Voltage-clamp analysis of the ionic conductances in a leech neuron with a purely calcium-dependent action potential

1. The purely calcium-dependent action potential of the anterior lateral giant (ALG) cell in the leech Haementeria was examined under voltage clamp. 2. Analysis with ion substitutions showed that the ALG cell action potential is generated by only two time- and voltage-dependent conductance systems, an inward Ca-dependent current (ICa) and an outward Ca-dependent K current IK(Ca). 3. The kinetic properties of the inward current were examined both in Cs-loaded neurons with Ca as the current carrier as well as in Ba-containing Ringer solutions with Ba as the current carrier, since Ba effectively blocked all time- and voltage-dependent outward current. 4. During a maintained depolarization, Ba and Ca currents activated with a time constant tau m, they then inactivated with the decay following a single exponential time course with a time constant tau h. The time constants for decay of both Ba and Ca currents were comparable, suggesting that the mechanism of inactivation of ICa in the ALG cell is largely voltage dependent. In the range of potentials from 5 to 45 mV, tau m varied from 8 to 2 ms and tau h varied from 250 to 125 ms. 5. The activation of currents carried by Ba, after correction for inactivation, could be described reasonably well by the expression I'Ba = I'Ba(infinity) [1--exp(-t/tau m)]. 6. The steady-state activation of the Ba-conductance mBa(infinity) increased sigmoidally with voltage and was approximated by the equation mBa(infinity) = (1 + exp[(Vh-6)/3])-1. The steady-state inactivation hBa(infinity) varied with holding potential and could be described by the equation hBa(infinity) = [1 + exp(Vh + 10/7)]-1. Recovery from inactivation of IBa was best described by the sum of two exponential time courses with time constants of 300 ms and 1.75 s, respectively. 7. The outward current IK(Ca) developed very slowly (0.5-1 s to half-maximal amplitude) and did not inactivate during a 20-s depolarizing command pulse. Tail current decay of IK(Ca) followed a single exponential time course with voltage-dependent time constants of between 360 and 960 ms. The steady-state activation n infinity of IK(Ca) increased sigmoidally with depolarization as described by the equation n infinity = [1 + exp(Vh-13.5)/-8)]-1. 8. The reversal potentials of IK(Ca) tail currents were close to the expected equilibrium potential for potassium and they varied linearly with log [K]o with a slope of 51 mV. These results suggest a high selectivity of the conductance for K ions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inactivation of a slow Ca2+ current in CA1 neurones of the adult rat hippocampal slice

CA1 neurones of the adult rat hippocampal slice preparation were voltage clamped at or near -40 mV membrane potential using a single electrode clamp method. Depolarizing voltage commands from a holding potential of -40 mV elicited voltage-dependent inward Ca2+ currents comprising a fast and a slow component. The latter one was investigated for its susceptibility to inactivation, which was maximally expressed at around 0 mV membrane potential. When extracellular Ca2+ was replaced by Ba2+, inward currents became much larger and were followed by long tail currents. Similar data were observed in neurones injected with the Ca2+ chelator BAPTA. It is suggested that inactivation of the slow Ca2+ current depends at least partly on the levels of intracellular free Ca2+ in hippocampal neurones.     

Kinetic properties of T-type Ca2+ currents in isolated rat hippocampal CA1 pyramidal neurons.

1. T-type Ca2+ channels producing a transient inward current were studied in pyramidal neurons acutely isolated from the ventral portion of rat hippocampal CA1 region. Membrane currents were recorded by the suction-pipette technique, which allows for internal perfusion under a single-electrode voltage clamp. 2. In all cells superfused with external solution containing 10 mM Ca2+, the T-type Ca2+ current was evoked by step depolarization to potentials more positive than -60 mV from a holding potential of -100 mV and reached a peak in the current-voltage relationship around -30 mV at 20-22 degrees C. 3. Activation and inactivation processes of T-type Ca2+ current were highly potential dependent, and the latter was fitted by a single exponential function. 4. Steady-state inactivation of T-type Ca2+ current could be fitted by a Boltzmann's equation with a slope factor of 6.0 and a half-inactivated voltage of -79 mV. 5. Recovery from inactivation of T-type Ca2+ current was not a single exponent. The major component of recovery (60-90% of total) was voltage sensitive with a time constant of 215 ms at -100 mV. 6. Amplitude of the T-type Ca2+ current depended on the external Ca2+ concentration. The ratio of peak amplitude in the individual current-voltage relationships of Ca2+, Ba2+, and Sr2+ currents passing through T-type Ca2+ channel was 1.0:0.85:1.32. The current kinetics were much the same. 7. All kinetic properties, including activation and inactivation, as well as the amplitude of T-type Ca2+ current, were temperature sensitive with Q10 (temperature coefficient) values of 1.7-2.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the dihydropyridine receptor subunits γ and α2δ on the kinetics of heterologously expressed L-type Ca2+ channels

