Delays in inactivation development and activation kinetics in myxicola giant axons

Na inactivation was studied in Myxicola (two-pulse procedure, 6-ms gap between conditioning and test pulses). Inactivation developed with an initial delay (range 130-817 microseconds) followed by a simple exponential decline (time constant tau c). Delays (deviations from a simple exponential) are seen only for brief conditioning pulses were gNa is slightly activated. Hodgkin-Huxley kinetics with series resistance, Rs, predict deviations from a simple exponential only for conditioning pulses that substantially activate gNa. Reducing INa fivefold (Tris substitution) had no effect on either tau c or delay. Delay in not generated by Rs or by contamination from activation development. The slowest time constant in Na tails is approximately 1 ms (Goldman and Hahin, 1978) and the gap was 6 ms. Shortening the gap to 2 ms had no effect on either tau c or delay. Delay is a true property of the channel. Delay decreased with more positive conditioning potentials, and also decreased approximately proportionally with time to peak gNa during the conditioning pulse, as expected for sequentially coupled activation and inactivation. In a few cases the difference between Na current values for brief conditioning pulses and the tau c exponential could be measured. Difference values decayed exponentially with time constant tau m. The inactivation time course is described by a model that assumes a process with the kinetics of gNa activation as a precursor to inactivation.     

Effects of membrane polarization on sarcoplasmic calcium release in skeletal muscle

Calcium release from the sarcoplasmic reticulum was investigated in voltage-clamped, tetrodotoxin-treated frog skeletal muscle fibres injected with arsenazo III. Short (5 ms) depolarizing pulses (test pulses) produced a transient change in arsenazo III absorption, signalling an increase in intracellular calcium in concentration (calcium transient). Conditioning subthreshold depolarizations, which preceded the test pulse, potentiated the calcium transient triggered by the test pulse. Conditioning hyperpolarizations, applied either before or after the test pulse, inhibited the calcium transient. These effects of conditioning polarizations on the calcium transient may explain similar effects of subthreshold polarizations on muscle contraction that have previously been reported. The potentiating effect of subthreshold depolarizations was observed only when the test pulse was short (5 ms). The potentiating effect develops at -48 mV with a time constant of about 7 ms at 6.5 degrees C; this seems to be slower than that predicted by the potential spread from the surface along the tubular system. Thus, part of the effect could arise from the coupling process between tubular depolarization and calcium release.     

Dynamics of aminopyridine block of potassium channels in squid axon membrane

Aminopyridines (2-AP, 3-AP, and 4-AP) selectively block K channels of squid axon membranes in a manner dependent upon the membrane potential and the duration and frequency of voltage clamp pulses. They are effective when applied to either the internal or the external membrane surface. The steady-state block of K channels by aminopyridines is more complete for low depolarizations, and is gradually relieved at higher depolarizations. The K current in the presence of aminopyridines rises more slowly than in control, the change being more conspicuous in 3-AP and 4-AP than in 2-AP. Repetitive pulsing relieves the block in a manner dependent upon the duration and interval of pulses. The recovery from block during a given test pulse is enhanced by increasing the duration of a conditioning depolarizing prepulse. The time constant for this recovery is in the range of 10-20 ms in 3-AP and 4-AP, and shorter in 2-AP. Twin pulse experiments with variable pulse intervals have revealed that the time course for re-establishment of block is much slower in 3-AP and 4-AP than in 2-AP. These results suggest that 2-AP interacts with the K channel more rapidly than 3-AP and 4-AP. The more rapid interaction of 2-AP with K channels is reflected in the kinetics of K current which is faster than that observed in 3-AP or 4-AP, and in the pattern of frequency-dependent block which is different from that in 3-AP or 4-AP. The experimental observations are not satisfactorily described by alterations of Hodgkin-Huxley n-type gating units. Rather, the data are consistent with a simple binding scheme incorporating no changes in gating kinetics which conceives of aminopyridine molecules binding to closed K channels and being released from open channels in a voltage-dependent manner.     

Two classes of gating current from L-type Ca channels in guinea pig ventricular myocytes.

