Biomedical subjects
Publications and source records attributed to H D Lux.
Inactivation of calcium channels has been attributed to a direct reaction of calcium ions entering the cell with the calcium channel. For a single channel this hypothesis predicts a correlation between the amount of calcium entering during an opening or a burst of openings and the subsequent closed times. No such correlation was found, and the possibility that, upon entry, calcium ions produce inactivation is excluded.
Adenosine 3',5'-cyclic monophosphate (cAMP) was injected by a fast and quantitative pressure injection method into voltage-clamped identified Helix neurons. The intracellular elevation of cAMP caused an inward current which was not accompanied by a significant change in membrane conductance in a negative potential range with little activation of voltage-dependent membrane conductances. Near resting potential Na+ ions were the main carrier of the cAMP-induced inward current as measured with ion-selective microelectrodes. TTX did not affect the Na+ influx. K+ and less effective Ca2+ could substitute for Na+ in carrying the inward current. In the presence of Na+, divalent cations such as Ca2+ and Mg2+, and also La3+ exerted an inhibitory influence on the cAMP-induced inward current, and Ca2+ as measured with ion-selective microelectrodes did not contribute significantly to the current. Thus, the inward current was of a non-specific nature. Simultaneously to this cAMP action, the membrane permeability for K+ ions was decreased by cAMP. This effect became particularly obvious when K+ currents were activated by long-lasting, depolarizing voltage steps. In this situation a reduced K+ efflux following cAMP injection was observed by means of K+-selective microelectrodes located near the external membrane surface. Outward K+ currents were less reduced by cAMP if external Ca2+ was replaced by Ni2+. The nearly compensatory increase and decrease of two membrane conductances in the same neuron explained the lack of change in the cell input resistance despite the considerable depolarizing action of intracellularly elevated cAMP.
Since activation of GABAA receptors is believed to open an associated Cl- channel, the intracellular Cl- activity (aiCl) must be lower than that predicted from a passive distribution, to account for hyperpolarizing responses, or higher, to account for depolarizing responses. The physiological and pharmacological properties of the implied Cl- extruding and accumulating mechanisms have been investigated by direct measurements of aiCl. A coupled K+-Cl- co-transport has been found in crayfish stretch receptor neurones and a predominating Cl(-)HCO3(-) exchange in guinea pig vas deferens. From the different ionic mechanisms involved in Cl- extrusion and accumulation, it is proposed that drugs which affect Cl- transport mechanisms will reduce GABA responses of both polarities only if their action is via interference with the Cl- recognition site, but not if it is via interference with the co- or counter-ion recognition site.
The conductance increase induced by bath application of GABA has been measured in voltage-clamped stretch-receptor neurones of crayfish. A rapid conductance increase was obtained only at GABA concentrations above 3.3 X 10(-4) M. The response to lower GABA concentrations (between 10(-4) and 10(-6) M) developed slowly over 30-60 min. Repetitive application of intermediate GABA concentrations induced postsynaptic conductance changes which were progressively enhanced in their onset and magnitude. In the presence of nipecotic acid or in Na+-free Ringer solutions, the response to all GABA concentrations was rapid and constant for each concentration. The time course of inhibitory postsynaptic currents was unaffected by nipecotic acid. These results suggest the presence of a saturable GABA uptake system which limits the access of bath-applied GABA to postsynaptic receptors. This system has little if any effect on the termination of response to synaptically released GABA.
K+ channels were studied in oligodendrocytes in cultures of mouse spinal cord. Single channel currents were measured using the gigaseal technique. The conductance of the channels varied greatly i.e. from 6 to 125 pS (38 +/- 28 SD, N = 21). In some patches there were up to three current levels of the same size. At -70 mV the open state probability was 0.51 +/- 0.17 and the average duration of an opening 70 +/- 20 ms for 4 channels with conductance from 16-57 pS. These analyses exclude brief flickering (less than 2 ms) or long closed periods (seconds to minutes). These times were not markedly affected by pulling the patch off the cell or by superfusing the isolated patch with media containing 10 mmol X 1(-1) TEA or EGTA without Ca2+. At membrane potentials between -90 and -30 mV there was a small but consistent effect of depolarization to increase the open state probability. Large positive or negative voltage steps decreased the open state probability. Current voltage measurements on intact cells showed a striking decrease in membrane conductance at these large membrane potentials. The leakage conductance of the patch also exhibited some K+ selectivity. The oligodendrocyte membrane appears to contain about one K+ channel per 5 micron 2. The known electrical properties of cultured oligodendrocytes can essentially be explained by the distribution and properties of these K+ channels.