Ba2+ currents through L-type Ca2+ channels were measured in tsA201 cells transiently transfected with expression vectors encoding the dihydropyridine (DHP) receptor subunits alpha1C, beta1a-GFP, alpha2delta and gamma. The subunit effect on channel function was studied by omitting either alpha2delta or gamma from the transfection mixture and analyzing the voltage dependence and kinetics of activation, inactivation and recovery from inactivation. Activation could be described by a single exponential function while the time course of inactivation of the Ba2+ current followed a double exponential function. Progressively longer depolarization led to increasingly slower recovery, indicating the successive occupancy of several inactive states. Activation parameters remained largely unaffected in y-deficient cells whereas the voltage dependence of inactivation was shifted by 16 mV to more positive potentials and the larger one of the two inactivation time constants was increased by one-third. On the other hand, alpha2delta-deficient cells showed decreased current density and slowed activation and inactivation. Recovery from inactivation was significantly slowed by gamma coexpression. This and the effect of the gamma subunit on steady-state inactivation were independent of the presence of alpha2delta. We conclude that y stabilizes L-type Ca2+ channel inactivation in a way similar to certain Ca(2+)-antagonistic drugs. Alpha2delta is not needed for this effect.     

Properties of persistent sodium conductance and calcium conductance of layer V neurons from cat sensorimotor cortex in vitro

Properties of the persistent sodium conductance and the calcium conductance of layer V neurons from cat sensorimotor cortex were examined in an in vitro slice preparation by use of a single microelectrode, somatic voltage clamp, current clamp, intra- and extracellular application of blocking agents, and extracellular ion substitution. The persistent sodium current (INaP) attained its steady level within 2-4 ms of a step change in voltage at every potential where it could be examined directly [to about 40 mV positive to resting potential (RP)]. Because of its fast onset INaP can be activated during a single excitatory postsynaptic potential (EPSP) and can influence the subsequent voltage time course and cell excitability. Application of a depolarizing holding potential greater than or equal to 20 mV positive to RP could inactivate spikes, thus allowing examination of INaP at voltages positive to spike threshold. At every potential where INaP was visible, it was mixed with a slow outward current. After depressing potassium currents with blocking agents, INaP could be observed during depolarizations to about 40 mV positive to RP where it is normally hidden by the larger outward currents. Indirect evidence suggests that INaP is present and large during prolonged depolarizations greater than 50 mV positive to RP. INaP was blocked by intracellular injection of the lidocaine derivative QX-314, as well as by extracellular tetrodotoxin (TTX). INaP was much more sensitive to QX-314 than was the height and rate of rise of the spike. This observation and the results in paragraph 3 above are best explained by separate INaP and spike sodium channels. After blockade of INaP and sodium spikes, Ca2+ spikes could be evoked only if potassium currents were first depressed. The Ca2+-dependent nature of the regenerative potentials was indicated by their disappearance when Co2+ or Mn2+ was substituted for Ca2+ in the perfusate and by the appearance of greatly enhanced potentials of similar form when Ba2+ was substituted for Ca2+. Ba2+ substitution greatly enhanced evoked and spontaneous synaptic potentials. Prolonged-plateau action potentials could be evoked in the presence of TTX and Ba2+. Ca2+ spike threshold was 30-40 mV positive to RP, which is significantly more positive than sodium spike threshold. Results of voltage clamp in the normal perfusate and in the presence of Ca2+-blockers or Ba2+ indicated that little or no Ca2+ conductance is activated in the voltage range 25 mV positive to RP where INaP is the dominant ionic current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Currents under voltage clamp of burst-forming neurons of the cardiac ganglion of the lobster (Homarus americanus)