Intramembrane charge movement was recorded in guinea pig ventricular myocytes at 19-22 degrees C using the whole-cell patch clamp technique. From a holding potential of -110 mV, the dependence of intramembrane charge moved on test voltage (Q(V)) followed the sum of two Boltzmann components. One component had a transition voltage (V) of -48 mV and a total charge (Qmax) of congruent to 3 nC/microF. The other had a V of -18 mV and a Qmax of 11 nC/microF. Ba2+ currents through Ca channels began to activate at -45 mV and peaked at congruent to -15 mV. Na+ current peaked at -35 to -30 mV. Availability of charge (in pulses from -70 to +10 mV) depended on the voltage of conditioning depolarizations as two Boltzmann terms plus a constant. One term had a V of -88 mV and a Qmax of 2.5 nC/microF; the other had a V of -29 mV and a Qmax of 6.3 nC/microF. From the Q(V) dependence, the voltage dependence of the ionic currents, and the voltage dependence of the availability of charge, the low voltage term of Q(V) and availability was identified as Na gating charge, at a total of 3.5 nC/microF. The remainder, 11 nC/microF, was attributed to Ca channels. After pulses to -40 mV and above, the OFF charge movement had a slow exponentially decaying component. Its time constant had a bell-shaped dependence on OFF voltage peaking at 11 ms near -100 mV. Conditioning depolarizations above -40 mV increased the slow component exponentially with the conditioning duration (tau approximately equal to 480 ms). Its magnitude was reduced as the separation between conditioning and test pulses increased (tau approximately equal to 160 ms). The voltage distribution of the slow component of charge was measured after long (5 s) depolarizations. Its V was -100 mV, a shift of -80 mV from the value in normally polarized cells. This voltage was the same at which the time constant of the slow component peaked. Qmax and the steepness of the voltage distribution were unchanged by depolarization. This indicates that the same molecules that produce the charge movement in normally polarized cells also produce the slow component in depolarized cells. 100 microns D600 increased by 77% the slow charge movement after a 500-ms conditioning pulse. These results demonstrate two classes of charge movement associated with L-type Ca channels, with kinetics and voltage dependence similar to charge 1 and charge 2 of skeletal muscle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Delayed kinetics of squid axon potassium channels do not always superpose after time translation.

The activation of potassium ion conductance in squid axons by voltage-clamp depolarization is delayed when the depolarizing step is preceded by a conditioning hyperpolarization of the axonal membrane. Moreover, the control conductance kinetics superpose with the delayed kinetics when they are translated along the time axis by an amount equal to the delay. We have found that the degree of superposition with internally perfused axons depends upon voltage-clamp protocol. The kinetics superpose almost exactly for modest test depolarizations, whereas they clearly fail to superpose completely for more positive levels of membrane depolarization. We have modeled these results by incorporating a time dependence into the rate constant of activation of potassium channel gates in the Hodgkin and Huxley model of potassium ionic conductance.     

Further studies of activation-inactivation coupling in Myxicola axons. Insensitivity to changes in calcium concentration.

In Myxicola axons subjected to moderate depolarizations the sodium inactivation time constants obtained from the decay of sodium current during a maintained depolarizatin (TSh) are substantially smaller than inactivation time constants determined at the same potential from the effect of changes in the duration of conditioning prepulses (Tph). This report extends these observations to positive membrane potentials and demonstrates that for sufficiently large depolarizations TSh and Tph become comparable. The ratio of inactivation time constants, Tph/TSh, is unaffected by changes in [Ca++] provided total divalent cation concentration is maintained constant, while changes in total divalent ion concentrations produce simple voltage shifts comparable to those obtained from measurement of membrane sodium or potassium conductances. Sodium inactivation delay was quantitatively determined as a function of membrane potential, and found to be similarly unaffected by changes in [Ca++] at constant total divalent ion concentration. Inactivation delay is, however, directly proportional to the activation rate constant over a wide range of potentials.     

Potassium ion currents in the crayfish giant axon. Dynamic characteristics.

The kinetics of the voltage-sensitive potassium channel in crayfish axon have been examined. The conductance increase after a step depolarization from rest can be described by a first-order kinetic process raised to the third power. When conditioning voltage levels preceded the test pulse, the steady-state conductance was found to be independent of initial conditions. Depolarizing conditioning voltages in general allowed superposition of test voltage potassium currents by a shift along the time axis. Hyperpolarizing conditioning voltages produced a delay in onset of conductance during the test pulse and changed the kinetics so that superposition was not possible. The delay increased during the hyperpolarization with a first-order lag having a time constant in the range of 1.5-3 ms. Return to the resting level caused recovery from the delayed state to follow a single exponential decay with a time constant of 1.9-2.2 ms. The steady state delay vs. voltage curves were not saturated at potentials as negative as -180 mV.     