Isolated Ca currents in cultured dorsal root ganglion (DRG) cells were studied using the patch clamp technique. The currents persisted in the presence of 30 microM tetrodotoxin (TTX) or when external Na was replaced by choline. They were fully blocked by millimolar additions of Cd2+ and Ni2+ to the bath. Two components of an inward-going Ca current were observed. In 5 mM external Ca, a current of small amplitude, turned on already during steps changes to -60 mV membrane potential, leveled off at -30 mV to a value of approximately 0.2 nA. A second, larger current component, which resembled the previously described Ca current in other cells, appeared at more positive voltages (-20 to -10 mV) and had a maximum approximately 0 mV. The current component activated at the more negative membrane potentials showed the stronger dependence on external Ca. The presence of a time- and a voltage-dependent activation was indicated by the current's sigmoidal rise, which became faster with increased depolarization. Its tail currents were generally slower than those associated with the Ca currents of larger amplitude. From -60 mV holding potential, the maximum obtainable amplitude of the low depolarization-activated current was only one-tenth of that achieved from a holding potential of -90 mV. Voltage-dependent inactivation of this current component was fast compared with that of the other component. The properties of this low voltage-activated and fully inactivating Ca current suggest it is the same as the inward current that has been postulated in several central neurons (Llinas, R., and Y. Yarom, 1981, J. Physiol. (Lond.), 315:569-584), which produce depolarizing potential waves and burst-firing only when membrane hyperpolarization precedes.
The action of lithium on cellular calcium regulation has been observed in snail neurons using electrophysiological methods. Recently, calcium-dependent mechanisms are thought to play a central role in the integrative functions of the CNS. The coupling between humoral and electrical effects of neurons by calcium iones appears to be especially interesting in respect of the clinical actions of lithium. Here we discuss clinical and experimental studies considering the question if the well known effects of lithium therapy could be produced by an action of lithium on the calcium regulation of nerve cells.
The flow of Ca ions through single Ca channels has been examined. The gigaseal method was used on identifiable snail neurons that were voltage clamped using a two-microelectrode voltage clamp method. Average Ca patch currents and whole cell currents have similar time courses. They are affected similarly by changes in temperature. The differences in amplitude and inactivation between Ba and Ca whole cell currents were present in the patch records. The stationary noise spectra recorded from ensembles of multichannel patches have two components with fast and slow time constants equivalent to two components in the whole cell tail current relaxations. Elementary current amplitudes measured from the variance-mean relationship and from noise spectra gave values comparable to measurements from single channels. The single channel I-V relationship was curvilinear and the maximum slope conductance in 40 mM Cao was 7 pS. The amplitude of unitary currents was unchanged at long times when inactivation had occurred; hence depletion is not involved in this process. Channel density was approximately 3 microns-2 and was the same for Ba and Ca currents. The whole cell asymmetry currents gave very large values for the gating charge per channel. Changes in temperature from 29 to 9 degrees C had only a slight effect on the two Ca tail current tau's at potentials where turn-on of patch and whole cell currents was markedly slowed and the peak amplitudes were reduced by one-third. Single channel recordings were obtained at these two temperatures, and the mean open time and the fast component of the closed times were scarcely affected. Unit amplitudes were reduced by 30% and the slow closed time component was doubled. Therefore, peak currents and the slow closed time component was doubled. Therefore, peak currents were reduced partly as a result of the reduction in unit amplitude, but mainly as a result of a reduction in opening probability, the latter arising from an increase of the long closed times. It is concluded that the behavior of single Ca channels in membrane patches is the same as it is in whole cells. Cooling from 29 to 9 degrees C acts primarily on transitions among closed states and has little effect on the open to closed transition.
Activation and inactivation properties of Ca currents were investigated by studying the behavior of single Ca channels in snail neurons. The methods described in the previous paper were used. In addition, a zero-phase digital filter has been incorporated to improve the analysis of latencies to first opening, or waiting times. It was found that a decrease in the probability of single channel opening occurred with time. This was especially marked at 29 degrees C and paralleled the inactivation observed in macroscopic currents. The fact that a single channel was observed means that there is a significant amount of reopening from the "inactivated" state. Small depolarizations at 18 degrees C showed little inactivation. From these measurements, histograms of single channel open, closed, and waiting times were analyzed to estimate the rate constants of a three-state model of activation. Two serious discrepancies with the model were found. First, waiting time distributions at -20 mV were slower than those predicted by parameters obtained from an analysis of the single channel closed times. Second, it was shown that the time and the magnitude of the peak of the waiting time histogram were inconsistent with a three-state model. It is concluded that a minimum of four states are involved in activation. Some four-state models may be eliminated from further consideration. However, a comprehensive model of Ca channel kinetics must await further measurements.