Crustacean cardiac ganglion neuronal somata, although incapable of generating action potentials, produce regenerative, slow (greater than 200 ms) depolarizing potentials reaching -20 mV (from -50 mV) in response to depolarizing stimuli. These potentials initiate a burst of action potentials in the axon and are thus termed driver potentials. The somata of the anterior-most neurons (cells 1 or 2) were isolated by ligaturing for study of their membrane currents with a two-electrode voltage clamp. Inward current is attributed to Ca2+ by reason of dependence of driver potential amplitude on [Ca2+]0, independence of [Na+]0, resistance to tetrodotoxin, and inhibition by Cd (0.2 mM) and Mn (4 mM). Ca-mediated current (ICa) is present at -40 mV. It is optimally activated by a holding potential (Vh) of -50 to -60 mV and by clamps (command potential, Vc) to -10 mV. Time to peak (10-30 ms) and amplitude are strongly voltage dependent. Maximum tail-current amplitudes observed at -70 to -85 mV are ca. 100 nA. Inward tail peaks may not be resolved by our clamp (settling time, 2 ms). Tails relax with a time constant (tau) of approximately equal to 12 ms (at -70 to -85 mV). ICa exhibits inactivation in double pulse regimes. Recovery has a tau of approximately equal to 0.7 s. Tail current analyses indicate an exponential decline (tau approximately equal to 23 ms at -20 mV) toward a maintained amplitude of inward current tails. Analysis of outward currents indicates the presence of three conductance mechanisms having voltage dependences, time courses, and pharmacology similar to those of early outward current (IA), delayed outward current (IK), and outward current (IC) of molluscan neurons. Analysis of tail currents indicates a reversal potential for each of these near -75 mV, indicating that they are K currents. Early outward current, IA, shows a peak at 5 ms followed by rapid decline. Response to a second clamp given within 0.4 s is reduced; recovery is exponential, with a tau of approximately equal to 200 ms (at Vh = -50 mV). The amplitude of IA tested at 0 mV shows activation or deactivation by subthreshold shifts of Vh. The extent and rate of these changes shows voltage dependence (tau approximately equal to 100-500 ms for subthreshold prepulses). At the normal cell resting potential of -50 mV the amplitude of IA is 25% of that tested from -80 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fast and slowly inactivating components of Ca-channel current and their sensitivities to nicardipine in isolated smooth muscle cells from rat vas deferens

(1) Fast and slowly inactivating components of Ca-channel current were compared to clarify whether more than one type of Ca-channel exists in smooth muscle cells from rat vas deferens using the whole cell variant of the patch clamp technique. The pipette was filled with 150 mM Cs solution to eliminate outward current and Ba was used as the charge carrier for Ca-channel current. (2) When activated by a 5 s test pulse to 0 mV from a holding potential of −60 mV, the inactivation process of Ba-current was well fitted by the sum of two exponentials. The time constant of the faster inactivating component was 100–300 ms and that of the slower inactivating component was 1.5–3 s. Steadystate inactivation curves of the fast- and slow-components were very similar. (3) The inward current activated at 0 mV from −80 mV was inactivated faster than that from −30 mV. The voltage-dependencies of the peak current from holding potentials of −30 mV and −80 mV were similar. Both had voltage threshold at −30 mV and were maximal at +10 mV. (4) Low concentrations of nicardipine (10⁻⁹ to 10⁻⁷ M) preferentially inhibited the slow component while higher concentration (10⁻⁶ to 10⁻⁵ M) were required to block the fast component. The current activated from a holding potential of −30 mV was almost fully suppressed by 10⁻⁷ M nicardipine whereas that from −80 mV was blocked only slightly. The voltage dependencies of the peak currents before and during the superfusion with nicardipine (10⁻⁷ M) were similar although the peak amplitude was suppressed in the presence of the drug. (5) These results suggest that the existence of either (a) two populations of Ca channels that differ in the time course of inactivation and the sensitivity to nicardipine, but have nearly identical dependence on membrane potential or (b) one population of Ca channel having two different states of inactivation and the sensitivity of nicardipine, in rat vas deferens.     

Characterization of voltage-dependent calcium currents in mouse motoneurons.