Depolarization-activated potentiation of the T fiber synapse in the blue crab

The blue crab T fiber synapse, associated with the stretch receptor of the swimming leg, has a nonspiking presynaptic element that mediates tonic transmission. This synapse was isolated and a voltage clamp circuit was used to control the membrane potential at the release sites. The dependence of transmitter release on extracellular calcium, [Ca]o, was studied over a range of 2.5-40 mM. A power relationship of 2.7 was obtained between excitatory postsynaptic potential (EPSP) rate of rise and [Ca]o. Brief presynaptic depolarizing steps, 5-10 ms, presented at 0.5 Hz activated EPSP's of constant amplitude. Inserting a 300-ms pulse (conditioning pulse) between these test pulses potentiated the subsequent test EPSPs. This depolarization-activated potentiation (DAP) lasted for 10-20 s and decayed with a single exponential time course. The decay time course remained invariant with test pulse frequencies ranging from 0.11 to 1.1 Hz. The magnitude and decay time course of DAP were independent of the test pulse amplitudes. The magnitude of DAP was a function of conditioning pulse amplitudes. Large conditioning pulses activated large potentiations, whereas the decay time constants were not changed. The DAP is a Ca-dependent process. When the amplitude of conditioning pulses approached the Ca equilibrium potential, the magnitude of potentiation decreased. Repeated application of conditioning pulses, at 2-s intervals, did not produce additional potentiation beyond the level activated by the first conditioning pulse. Comparison of the conditioning EPSP waveforms activated repetitively indicated that potentiation lasted transiently, 100 ms, during a prolonged release. Possible mechanisms of the potentiation are discussed in light of these new findings.     

Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. Interactions between toxin binding and channel gating

Tetrodotoxin (TTX) block of cardiac sodium channels was studied in rabbit Purkinje fibers using a two-microelectrode voltage clamp to measure sodium current. INa decreases with TTX as if one toxin molecule blocks one channel with a dissociation constant KD approximately equal to 1 microM. KD remains unchanged when INa is partially inactivated by steady depolarization. Thus, TTX binding and channel inactivation are independent at equilibrium. Interactions between toxin binding and gating were revealed, however, by kinetic behavior that depends on rates of equilibration. For example, frequent suprathreshold pulses produce extra use-dependent block beyond the tonic block seen with widely spaced stimuli. Such lingering aftereffects of depolarization were characterized by double-pulse experiments. The extra block decays slowly enough (tau approximately equal to 5 s) to be easily separated from normal recovery from inactivation (tau less than 0.2 s at 18 degrees C). The amount of extra block increases to a saturating level with conditioning depolarizations that produce inactivation without detectable activation. Stronger depolarizations that clearly open channels give the same final level of extra block, but its development includes a fast phase whose voltage- and time-dependence resemble channel activation. Thus, TTX block and channel gating are not independent, as believed for nerve. Kinetically, TTX resembles local anesthetics, but its affinity remains unchanged during maintained depolarization. On this last point, comparison of our INa results and earlier upstroke velocity (Vmax) measurements illustrates how much these approaches can differ.     

Modulation by dopamine of population spikes in area CA1 hippocampal neurons elicited by paired stimulus pulses

Extracellular recording techniques were used to study the effects of dopamine on postactivation excitability of rat area CA1 hippocampal neurons maintained in vitro. Population spikes were elicited by delivery of conditioning and test stimulus pulses to afferent fibers. The interval between the conditioning and test volley was set to separate delivery of stimuli by 10 to 80 msec. The effect of superfusion or microtopical application of dopamine (DA) on population responses to test stimulus pulses was studied. When paired stimulus volleys, separated by brief intervals (up to 40 msec), were delivered to afferent fibers, paired-pulse suppression (PPS) was indicated by the amplitude of the population spike elicited by the test volley being smaller than that elicited by the conditioning volley. When paired volleys were separated by longer intervals (40 to 80 msec), the response elicited by the test volley was larger in amplitude than that elicited by the conditioning volley, indicating paired-pulse facilitation (PPF). Following exposure to DA, the amplitude of the population response elicited by the conditioning volley was larger than the amplitude before exposure to DA. This effect was long-lasting, enduring for tens of minutes. However, when the amplitude of the conditioning population response was held constant, the PPS was decreased, indicating disinhibition. It is suggested that dopamine produces a long-lasting attenuation of an intervening inhibitory influence onto CA1 pyramidal neurons.     

