Block of potassium outward currents in the crayfish stretch receptor neurons by 4-aminopyridine,tetraethylammonium chloride and some other chemical substances

The effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the outward potassium currents in the rapidly and slowly adapting stretch receptor neurons (SRNs) of the crayfish (Pacifastacus leniusculus) were studied using a two micro-electrode voltage-clamp technique. The leakage current was not affected by either 4-AP or TEA. External 4-AP blocked the peak outward current in a dose-dependent manner (1:1 stoichiometry) with an apparent dissociation constant (K_d) of 2.3 ± 0.2 mm (mean ± SEM) in the slowly and 1.4 ± 0.2 mm in the rapidly adapting SRN, the block being voltage dependent. External application of TEA resulted in a block of the steady state current enhancing the transient characteristics of the current response. The block appeared to deviate from a 1: 1 stoichiometry and the apparent K_d for TEA was 9.6 ± 3.4 mm with a cooperativity factor n= 0.43 ± 0.03 in the slowly adapting SRN and 34.5 ± 9.2 mm and 0.37 ± 0.03 respectively in the rapidly adapting SRN. Low Ca²⁺, apamin and charybdotoxin, which are known to block Ca²⁺-dependent K-currents, had no effects on the outward current as was also the case with catechol. It is concluded that the different effects of TEA and 4-AP on the outward current in the two types of SRNs can be explained by the presence of at least two, probably heteromultimeric, channel populations having similar sensitivity to 4-AP but different sensitivity to TEA. One channel has a high affinity (K_d= 0.8–1.6 mm) for TEA and the other a low affinity (K_d= 173–213 mm) for TEA. The low-affinity channel seems to dominate in the slowly adapting SRN while both channels are equally common in the rapidly adapting SRN. Further, the present results do not support the existence of a macroscopic Ca²⁺-dependent K⁺ current in the SRNs.     

Modulation of Ca(2+) signaling by K(+) channels in a hypothalamic neuronal cell line (GT1-1)

The pulsatile release of gonadotropin releasing hormone (GnRH) is driven by the intrinsic activity of GnRH neurons, which is characterized by bursts of action potentials correlated with oscillatory increases in intracellular Ca(2+). The role of K(+) channels in this spontaneous activity was studied by examining the effects of commonly used K(+) channel blockers on K(+) currents, spontaneous action currents, and spontaneous Ca(2+) signaling. Whole-cell recordings of voltage-gated outward K(+) currents in GT1-1 neurons revealed at least two different components of the current. These included a rapidly activating transient component and a more slowly activating, sustained component. The transient component could be eliminated by a depolarizing prepulse or by bath application of 1.5 mM 4-aminopyridine (4-AP). The sustained component was partially blocked by 2 mM tetraethylammonium (TEA). GT1-1 cells also express inwardly rectifying K(+) currents (I(K(IR))) that were activated by hyperpolarization in the presence of elevated extracellular K(+). These currents were blocked by 100 microM Ba(2+) and unaffected by 2 mM TEA or 1.5 mM 4-AP. TEA and Ba(2+) had distinct effects on the pattern of action current bursts and the resulting Ca(2+) oscillations. TEA increased action current burst duration and increased the amplitude of Ca(2+) oscillations. Ba(2+) caused an increase in the frequency of action current bursts and Ca(2+) oscillations. These results indicate that specific subtypes of K(+) channels in GT1-1 cells can have distinct roles in the amplitude modulation or frequency modulation of Ca(2+) signaling. K(+) current modulation of electrical activity and Ca(2+) signaling may be important in the generation of the patterns of cellular activity responsible for the pulsatile release of GnRH.     