1. Calcium channel currents were measured with the whole-cell patch clamp technique in cultured, identified mouse motoneurons. Three components of current were operationally defined on the basis of voltage dependence, kinetics, and pharmacology. 2. Test potentials to -50 mV or greater (10 mM external Ca2+) elicited a low-voltage activated T-type current that was transient (decaying to baseline in less than 200 ms) and had a relatively slow time to peak (20-50 ms). A 1-s prepulse to -45 mV produced approximately half-maximal inactivation of this T current. 3. Two high-voltage activated (HVA) components of current (1 transient and 1 sustained) were activated by test potentials to -20 mV or greater (10 mM external Ca2+). A 1-s prepulse to -35 mV produced approximately half-maximal inactivation of the transient component without affecting the sustained component. 4. When Ba2+ was substituted for Ca2+ as the charge carrier, activation of the HVA components was shifted in the hyperpolarizing direction, and the relative amplitude of the transient HVA component was reduced. 5. Amiloride (1-2 mM) caused a reversible, partial block of the T current without affecting the HVA components. 6. The dihydropyridine agonist isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3- pyridine-carboxylate [(+)-SDZ 202-791, 100 nM-1 microM)] shifted the activation of the sustained component of HVA current to more negative potentials and increased its maximal amplitude. Additionally, (+)-SDZ 202-791 caused the appearance of a slowed component of tail current.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-channel current and its modification by the dihydropyridine agonist BAY k 8644 in isolated smooth muscle cells

The electrophysiological properties of single smooth muscle cells isolated from the longitudinal layer of the guinea-pig ileum were studied with the whole-cell patch-clamp technique. The finding of resting potentials between -45 and -50 mV and the occurrence of spontaneous electrical activity when K+ was the predominant intracellular cation indicated that the cells were not leaky or hyperpermeable. The existence of an inward Ca2+ current overlapping in time with an outward rectifying K+ current was demonstrated. The latter could be selectively blocked by replacing internal K+ with Cs+ and external Ca2+ with Ba2+. Depolarizations to potentials between -40 and +50 mV evoked time-dependent inward currents, with a maximum peak value between -20 and 0 mV. For depolarizations beyond +50 mV time-dependent outward currents appeared. These currents were inhibited by 0.1 mM CdCl2. The activation of the inward current showed a sigmoidal time course, and the rate of onset of the current increased at more positive potentials. Inactivation could be described by two exponentials. The threshold for activation was about -40 mV, and full activation was reached at 0 mV. Inactivation was complete near 0 mV, whereas the channels were fully available at -80 mV. The fully-activated Ca2+-channel current was strongly voltage dependent. The conductance decreased for potentials close to the reversal potential, and showed rectification for hyperpolarizing potentials. The Ca2+ agonist BAY k 8644 enhanced the Ca2+-channel current without a significant effect on its kinetics. The fully-activated current and the steady-state activation were enhanced in a rather voltage-independent way.     

Intracellular N-methyl-d-glucamine modifies the kinetics and voltage-dependence of the calcium current in guinea pig ventricular heart cells

The effects of internal substitution of the impermeant cation N-methyl-d-glucamine (NMG) for Cs ion on the properties of the Ca-current (l-type channel) were examined in single guinea pig cardiac myocytes with the whole-cell clamp technique. The properties of the cobaltsensitive Ca current recorded in the presence of internal NMG or Cs were compared and the results were as follows. (1) The overall duration of the Ca-dependent slow action potential was markedly increased in the presence of internal NMG (6-fold at 0 mV) when compared to action potentials recorded with internal Cs. (2) The cobalt-sensitive Ca currents recorded with internal NMG or Cs had similar reversal potentials. However, in the presence of internal NMG, the maximum current density of the cobalt-sensitive Ca current was decreased and both the threshold and potential at which maximum current occurred were negatively shifted. (3) Voltage-dependence of steady-state activation, but not inactivation, of the cobalt-sensitive Ca current was shifted by −11.8 mV with internal NMG. (4) NMG increased the halftime of activation and inactivation of the cobalt-sensitive Ca current. The voltage-dependence of the half-time of inactivation was shifted by about −30 mV between 0 and +60 mV. Time constants measurements showed that NMG affected more the slow phase of inactivation of the Ca current. (5) When Ba was the charge carrier, NMG removed most of the inactivation of the current, suggesting a slowing of the voltage-dependent process of inactivation. (6) The results are consistent with a modification of the properties of the Ca channel by internal NMG. This work was supported by the Deutsche Forschungsgemeinschaft SFB 246 Project A1     

A-type potassium currents dominate repolarisation of neonatal rat primary auditory neurones in situ.