Does it pay to delay? Flesh flies show adaptive plasticity in reproductive timing

Life-history plasticity is widespread among organisms. However, an important question is whether it is adaptive. Most models for plasticity in life-history timing predict that animals, once they have reached the minimal nutritional threshold under poor conditions, will accelerate development or time to reproduction. Adaptive delays in reproduction are not common, especially in short-lived species. Examples of adaptive reproductive delays exist in mammalian populations experiencing strong interspecific (e.g., predation) and intraspecific (e.g., infanticide) competition. But are there other environmental factors that may trigger an adaptive delay in reproductive timing? We show that the short-lived flesh fly Sarcophaga crassipalpis will delay reproduction under nutrient-poor conditions, even though it has already met the minimal nutritional threshold for reproduction. We test whether this delay strategy is an adaptive response allowing the scavenger time to locate more resources by experimentally providing supplemental protein pulses (early, mid and late) throughout the reproductive delay period. Flies receiving additional protein produced more and larger eggs, demonstrating a benefit of the delay. In addition, by tracking the allocation of carbon from the pulses using stable isotopes, we show that flies receiving earlier pulses incorporated more carbon into eggs and somatic tissue than those given a later pulse. These results indicate that the reproductive delay in S. crassipalpis is consistent with adaptive post-threshold plasticity, a nutritionally linked reproductive strategy that has not been reported previously in an invertebrate species.     

Adaptation and cross-adaptation to odor stimulation of olfactory receptors in the tiger salamander

We have used the effects of self- and cross-adaptation on the unitary responses of olfactory receptors of the tiger salamander to odor stimulation to investigate the stimulus-specific components of these responses and to provide information about the cross-cell variations in the numbers and numbers of types of constitutent receptive sites. An olfactometer delivered sequential odorous pulses, either juxtaposed or separated by a variable time delay. We used four pairs of odorants judged to be similar within a given pair. The unitary response to the test stimulation relative to that of the conditioning stimulation varied from being unchanged to being completely eliminated. We sometimes observed substantial poststimulus increases in the firing rate following stimulation with juxtaposed odorous pulse. Except in the case of one odorant pair, cross-adaptation occurred both with juxtaposed pulses and with pulses separated in time. With the methyl butyrate/ethyl butyrate odorant pair, however, statistically significant cross-adaptation appeared only with juxtaposed pulses. We propose a simple model to aid in explaining these phenomena. The experimental observations in conjunction with this model are used to obtain estimates of the maximal and minimal number of receptive site types available for interaction with the chosen odorants.     

Gating currents in th intact crayfish giant axon.

Both single-sweep and signal-averaged asymmetry current are measured from intact crayfish axons after ionic currents are blocked with tetrodotoxin and 4-aminopyridine. The ON asymmetry charge saturates at about 0 mV and no ON charge movement is detectable at voltages negative to -140 mV. The areas of ON and OFF asymmetry charge are equal for short depolarizations but the ratio QOFF/QON decreases for longer depolarizing pulses. Sodium and asymmetry current magnitudes can be changed in parallel by lowering the hold potential or by imposing conditioning prepulses. Our results are consistent with the concept that asymmetry current in generated by movement of trapped charge in association with Na channel gating.     

Dynamics of potassium ion currents in squid axon membrane. A re-examination.

The original experiments of Cole and Moore (1960. Biophys. J. 1:161-202.), using conditioning and test membrane potentials to examine the dynamics of the potassium channel conductance in the squid axon, have been extended to test voltage levels by the use of tetrodotoxin to block the sodium conductance. The potassium currents for test voltage levels from -20 to +85 mV were superposable by translation along the time axis for all conditions tested: (a) with depolarizing conditioning voltages; (b) with hyperpolarizing conditioning voltages; and (c) in normal and in high potassium external media. The only deviations from superposition seen were when the internal sodium concentration was abnormally high and the potassium currents showed saturation at high levels of depolarization. Some restoration toward normal kinetics could be obtained by rapidly repeated depolarizations.     

