channels "remembers" its previous existence. A simple model can be used to explain this phenomenon.     

The Role of Pulse Shape in Motor Cortex Transcranial Magnetic Stimulation Using Full-Sine Stimuli

A full-sine (biphasic) pulse waveform is most commonly used for repetitive transcranial magnetic stimulation (TMS), but little is known about how variations in duration or amplitude of distinct pulse segments influence the effectiveness of a single TMS pulse to elicit a corticomotor response. Using a novel TMS device, we systematically varied the configuration of full-sine pulses to assess the impact of configuration changes on resting motor threshold (RMT) as measure of stimulation effectiveness with single-pulse TMS of the non-dominant motor hand area (M1). In young healthy volunteers, we (i) compared monophasic, half-sine, and full-sine pulses, (ii) applied two-segment pulses consisting of two identical half-sines, and (iii) manipulated amplitude, duration, and current direction of the first or second full-sine pulse half-segments. RMT was significantly higher using half-sine or monophasic pulses compared with full-sine. Pulses combining two half-sines of identical polarity and duration were also characterized by higher RMT than full-sine stimuli resulting. For full-sine stimuli, decreasing the amplitude of the half-segment inducing posterior-anterior oriented current in M1 resulted in considerably higher RMT, whereas varying the amplitude of the half-segment inducing anterior-posterior current had a smaller effect. These findings provide direct experimental evidence that the pulse segment inducing a posterior-anterior directed current in M1 contributes most to corticospinal pathway excitation. Preferential excitation of neuronal target cells in the posterior-anterior segment or targeting of different neuronal structures by the two half-segments can explain this result. Thus, our findings help understanding the mechanisms of neural stimulation by full-sine TMS.     

Altered ion channel conductance and ionic selectivity induced by large imposed membrane potential pulse.

The effects of large magnitude transmembrane potential pulses on voltage-gated Na and K channel behavior in frog skeletal muscle membrane were studied using a modified double vaseline-gap voltage clamp. The effects of electroconformational damage to ionic channels were separated from damage to lipid bilayer (electroporation). A 4 ms transmembrane potential pulse of -600 mV resulted in a reduction of both Na and K channel conductivities. The supraphysiologic pulses also reduced ionic selectivity of the K channels against Na+ ions, resulting in a depolarization of the membrane resting potential. However, TTX and TEA binding effects were unaltered. The kinetics of spontaneous reversal of the electroconformational damage of channel proteins was found to be dependent on the magnitude of imposed membrane potential pulse. These results suggest that muscle and nerve dysfunction after electrical shock may be in part caused by electroconformational damage to voltage-gated ion channels.     

Wavelet analysis of nonequilibrium ionic currents in human heart sodium channel (hH1a)

Nonequilibrium response spectroscopy (NRS), the technique of using rapidly fluctuating voltage pulses in the study of ion channels, is applied here. NRS is known to drive an ensemble of ion channels far from equilibrium where, it has been argued, new details of ion channel kinetics can be studied under nonequilibrium conditions. In this paper, a single-pulse NRS technique with custom-designed waveforms built from wavelets is used. The pulses are designed to produce different responses from two competing models of a human heart isoform of the sodium channel (hH1a). Experimental data using this new type of pulses are obtained through whole-cell recordings from mammalian cells (HEK 293). Wavelet analysis of the model response and the experimental data is introduced to show how these NRS pulses can aid in distinguishing the better of the two models and thus introduces another important application of this new technique.     

Inactivation of voltage-gated delayed potassium current in molluscan neurons. A kinetic model.

Voltage-gated delayed potassium current in molluscan neurons is characterized by a marked inactivation. Inactivation can accumulate between repetitive pulses, giving rise to current patterns in which the maximum current during a second voltage pulse is less than the current at the end of the preceding pulse (cumulative inactivation). Other features of inactivation of this current include an onset time-course that can be characterized by the sum of two exponential processes and an early minimum in the recovery-vs.-time curve. A simple four-state model is developed that can, when supplied with rate constants derived from voltage-clamp experiments, reproduce these features of inactivation. The model incorporates state-dependent inactivation rates. Upon depolarization, both open and closed channels can be inactivated, although inactivation of closed channels is much faster. Upon repolarization, recovery from inactivated states is sufficiently slow that little recovery occurs during a short interpulse interval. Cumulative inactivation comes about as a result of fast inactivation during the second pulse, further limiting the peak current from the level at the end of the previous pulse.     

Measurement of calcium channel inactivation is dependent upon the test pulse potential.

We have developed two methods to measure Ca2+ channel inactivation in Lymnaea neurons-one method, based upon the conventional double-pulse protocol, uses currents during a moderately large depolarizing pulse, and the other uses tail currents after a very strong activating pulse. Both methods avoid contamination by proton currents and are unaffected by rundown of Ca2+ current. The magnitude of inactivation measured differs for the two methods; this difference arises because the measurement of inactivation is inherently dependent upon the test pulse voltage used to monitor the Ca2+ channel conductance. We discuss two models that can generate such test pulse dependence of inactivation measurements-a two-channel model and a two-open-state model. The first model accounts for this by assuming the existence of two types of Ca2+ channels, different proportions of which are activated by the different test pulses. The second model assumes only one Ca2+ channel type, with two closed and open states; in this model, the test pulse dependence is due to the differential activation of channels in the two closed states by the test pulses. Test pulse dependence of inactivation measurements of Ca2+ channels may be a general phenomenon that has been overlooked in previous studies.     