Spiral ganglion neurones provide the afferent innervation to cochlear hair cells. Little is known of the molecular physiological processes associated with the differentiation of these neurones, which occurs up to and beyond hearing onset. We have identified novel A-type (inactivating) potassium currents in neonatal rat spiral ganglion neurones in situ, which have not previously been reported from the mammalian cochlea, presumably as a consequence of altered protein expression associated with other preparations. Under whole-cell voltage clamp, voltage steps activated both A-type and non-inactivating outward currents from around -55 mV. The amplitude of the A-type currents was dependent on the holding potential, with steady-state inactivation relieved at hyperpolarised potentials. At -60 mV (close to the resting potential in situ) the currents were approximately 30% enabled. The inactivation kinetics and the degree of inactivation varied between cells, suggesting heterogeneous expression of multiple inactivating currents. A-type currents provided around 60% of total conductance activated by depolarising voltage steps from the resting potential, and were very sensitive to bath-applied 4-aminopyridine (0.01-1 mM). Tetraethylammonium (0.1-30 mM) also blocked the majority of the A-type currents, and the non-inactivating outward current, but left residual fast inactivating A-type current. Under current clamp, neurones fired single tetrodotoxin-sensitive action potentials. 4-Aminopyridine relieved the A-type current mediated stabilisation of membrane potential, resulting in periodic small amplitude action potentials.This study provides the first electrophysiological evidence for A-type potassium currents in neonatal spiral ganglion neurones and shows that these currents play an integral role in primary auditory neurone firing.     

Kinetic properties of a transient outward current in rat neocortical neurons.

1. Whole-cell patch-clamp techniques were used to record outward currents in embryonic rat neocortical neurons maintained in culture. In the presence of tetrodotoxin and cadmium, depolarization evoked an outward current with a complex waveform. This outward current consisted of an initial fast transient component and a late, slowly inactivating component. 2. The two outward current components could be separated pharmacologically with the use of tetraethylammonium (TEA) and 4-aminopyridine (4-AP). TEA (20 mM) applied extracellularly completely blocked the late component, unmasking a fast transient outward current (TOC). 4-AP (5 mM) applied extracellularly blocked the early component while reducing the late component by 27.8 +/- 9.7% (mean +/- SE). 3. The TOC activated after a short delay and rose rapidly to a peak. The time to peak was voltage dependent and decreased with depolarization. In the presence of 200 microM extracellular cadmium, activation threshold was around -25 mV, and current amplitude increased with depolarization. The voltage-conductance relationship was well fitted by the use of the Boltzmann equation with a Vm of +19 mV for half activation and a slope factor of +6 mV. 4. On sustained depolarization the TOC rapidly inactivated and decayed to baseline within 500-600 ms. The decay phase followed a single exponential time course with a time constant of 55-65 ms. The decay time was most rapid at potentials from +5 to +20 mV and increased slightly with further depolarization. 5. Steady-state inactivation of the TOC, in the presence of cadmium, was complete near -10 mV and was totally relieved at potentials more negative than -75 mV. With the use of the Boltzmann equation, a Vm of -34 mV for half inactivation and a slope factor of -8.6 mV were found. 6. Recovery of the TOC from steady-state inactivation followed a single exponential time course and was voltage dependent. When the membrane potential was held at -84 mV during the conditioning pulse, the time constant of recovery was 17 ms, increasing to 45.2 and 58.1 ms at holding potentials of -64 and -44 mV, respectively. Holding at potentials more negative than -84 mV produced no further change in the recovery time course. 7. The presence of 200 microM external cadmium altered the TOC activation and inactivation curves. Removal of cadmium produced a -16-mV shift in the Vm for half activation and a -25-mV shift in the inactivation curve. This sensitivity to cadmium is higher than that reported in other systems.(ABSTRACT TRUNCATED AT 400 WORDS)     