Properties of an early outward current in single cells of the mouse ventricle

Single ventricular myocytes of adult mice were prepared by enzymatic dissociation for voltage clamp experiments with the one suction pipette dialysis method. After blocking the Na current by 10(-4) mol/l TTX early outward currents (IEO) with incomplete inactivation could be elicited by clamping from -50 mV to test potentials (VT) positive to -30 mV. Interfering Ca currents were very small (less than 0.6 nA at VT = 0 mV). The approximation of IEO by the q4r-model showed a pronounced decrease in the time constant of activation (tau q) to more positive potentials. At 50 ms test pulses the time course of the incomplete inactivation could be described by two exponentials and a constant. The time constant of the fast exponential (tau r1) showed a slight decline towards more positive test potentials (8.1 +/- 1.0 ms at -10 mV; 5.8 +/- 1.2 ms at +50 mV, mean +/- SD, n = 5) whereas the time constant of the slow exponential (tau r2) was voltage independent (41.1 +/- 7.9 ms, mean +/- SD, n = 5). The contributions of the fast exponential and the pedestal increased towards positive test potentials. The Q10 value for the time constants of activation and fast inactivation was 2.36 +/- 0.19 and 2.51 +/- 0.09 (mean +/- SD, n = 3), respectively. After an initial delay the recovery of IEO at a recovery potential of -50 mV could be fitted monoexponentially with a time constant of 16.3 +/- 2.9 ms (mean +/- SD, n = 3). The time course of the onset of inactivation determined with the double pulse protocol was slower than the decay at the same potential, and could be described as sum of a fast (tau = 18.4 +/- 6.0 ms) and a slow (tau = 62.1 +/- 19.9ms, mean +/- SD, n = 3) exponential. IEO could be blocked completely by 1 mmol/l 4-aminopyridine at potentials up to +20 mV. Stronger depolarizations had an unblocking effect.     

Cycloheximide-induced mitotic delay in Physarum polycephalum

Cycloheximide pulses applied to Physarum polycephalum surface plasmodia delay mitosis. Pulses applied in G2 cause a delay of mitosis which is linearly dependent on the phase in the cell cycle at which the pulse is applied. A 30 min pulse of 10 micrograms/ml cycloheximide starting in G2 at time t after mitosis induces an excess delay (delay in excess of pulse duration) of the next mitosis of (0.55) t-1.3 h. The excess delays induced by 7 h pulses during G2 are at most 1 h larger. Pulses applied less than 30 min before mitosis induce only small delays.     

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)     

U-type inactivation of Kv3.1 and Shaker potassium channels.

We previously concluded that the Kv2.1 K(+) channel inactivates preferentially from partially activated closed states. We report here that the Kv3.1 channel also exhibits two key features of this inactivation mechanism: a U-shaped voltage dependence measured at 10 s and stronger inactivation with repetitive pulses than with a single long depolarization. More surprisingly, slow inactivation of the Kv1 Shaker K(+) channel (Shaker B Delta 6--46) also has a U-shaped voltage dependence for 10-s depolarizations. The time and voltage dependence of recovery from inactivation reveals two distinct components for Shaker. Strong depolarizations favor inactivation that is reduced by K(o)(+) or by partial block by TEA(o), as previously reported for slow inactivation of Shaker. However, depolarizations near 0 mV favor inactivation that recovers rapidly, with strong voltage dependence (as for Kv2.1 and 3.1). The fraction of channels that recover rapidly is increased in TEA(o) or high K(o)(+). We introduce the term U-type inactivation for the mechanism that is dominant in Kv2.1 and Kv3.1. U-type inactivation also makes a major but previously unrecognized contribution to slow inactivation of Shaker.     

The mechanism of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor desensitization after removal of glutamate.

We have examined responses of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptors in the chick nucleus magnocellularis to pairs of pulses of glutamate and determined the extent of desensitization and the rate of recovery. Receptors recovered from desensitization with a time constant of 16 ms, regardless of the concentration or duration of the conditioning pulse. Even with very brief conditioning pulses, evoking submaximal currents, desensitization occurred at a consistent rate after the removal of free ligand. A quantitative kinetic model based on these data shows that receptors must desensitize from a closed state. The results provide evidence that very brief exposure to glutamate, on the time scale of uniquantal synaptic transmission, will result in a significant reduction in sensitivity of postsynaptic receptors.     

Training history affects magnitude of spontaneous recovery from extinction of appetitive conditioned responding

Three experiments examined spontaneous recovery from extinction of appetitive conditioned responding (CR) as a function of training history. Rats first received reinforced and non-reinforced conditioning trials. Groups of rats were equated for total number of reinforced trials but differed in the number of non-reinforced trials, or in the order of reinforced and non-reinforced trials. CR was then extinguished in all groups. Subsequently, the extent of recovery of CR was assessed in a test session performed either 1 or 17 days after the last extinction session. After a 17-day delay, rats that had received all reinforced trials immediately prior to the first extinction session showed stronger recovery than did rats having received all reinforced trials at the beginning of training, or interspersed among non-reinforced trials. No significant spontaneous recovery was observed after a 1-day test delay. These results, which may be of clinical relevance with respect to relapse after therapy, are explained in terms of the training schedules generating differences in strength of inhibitory associations, and a relatively long, but not a short, test delay attenuating these associations.