Brief pressure injections of aqueous solutions of cAMP in identified neurons of Helix pomatia caused depolarizations which lasted for tens of seconds. In voltage-clamped neurons an inward current of similar duration was induced which saturated at 10 microA/cm2 cell surface. In the range of negative membrane potentials with little voltage-dependent activation, this current was not accompanied by a change in membrane conductance. The inward current was not produced by injection of ATP, ADP, adenosine, inosine or cGMP. cAMP derivatives produced longer-lasting effects. Prolongation of the inward current was also observed after inhibition of the phosphodiesterase by IBMX. Drugs which block active transport had no effect on the response to cAMP injection. The inward current depended on extracellular sodium, and was maximal when all other mono- and divalent cations were replaced by Na+. The cAMP-induced current was accompanied by a transient increase in [Na+]i, but there was no change in [Cl-]i. Li+ could largely substitute for Na+; Ca2+ was less effective. Addition of Mg2+ or Ca2+ to solutions containing a high Na+-concentration inhibited the response. Internal acidification with HCl reversibly enhanced the inward current. These data indicate that the depolarizing effect of cAMP can be accounted for by an inward movement of Na-ions, and that the effect is augmented by H+-ions.
Single channel currents were recorded from isolated membrane patches of cultured mouse oligodendrocytes using the giga-seal technique. The observed conductance of the channel was 71 +/- 34 pS. Isolated patches contained 1 to 4 channels with similar conductances and kinetics. Closed times of the channel varied from less than 1 msec to many minutes. The open state was always interrupted by flickering to the closed state. The kinetics of opening and closing appeared insensitive to voltage steps of up to +/- 75 mV from the resting level of the membrane potential, but could be affected by very large voltage steps. The observed changes in channel current in response to changes of potassium concentration on either side of the membrane indicate a high selectivity for potassium. The results show a membrane with constant macroscopic permeability that contains channels which open and close.
Membrane Ca2+ currents are the triggers for numerous cellular activities such as secretions, contraction, and oscillations in neural discharge. Different Ca2+ channels are assumed to subserve the various functions; this does not apply to Na+ currents for which there is thought to be only one type of channel. We have examined single Ca2+ channels in cells from three very different species (bird, snail and rat) using the patch-clamp method, and report here that unitary events consisted of small, brief current pulses that often occurred in bursts and were similar for all three species. Thus, reported differences among various Ca2+ current must have other origins.
Current fluctuations were measured in small, 3-6 micrometers-diameter patches of soma membrane in bursting neurons of the snail, Helix pomatia. The fluctuations dramatically increased in magnitude with depolarization of the membrane potential under voltage clamp conditions. Two components of conductance noise were identified in the power spectra calculated from the membrane currents. One component had a corner frequency which increased with depolarization. This component was blocked by intracellular injection of TEA and was relatively insensitive to extracellular calcium levels (as long as the total number of effective divalent cations remained constant). It was identified as fluctuations of the voltage-dependent component of delayed outward current. The second component of conductance noise had a corner frequency which decreased with depolarization. It was relatively unaffected by TEA injection and was reversibly blocked by substitution of extracellular calcium with magnesium, cobalt, or nickel. This second component of noise was identified as fluctuations of the calcium-dependent potassium current. The results suggest that the two components of delayed outward current are conducted through physically distinct channels.
Extracellular Na+ - and Cl- -concentrations ([Na+]o, [Cl-]o) were recorded with ion-selective microelectrodes during repetitive stimulation and stimulus-induced self-sustained neuronal afterdischarges (SAD) in the sensorimotor cortex of cats. In all cortical layers [Na+]o initially decreased by 4-7 mM. In depths of more than 600 micrometer below the cortical surface such decreases usually turned into increases of 2-6 mM during the course of the SADs, whereas in superficial layers [Na+]o never rose above its resting level. [Cl-]o always showed an increase in the course of the SADs often preceded by an initial small decrease. The average increase at a depth of 1,000 micrometers was about 7 mM. [Cl-]o reached peak values at about the end of the ictal period, whereas [Na+]o reached its maximum shortly after the end of the SAD, at times when [K+]o was still elevated above the baseline concentration. These data indicate that the extracellular osmolarity can increase during SAD by up to 30 mM. Such an increase in osmolarity can be explained by an increase in the number of intracellular particles, caused by cleavage of larger molecules during enhanced metabolism. This could lead to cell-swelling due to passive water influx from the extracellular space (ES). However, the resulting reduction of the size of the ES is calculated to be less than 10% for an increase in intracellular osmolarity by 30 mOsm. This value is too small as compared to previously measured ES-reductions under similar conditions (i.e., 30% reduction at 1,000 micrometers; Dietzel et al. 1980). Reductions of the size of the ES that accompany the observed changes in the ionic environment, are quantitatively explained on the basis of the extended glial buffering mechanism described in the preceding paper.