Removal of Ca current inactivation in dialysed guinea-pig atrial cardioballs by Ca chelators

Ca currents flowing during voltage clamp depolarizations were studied in cultured guinea-pig atrial cardioballs by means of single low resistance patch clamp pipettes. The pipettes were filled with solutions containing Cs+ as major cation in order to block K+ currents and high concentrations of various Ca chelating agents (EGTA, nitrilotriacetic acid, citrate, dipicolinic acid) to prevent rises of the intracellular Ca-activity by Ca-entry. Ca currents of myocytes loaded with 20 mM of either EGTA [(ethylenedioxy)-diethylenedinitrilo)tetra-acetic acid] or NTA (nitrilotriacetic acid) display a biphasic time course of inactivation at membrane potentials between -25 and +45 mV. The fast phase is reduced with increasingly positive membrane potentials. In cells loaded with either citrate or DPA (dipicolinic acid, pyridine-2,6-dicarboxylic acid) inactivation is negligible or absent for small depolarizations. In the range of membrane potentials where maximum current flows (0-+10 mV) a monophasic slow time course of inactivation is observed. At more positive membrane potentials inactivation is slowed. The amount of inactivation under this condition is related to the current density of the cell. Conditions, which for a given membrane potential reduce the amplitude of ICa such as extracellular application of blocking ions (Co2+, Cd2+), a conditioning depolarization, or 'rundown' of Ca-channels lead to a slowing or a complete removal of inactivation in cells dialysed with citrate or DPA respectively. Cells loaded with these Ca chelators did not show any symptom of voltage dependent inactivation of ICa. Under the conditions described action potentials were recorded in the current clamp mode. Upon dialysis with EGTA the typical 'triangular shaped' atrial action potential develops a plateau of 500 to 800 ms in duration. With citrate-containing pipette solutions the action potential duration usually is several seconds. The results for the first time demonstrate that inactivation of cardiac ICa can be considerably slowed or even removed. They provide further strong support for the hypothesis that inactivation of this current depends on Ca entry rather than membrane potential. The fast phase of inactivation observed with EGTA (NTA) possibly reflects the slow kinetics of the binding reaction of this type of Ca chelators.     

Transient K current in the somatic membrane of cultured central neurons of embryonic rat.

1. Somatic K currents of cultured hippocampal, striatal, and spinal cord neurons of embryonic rat were recorded under voltage clamp in membrane spheres ("blebs") excised by means of a tight-seal pipette. 2. The somatic K current in blebs was subject to rapid and near complete inactivation during 300-ms depolarizations, whereas whole-cell K currents included a substantial maintained component. Size and kinetic properties of bleb and whole-cell currents were stable throughout the recording period. 3. The steady-state inactivation of somatic A current was steeply voltage dependent and complete near voltage levels that activated current, whereas peak conductances did not saturate during depolarizations up to +90 mV. Activation started with a delay. Half-times of activation decreased with depolarization, but half-times of inactivation varied little with depolarization. Recovery from inactivation followed a sigmoidal time course with half-times of approximately 50 ms. 4. Half-times of activation and inactivation varied over more than an order of magnitude between individual neurons. Midpoint potentials of inactivation and peak conductance varied over approximately 40 mV. The parameter ranges of hippocampal, striatal, and spinal cord neurons overlapped. 5. Individual soma membranes revealed signs of K channel heterogeneity in their 4-aminopyridine block, current fluctuations, and current kinetics. On the other hand, currents elicited after conditioning pulses that established varied degrees of steady-state inactivation or of recovery from full inactivation had superimposable time courses. 6. The described characteristics of the somatic A channels are compared with those reported for the RCK4, Raw3, and mShal products expressed in Xenopus oocytes. Whereas the ranges of voltage dependencies and of most kinetic characteristics are compatible among native and cloned channels, these three cloned channels recover much more slowly from inactivation. In addition, inactivation in native channels, unlike that in RCK4 and Raw3 channels, was stable after excision in a subcellular fragment.     

Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pomatia.