Inactivation of the sodium channel. I. Sodium current experiments

Inactivation of sodium conductance has been studied in squid axons with voltage clamp techniques and with the enzyme pronase which selectively destroys inactivation. Comparison of the sodium current before and after pronase treatment shows a lag of several hundred microseconds in the onset of inactivation after depolarization. This lag can of several hundred microseconds in the onset of inactivation after polarization. This lag can also be demonstrated with double-pulse experiments. When the membrane potential is hyperpolarized to -140 mV before depolarization, both activation and inactivation are delayed. These findings suggest that inactivation occurs only after activation are delayed. These findings suggest that inactivation occurs only after activation; i.e. that the channels must open before they can inactivate. The time constant of inactivation measured with two pulses (τ(c)) is the same as the one measured from the decay of the sodium current during a single pulse (τ(h)). For large depolarizations, steady-state inactivation becomes more incomplete as voltage increases; but it is relatively complete and appears independent of voltage when determined with a two- pulse method. This result confirms the existence of a second open state for Na channels, as proposed by Chandler and Meves (1970. J. Physiol. [Lond.]. 211:653-678). The time constant of recovery from inactivation is voltage dependent and decreases as the membrane potential is made more negative. A model for Na channels is presented which has voltage-dependent transitions between the closed and open states, and a voltage-independent transition between the open and the inactivated state. In this model the voltage dependence of inactivation is a consequence of coupling to the activation process.     

Presynaptic facilitation at the crayfish neuromuscular junction. Role of calcium-activated potassium conductance

Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK(Ca), on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK(Ca) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with gK(Ca) not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK(Ca) by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltage-dependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gK(Ca) by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK(Ca) was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (greater than 50 ms) suggest either that residual free calcium does not account for facilitation at the crayfish NMJ or that the transmitter release mechanism has a markedly higher affinity or stoichiometry for internal free calcium than does gK(Ca). Finally, our data suggest that the calcium channels responsible for transmitter release at the crayfish NMJ are not of the L, N, or T type.     

Two-component structure of the current pulse of a ranaway electron beam generated during electric breakdown of elevated-pressure nitrogen

Conditions are investigated at which two current pulses of ranaway electron beams are generated in elevated-pressure nitrogen during one voltage pulse. It is shown that the regime with two runaway electron beam current pulses takes place at decreased values of the electric field strength E in the gap (or decreased values of the parameter E/p, where p is the gas pressure). The regime with two runaway electron beam current pulses is observed both at high (1500?C3000 Torr) and low (below 100 Torr) pressures. It is shown that, for the second runaway electron beam current pulse to form, the voltage across the gap should be partially reduced during the first pulse. At low nitrogen pressures (~10 Torr), the regime in which two runaway electron beams are generated can be implemented by increasing the breakdown strength of the gap and/or increasing the value of E/p. In experiments carried out in atmospheric-pressure air with a picosecond time resolution, a rather complicated structure of the beam current pulse is observed at a voltage rise time of ~300 ps.     

Voltage-activated outward currents in adult and nymphal locust photoreceptors

The membrane properties of dark-adapted green sensitive photoreceptors of adult and nymphal desert locust (Schistocerca gregaria) were investigated in situ with single electrode (discontinuous) current and voltage clamp techniques. The photoreceptors had a resting potential of about -70 mV. The membrane rectified strongly in response to depolarizing current pulses at and above the resting potential. Two outward currents could be distinguished on the basis of different kinetics. The first, a transient current, was inactivating between-40 mV and -100 mV, and activated above -90 mV. The second, a sustained current had a similar activation range. The inactivating current could be blocked by application of 50 mM TEA into the retinal extracellular space. Both currents were expressed in nymphal photoreceptors already in the 1st instar larva and have similar properties as in the adults, although the behaviour of adults and nymphs is different. The conductances underlying these currents could be shown to modify the frequency response of the photoreceptor membrane.The work was started in the Department of Zoology, University of Cambridge, Cambridge, UK.     

State-dependent inactivation of the Kv3 potassium channel.

Inactivation of Kv3 (Kv1.3) delayed rectifier potassium channels was studied in the Xenopus oocyte expression system. These channels inactivate slowly during a long depolarizing pulse. In addition, inactivation accumulates in response to a series of short depolarizing pulses (cumulative inactivation), although no significant inactivation occurs within each short pulse. The extent of cumulative inactivation does not depend on the voltage during the depolarizing pulse, but it does vary in a biphasic manner as a function of the interpulse duration. Furthermore, the rate of cumulative inactivation is influenced by changing the rate of deactivation. These data are consistent with a model in which Kv3 channel inactivation is a state-dependent and voltage-independent process. Macroscopic and single channel experiments indicate that inactivation can occur from a closed (silent) state before channel opening. That is, channels need not open to inactivate. The transition that leads to the inactivated state from the silent state is, in fact, severalfold faster then the observed inactivation of current during long depolarizing pulses. Long pulse-induced inactivation appears to be slow, because its rate is limited by the probability that channels are in the open state, rather than in the silent state from which they can inactivate. External potassium and external calcium ions alter the rates of cumulative and long pulse-induced inactivation, suggesting that antagonistic potassium and calcium binding steps are involved in the normal gating of the channel.     