A calcium- and voltage-dependent current was found to be the principal outward current in identified Helix neurons. The current depends on the presence of [Ca2+]0, with half maximal activation at 1 mM [Ca2+]0, and it saturates beyond about 5 mM. The current is termed IK(Ca) since the charge carried by it corresponds to the amount of potassium ions transferred from the cell interior, as determined from the increase in K+ concentration in the external volume with K+ liquid ion-exchanger microelectrodes. IK(Ca) is characterized by cell shaped isochronal I/V curves. The peaks of these curves move from +30 mV to about +70 mV with an increase of the time of measurement from 30-200 ms. IK(Ca) rise times have a minimum of 10-15 ms at low depolarization around 0 mV, but increase about exponentially with more positive potentials. A tenfold decrease in [Ca2+]0 over the range of 30 to 0.3 mM also produces an increase in rise time, equivalent to a positive shift of potential by 20 mV. On repolarization of the membrane IK(Ca) disappears much faster than the intracellularly accumulated Ca2+, with a time constant which is similar to the minimum activation time constant.
The activation of calcium-dependent outward potassium current [IK(Ca)] by shortlasting Ca2+ inward currents was studied. These Ca2+ currents were produced either by small depolarizing pulses preceding the larger depolarizations or by interposed repolarizations (I.R.s) starting from depolarized membrane potentials. IK(Ca) then develops with a potential-invariant time course (half time 6-12 ms) and the normally bell-shaped isochronal IK(Ca)/V curve, measured at between 30 and 300 ms, is straightened. However, there Ca2+ - injecting pulses, of any amplitude and duration, do not increase the steady-state conductance to values beyond those measured with single step depolarizations to lower potentials. varied in length, activation of IK(Ca) increased linearly with the during depolarization to near the supposed calcium equilibrium potential with no further Ca2+ influx. When I.R.s are varied in length, activation of I(Ca) increases linearly with the amount of Ca2+ current. Fading of activation during I.R. follows a time course nearly ten times slower than activation and is not expressed in tail currents. The time course of IK(Ca) is described by a function defined only by voltage parameters of activation combined with a minimum activation time constant which is similar to that found in tail currents. Peak location and general form of the IK(Ca)/V relationship for times up to several hundred milliseconds are well predicted without the necessity of explicitly account for actual calcium entry.
1. The intracellular Cl(-) activity (a(Cl) (i)) of isolated crayfish stretch receptor neurones was measured using liquid ion exchanger Cl(-)-selective micro-electrodes. The potential developed due to the difference between the normal extracellular Cl(-) activity (a(Cl) (o)) and a(Cl) (i) (V(Cl)) was compared with the simultaneously measured reversal potential of the inhibitory post-synaptic potential (E(i.p.s.p.)) to further clarify the ionic basis of the i.p.s.p..2. In normal Ringer solution, V(Cl) (63.3 +/- 2.3 mV) was found to be close to the resting membrane potential (E(m), 62.6 +/- 3.9 mV) while E(i.p.s.p.) (74.5 +/- 1.9 mV) was more negative than either. The V(Cl) value corresponds to an apparent a(Cl) (i) of 12.7 +/- 1.3 mM, which is about 4 mM more than required for a Cl(-) governed E(i.p.s.p.) of 74.5 mV.3. Reducing a(Cl) (o) caused smaller changes in V(Cl) than predicted for passive Cl(-) re-distributions. On complete removal of extracellular Cl(-) (Cl(o) (-)), V(Cl) increased to 84.6 +/- 2.7 mV, equivalent to an apparent a(Cl) (i) of about 5 mM-Cl(-). This value can be used as an estimate of the level of intracellular interference on the Cl(-)-selective micro-electrode.4. Increasing extracellular K(+) (K(0) (+)) decreased both V(Cl) and E(i.p.s.p.). Decreasing K(o) (+) had the converse effect. The time course of the changes in V(Cl) and E(i.p.s.p.) was much the same. The difference between V(Cl) and E(i.p.s.p.) decreased to about 3 mV in high K(o) (+), and increased to about 30 mV in low K(o) (+). This variation in the difference between E(i.p.s.p.) and V(Cl) is consistent with the assumption that anions other than Cl(-) contribute to the recorded V(Cl) rather than another ion contributes to the inhibitory current.5. Application of 5 mM-NH(4) (+) or of frusemide (6 x 10(-4) M) decreased V(Cl) and E(i.p.s.p.). The difference between V(Cl) and E(i.p.s.p.) was also decreased.6. We conclude that a(Cl) (i) is lower than predicted from a passive distribution and thus the chloride equilibrium potential (E(Cl)) is more negative than E(m). If a constant intracellular interference equivalent to about 4 mM-Cl(-) is assumed to contribute to the recorded V(Cl), E(Cl) was approximately equal to E(i.p.s.p.) in all the experimental conditions. Therefore we suggest that the i.p.s.p. is solely generated by Cl(-) ions.