The net outward current in bursting pace-maker neurones of the snail (Helix pomatia) during sustained and repeated voltage clamp pulses was studied. The properties of currents remaining in cobalt-Ringer or after TEA injection were compared with those in untreated cells. 2. With sustained voltage clamp depolarizations the net outward current first increases to a maximum at 150 msec and then declines to 60% or less of its peak intensity. This depression, which is greater during repetition of short pulses (e.g. 100 msec pulses at 0-5 sec intervals), represents a true decrease in the outward flow of K (designated IK) and is not due to a decreased driving force resulting from extracellular K accumulation. The steady-state current-voltage (I-V) relationship for IK is N-shaped (Heyer & Lux, 1976). 3. A component of IK persists when Ca and Mg in the medium are replaced by Co (ICo-res). With voltage clamp depolarizations ICo-res increases rapidly to a maximum and then partially inactivates with voltage dependent time constants of hundredths or tenths of seconds. Repolarization removes the inactivation. Thus, repeated stimulation with short pulses does not increase the depression of ICo-res-ICo-res (e.g. measured during voltage steps from holding potentials of -50 to near 0 mV) is smaller in test pulses preceded by depolarization and larger in pulses preceded by hyperpolarization. The steady state I-V relationship is not N-shaped. ICo-res is blocked by intracellular injection of tetraethylammonium (TEA). 4. Repeated voltage clamp depolarization to near 0 mV with 100 msec pulses for neurones with large Ca currents in normal Ringer produces a long-term depression which is maximal with 300-400 msec repolarizations (to -50 mV) between pulses. This corresponds with stimulus parameters for the maximum Ca current (Heyer & Lux, 1976). Intracellular injection of Ca2+ (also Ba2+ and Co2+) likewise reduces the total net outward current and especially the delayed outward current under voltage clamp. 5. The component of IK which is removed by Co is identified as Ca dependent and designated IK(Ca). With single voltage clamp pulses IK(Ca) follows the approximate time course and voltage dependence of the slow inward Ca current (Iin slow; Heyer & Lux, 1976). Several lines of evidence suggest that Ca ions moving through the membrane activate IK(Ca). 6. Part of IK cannot be blocked by intracellular TEA injection. In different neurones the magnitude of the IK component resistant to TEA (ITEA-res) is approximately proportional to the relative magnitudes of Iin slow.ITEA-res does not inactivate with sustained depolarization and shows pronounced long-term depression with repetitive stimulation at intermediate intervals and an increased outward current at the onset of the second and subsequent pulses following short repolarizations. The steady-state I-V relationship is N-shaped. ITEA-res is abolished by extracellular Co. 7. A net inward current with low depolarizations can be measured after TEA injection...     

Whole-cell K+ currents in isolated rabbit corneal epithelial cells.

Membrane currents were recorded from enzymatically isolated cells from basal layers of rabbit corneal epithelium by the whole-cell clamp technique. Pipettes contained 140.4 mM KCl and extracellular K+ concentration was varied. The membrane currents on step voltage changes were rectangular currents with some fluctuations. The fluctuations disappeared near the zero-current potential. The reversal potential in normal Tyrode's solution with 5.4 mM K+ was -57.8 +/- 6.2 mV (mean +/- S.D., n = 10). Increasing [K+]o from 5.4 to 140.4 mM shifted the reversal potentials in the positive direction with a slope of 41.0 mV/decade. Concomitant depolarization of the resting potential was observed on increasing [K+]o. The whole-cell currents were blocked by Cs+ or Ba2+. These suggest that the major current component in the corneal epithelial cells in K+.     

Characterization of Ca current underlying burst formation in lobster cardiac ganglion motorneurons