VI. Synaptic Fuel Cell Reactions in Vascular-Interstitial-Neuromuscular Closed Circuit: Theory of Neuromuscular Activation

Spontaneous pulses of voltage occur in the caval vein at voluntary contraction of a leg muscle both in the rat and in humans. Morphology and vascular reactions indicate that vascular-interstitial-neuromuscular circuits (VINMC) exist. They require redox reactions, which are likely to occur at proteins in the cellular membranes of the synapse. Metabolic degradation of ATP in the nerve cell is known to generate a flow of current out of the cell, creating the resting potential. It corresponds to an electrochemical equilibrium potential. An overpotential leads to a closed circuit flow of current in the VINMC, producing redox products at the electrode equivalent redox proteins. The VINMC is thereby charged. Brain impulses open ionic channels of the nerve cell body, short-circuiting the VINMC. Electrochemical reactions by the redox products produce an action potential and reverse the current in the charged VINMC. The charging and discharging of the synaptic membranes are explained as electrochemical analogs. The action potential and its height, production, transport, and disappearance of various synaptic products, including vesicles and the voltage pulses in the associated vessels such as the caval vein and the aorta, can be explained.     

The relationship between Q gamma and Ca release from the sarcoplasmic reticulum in skeletal muscle

Asymmetric membrane currents and fluxes of Ca2+ release were determined in skeletal muscle fibers voltage clamped in a Vaseline-gap chamber. The conditioning pulse protocol 1 for suppressing Ca2+ release and the "hump" component of charge movement current (I gamma), described in the first paper of this series, was applied at different test pulse voltages. The amplitude of the current suppressed during the ON transient reached a maximum at slightly suprathreshold test voltages (-50 to -40 mV) and decayed at higher voltages. The component of charge movement current suppressed by 20 microM tetracaine also went through a maximum at low pulse voltages. This anomalous voltage dependence is thus a property of I gamma, defined by either the conditioning protocol or the tetracaine effect. A negative (inward-going) phase was often observed in the asymmetric current during the ON of depolarizing pulses. This inward phase was shown to be an intramembranous charge movement based on (a) its presence in the records of total membrane current, (b) its voltage dependence, with a maximum at slightly suprathreshold voltages, (c) its association with a "hump" in the asymmetric current, (d) its inhibition by interventions that reduce the "hump", (e) equality of ON and OFF areas in the records of asymmetric current presenting this inward phase, and (f) its kinetic relationship with the time derivative of Ca release flux. The nonmonotonic voltage dependence of the amplitude of the hump and the possibility of an inward phase of intramembranous charge movement are used as the main criteria in the quantitative testing of a specific model. According to this model, released Ca2+ binds to negatively charged sites on the myoplasmic face of the voltage sensor and increases the local transmembrane potential, thus driving additional charge movement (the hump). This model successfully predicts the anomalous voltage dependence and all the kinetic properties of I gamma described in the previous papers. It also accounts for the inward phase in total asymmetric current and in the current suppressed by protocol 1. According to this model, I gamma accompanies activating transitions at the same set of voltage sensors as I beta. Therefore it should open additional release channels, which in turn should cause more I gamma, providing a positive feedback mechanism in the regulation of calcium release.     

Voltage Dependence of ATP Secretion in Mammalian Taste Cells

Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm–like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca²⁺ transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels.     

Rate of opening of a population of Na channels in frog skeletal muscle fibers

Linear Systems convolution analysis of muscle sodium currents was used to predict the opening rate of sodium channels as a function of time during voltage clamp pulses. If open sodium channel lifetimes are exponentially distributed, the channel opening rate corresponding to a sodium current obtained at any particular voltage, can be analytically obtained using a simple equation, given single channel information about the mean open-channel lifetime and current.Predictions of channel opening rate during voltage clamp pulses show that sodium channel inactivation arises coincident with a decline in channel opening rate.Sodium currents pharmacologically modified with Chloramine-T treatment so that they do not inactivate, show a predicted sustained channel opening rate.Large depolarizing voltage clamp pulses produce channel opening rate functions that resemble gating currents.The predicted channel opening rate functions are best described by kinetic models for Na channels which confer most of the charge movement to transitions between closed states.Comparisons of channel opening rate functions with gating currents suggests that there may be subtypes of Na channel with some contributing more charge movement per channel opening than others.Na channels open on average, only once during the transient period of Na activation and inactivation.After transiently opening during the activation period and then closing by entering the inactivated state, Na channels reopen if the voltage pulse is long enough and contribute to steady-state currents.The convolution model overestimates the opening rate of channels contributing to the steady-state currents that remain after the transient early Na current has subsided.