1. The reversal potential of the Cl(-)-dependent, inhibitory post-synaptic potential (E(i.p.s.p.)) was measured in the isolated crayfish stretch receptor neurone using two intracellular micro-electrodes. The difference between E(i.p.s.p.) and the resting membrane potential (E(m)), the i.p.s.p. driving force, was reversibly decreased by addition of NH(3)/NH(4) (+), and the mechanism of this decrease was investigated.2. The NH(3)/NH(4) (+)-induced decrease in i.p.s.p. driving force was dose-dependent with an onset at about 0.2 mM. E(i.p.s.p.) always remained more negative than E(m) or, when the neurone was spontaneously firing, the threshold potential. E(m) and resting membrane resistance (R(m)) also decreased in a dose-dependent fashion. Synaptic conductance (g(s)) increased with low doses, but decreased on application of 20 mM-NH(3)/NH(4) (+). All the effects were fully reversible on return to normal Ringer solution.3. Intracellular acidification (substitution of 50% Cl(-) by acetate compared with isethionate) considerably reduced the i.p.s.p. driving force. Simultaneous application of NH(3)/NH(4) (+) and acetate-substituted Ringer solution caused a similar decrease in the driving force to application of the same concentration of NH(3)/NH(4) (+) under normal conditions. Increasing the extracellular pH at which a given concentration of NH(3)/NH(4) (+) was applied caused a smaller decline in the i.p.s.p. driving force. These results suggest that intracellular acidification decreases the i.p.s.p. driving force and that the NH(3)/NH(4) (+)-induced decline is caused by an action of the ammonium ion.4. Elevation of extracellular K(+) (K(+) (0)) decreased the i.p.s.p. driving force, E(m) and R(m), and increased g(s). Reduction of K(+) (0) had the converse effects on all parameters.5. Application of Rb(+) or Cs(+) mimicked the effects of NH(3)/NH(4) (+). Substitution of K(+) (0) by Rb(+), Cs(+) or NH(3)/NH(4) (+) opposed or even reversed the increase in i.p.s.p. driving force when Na(+) was used as the substitute. The effectiveness of the various cations in decreasing the driving force was in the following order: Rb(+) > NH(4) (+) > K(+) > Cs(+).6. Inhibition of the Na pump by ouabain or K(+)-free Ringer solution caused a gradual reduction in the i.p.s.p. driving force. Since the driving force also decreased when the Na(+) gradient probably was increased (elevated K(+) (0)), this suggests a dependence on the K(+) gradient rather than the Na(+) gradient or the Na pump itself.7. Frusemide (6 x 10(-4) M) reversibly decreased the i.p.s.p. driving force and E(m), and increased g(s). R(m) was not significantly affected. Application of frusemide in the presence of 5 mM-Rb(+) and vice versa, caused a further reduction in the driving force. The recovery of the driving force on removal of either agent was slowed by the presence of the other.8. Application of 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS; 10(-4) M) caused spontaneous firing and reduced E(i.p.s.p.) to the threshold potential. R(m) and g(s) increased. The effects were slowly reversible on removal of the drug.9. It is proposed that the i.p.s.p. driving force is maintained by a K(+)-Cl(-) co-transport mechanism, driven by the K(+) gradient. The K(+) site exhibits the binding selectivity: Rb(+) > NH(4) (+) > K(+) > Cs(+) and the mechanism is inhibited partially by frusemide and completely by DIDS.