Pharmacology of a slowly inactivating outward current in hippocampal CA3 pyramidal neurons

The pharmacology of a slowly inactivating outward current was examined using whole cell patch-clamp recordings in CA3 pyramidal cells of guinea pig hippocampal slices. The current had a low activation threshold (about -60 mV) and inactivated slowly (time constant of 3.4 +/- 0.5 s at -50 mV) and completely at membrane voltages depolarized to -50 mV. The slowly inactivating outward current was mainly mediated by K+ with a reversal potential close to the equilibrium potential for K+. The slowly inactivating outward current had distinct pharmacological properties: its time course was not affected by extracellular Cs+ (1 mM) or 4-AP (1-5 mM)-broad spectrum inhibitors of K+ currents and of inactivating K+ currents, respectively. The presence of extracellular Mn2+ (0.5-1 mM), which suppresses several Ca2+ -dependent K+ currents, also did not affect the slowly inactivating outward current. The current was partially suppressed by TEA (50 mM) and was blocked by intracellular Cs+ (134 mM). In addition, intracellular QX-314 (5 mM), a local anesthetic derivative, inhibited this current. The slowly inactivating outward current with its low activation threshold should be operational at the resting potential. Our results suggest that the transient outward current activated at subthreshold membrane potentials in hippocampal pyramidal cells consists of at least three components. In addition to the well-described A- and D-currents, the slowest decaying component reflects the time course of a distinct current, suppressible by QX-314.     

Characteristics of potassium and calcium currents of hepatic stellate cells (ito) in rat

Hepatic stellate cells (HSCs) are known to play a role in the pathogenesis of the increased intrahepatic vascular resistance found in chronic liver diseases. The aim of this study was to evaluate the K+ and Ca2+ currents in cultured HSCs from rat liver, through the patch-clamp technique. Most cells were positive for desmin immunostain after isolation and in alpha-smooth muscle actin immunostain after 10 - 14 days of culturing. Outward and inward rectifying K+ currents were confirmed. Two different types of K+ currents were distinguished: one with the inward rectifying current and the other without. The outward K+ currents consisted of at least four components: tetraethylammonium (TEA)-sensitive current, 4-aminopyridine (4-AP)-sensitive current, pimozide-sensitive current and three blocker-resistant current. The peaks of the outward K+ currents evoked by a depolarizing pulse were decreased to 32.0 +/- 3.0, 62.8 +/- 3.7 and 32.8 +/- 3.5% by 5 mM TEA, 2 mM 4-AP and 15 micro M pimozide, respectively. Moreover, the combined application of three blockers caused 86.6 +/- 4.8% suppression. The inward currents evoked hyperpolarizing pulses were inwardly rectifying and almost blocked by Ba2+. Elevation of external K+ increased the inward current amplitude and positively shifted its reversal potential. Voltage- dependent Ca2+ currents which were completely abolished by Cd2+ and nimodipine were detected in 14 day cultured HSCs. In this study, the cultured HSCs were found to express outward K+ currents composed of multiple pharmacological components, Ba2+-sensitive inward rectifying K+ current and L-type Ca2+ current.     

Three pharmacologically distinct potassium channels in molluscan neurones.

1. Potassium currents were studied under voltage-clamp conditions in nerve cell bodies of the nudibranch Tritonia diomedia. 2. Potassium currents could be separated into three distinct components on the basis of their sensitivity to 4-aminopyridine (4-AP), tetraethyl-ammonium (TEA) and to Co2+ and Mn2+ ions. 3. A transient potassium current, similar to the fast outward current described by Connor & Stevens (1971b) and Neher (1971), was blocked by externally applied 4-AP but was much less sensitive to TEA or to Co2+ or Mn2+. A single 4-AP ion binds each receptor with an apparent dissociation constant of 1-5 X 10(-3) M. 4-AP decreases the rates of activation and inactivation and reduces the maximum conductance of transient current channels. 4. Delayed outward current was not effected by 4-AP at concentrations which blocked the transient current, but it could be divided into two components by external application of TEA and Co2+ or Mn2+. 5. A voltage-dependent component of delayed current, termed K-current, was blocked by TEA. Each K-current receptor binds a single TEA ion with an apparent dissociation constant of 8 X 10(-3) M. Co2+ and Mn2+ have little or no effect on K-current. 6. A second component of delayed outward current, termed C-current, depends on Ca2+ entry for its activation. It is similar to the Ca2+ dependent potassium current reported by Meech & Stranden (1975) in Helix cells. C-current is essentially blocked by 30 mM external Co2+ or Mn2+. It is little affected by TEA, however, being reduced by about 20% at a TEA concentration of 100 mM. 7. It is concluded that three sets of potassium selective channels contribute to the outward current and that these channels can be separated pharmacologically.     