1. The anterior motorneurons of the cardiac ganglion of Homarus americanus were ligated less than 300 microns from the soma. This removes impulse-generating membrane and sites of synaptic input while preserving the ability of the soma to generate the burst-forming potentials termed "driver potentials" regenerative, slow (250-ms duration) depolarizations (to -20 mV) in response to brief, depolarizing stimuli. At stimulus intervals corresponding to rates of bursting observed in spontaneously active, intact ganglia (0.3-1.2/s), driver potential amplitude increases with increasing stimulus interval. 2. A two-electrode voltage clamp was used to characterize inward current observable from the ligated neurons in tetrodotoxin (TTX)-tetraethylammonium (TEA)-containing salines. The amplitude of inward current shows a hyperbolic relation to [Ca]o that is well fitted by a form of the Michaelis-Menten equation. Inward current is maintained but not augmented when Ca2+ is replaced by Ba2+ or Sr2+. It is concluded that the inward current, to be referred to as ICa, is mediated by voltage-dependent Ca channels. 3. Contamination of ICa by early outward current (IA) was evaluated by addition of 4-aminopyridine (4-AP, 4 mM). In the presence of 4-AP, the net inward current is increased and the potential at which maximum ICa occurs is shifted 10 mV more positive. 4. Subtraction of outward currents recorded in Mn2(+)-containing saline from overall currents in the absence of Mn2+ provided another means to separate inward from outward current. I-V curves from such "Mn-subtracted" records show ICa approaches a saturating value for steps to -5 mV and more depolarized. The time to peak ICa is voltage dependent. The largest inward currents (up to 240 nA) and minimal time to peak (4 ms) are observed for steps from holding potentials of -50 to -60 mV. 5. Decline of ICa during depolarized steps observed in Mn-subtracted records represents inactivation rather than development of competing outward current. Inactivation is slow and incomplete; the rate and fractional amount of inactivation are not directly voltage dependent. Nonsubtracted responses to 500-ms depolarizations to potentials evoking little outward current show that an initial rapid decline of ICa (tau approximately 40 ms) is followed at approximately 80 ms by a slower phase of decline (tau approximately 180 ms). With repetitive clamps, the early phase proved labile.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modification of sodium channel gating and kinetics by versutoxin from the Australian funnel-web spiderHadronyche versuta

The effects of a neurotoxin (versutoxin VTX), purified from the venom of the Australian Blue Mountains funnel-web spiderHadronyche versuta, on the ionic currents in rat dorsal root ganglion cells were investigated under voltage-clamp conditions using the whole-cell patch-clamp technique. VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. In contrast VTX produced a dosedependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation, a reduction in peak TTX-S sodium current but did not markedly slow tail current kinetics of TTX-S sodium currents. This steady-state sodium current was maintained during prolonged depolarizations at all test potentials and the reduction in sodium current amplitude produced by VTX had an apparentK _i of 37 nM. In the presence of 32 nM VTX the voltage dependence of steady-state sodium channel inactivation (h ) also showed a significant 7 mV shift in the voltage midpoint in the hyperpolarizing direction, with no change in the slope factor. In addition there was a steady-state or non-inactivating component present (14±2% of maximal sodium current) at prepulse potentials more depolarized than –40 mV, potentials which normally inactivate all TTX-S sodium channels. Finally, there was an observed increase in the rate of recovery from inactivation in the presence of VTX. These selective actions of VTX on sodium channel gating and kinetics are similar to those of-scorpion and sea anemone toxins.     

Outward membrane currents activated in the plateau range of potentials in cardiac Purkinje fibres

1. The membrane currents in Purkinje fibres under voltage clamp conditions have been investigated in the range of potentials at which the action potential plateau occurs. The results show that in this range slow outward current changes occur which are quite distinct from the potassium current activated in the pace-maker range of potentials.2. The time course of current change in response to step voltage changes is non-exponential. At each potential the current changes may be analysed in terms of the sum of two exponential changes and this property has been used to dissect the currents into two components, i(x1) and i(x2), both of which have been found to obey kinetics of the Hodgkin-Huxley type.3. The first component, i(x1), is activated with a time constant of about 0.5 sec at the plateau. At more positive and more negative potentials the time constants are shorter. The steady-state degree of activation varies from 0 at about -50 mV to about 1 at +20 mV. The instantaneous current-voltage relation is an inward-going rectifier but shows no detectable negative slope. In normal Tyrode solution ([K](0) = 4 mM) the reversal potential is about -85 mV.4. The second component, i(x2), is activated extremely slowly and the time constant at the plateau is about 4 sec. The steady-state activation curve varies from 0 at about -40 mV to 1 at about +20 mV. The instantaneous current-voltage relation is nearly linear. The reversal potential occurs between -50 and -20 mV in different preparations.5. It is suggested that these currents are carried largely by K ions, but that some other ions (e.g. Na) also contribute so that the reversal potentials are positive to E(K).6. The relation of these results to previous work on delayed rectification in cardiac muscle is discussed.