Transient voltage and calcium-dependent outward currents in hippocampal CA3 pyramidal neurons

Membrane currents activated by step changes in membrane potential were studied in hippocampal pyramidal neurons of region CA3 using the single microelectrode voltage-clamp technique. The transient outward current activated by depolarizing steps appeared to be composed of two transient currents that could be distinguished by differences in voltage sensitivity, time course, and pharmacological sensitivity. The more slowly decaying current was activated by voltage steps positive to -60 mV and declined exponentially with a time constant between 200 and 400 ms. This current inactivated as the holding potential was made more positive over the range of -75 to -45 mV and was 50% inactivated near -60 mV. The more slowly decaying transient current was selectively blocked by 0.5 mM 4-aminopyridine (4-AP) but not by 5-10 mM tetraethylammonium (TEA) or 2-5 mM Mn2+. The second transient current had a much faster time course than the 4-AP-sensitive current, having a duration of 5-20 ms. This very fast transient current was observed during potential steps positive to -45 mV. The fast transient current was inactivated when the holding potential was made positive to -45 mV. The amplitude of the fast transient current was greatly reduced by the application of 4 mM Mn2+ or Ca2+-free artificial cerebrospinal fluid (CSF). The fast transient current appeared to be unaffected by 0.5 mM 4-AP but was greatly reduced by 10 mM TEA. These results suggest that the transient outward current observed during depolarizing steps is composed of at least two distinct transient currents. The more slowly decaying current resembles the A-current originally described in molluscan neurons (9, 32, 42) in voltage sensitivity, time course, and pharmacological sensitivity. The faster transient current resembles a fast, Ca2+-dependent transient current previously observed in bull-frog sympathetic neurons (5, 27).     

Permeability characteristics of monovalent cations in atrial myocytes of the rat heart

We investigated the permeability of Cs+ and Na+ through various ion channels in rat atrial myocytes using the whole-cell voltage-clamp technique. With isotonic CsCl (140 mM) on both sides of the membrane and nominally [Ca2+]o-free conditions, depolarising clamp pulses induced an increase of outward currents which showed a biphasic time course. Repolarisation to the holding potential induced inward tail currents. With isotonic NaCl, depolarisation also induced outward currents which showed a monotonic decay, but inward tail currents were not observed. Both in NaCl and CsCl, currents were hardly affected by TEA (10 mM), 4-AP (5 mM) and DIDS (100 microM). Nicardipine (1 M) almost completely blocked time-dependent outward currents in isotonic NaCl solution, leaving only time-independent currents which showed linear I-V relationship. In isotonic CsCl conditions, nicardipine blocked outward current considerably, but there still remained time-dependent outward currents and inward tail currents. Addition of E-4031 (2-20 M) which is known as a specific blocker of the rapidly activating delayed rectifier K+ current (IKr) completely blocked these time-dependent outward and inward currents, leaving only a time-independent current. Time-independent currents recorded in the presence of nicardipine and E-4031 were inhibited by GdCl3, which is known to block non-selective cation (NSC) currents. From these results, it was suggested that NSC current in atrial myocytes can be investigated in isotonic Cs+ or Na+ solution in the presence of Ca2+ channel and IKr blockers.     

Serotonergic modulation of two potassium currents in the pleural sensory neurons of Aplysia

1. The properties of membrane currents that were modulated by serotonin (5-HT) were investigated with two-electrode voltage-clamp techniques in sensory neuron somata isolated from the pleural ganglion of Aplysia californica. The modulatory effects of 5-HT were revealed by computer subtraction of current responses elicited in the presence of 5-HT from current responses elicited prior to the application of 5-HT. The complexities of the resulting 5-HT difference currents (I5-HT) suggested that 5-HT modulated more than one component of membrane current. 2. The 5-HT difference currents appeared to have at least two distinct components. One component was clearly evident at membrane potentials more negative than -10 mV was relatively voltage independent and did not inactivate. A second component was activated at membrane potentials more positive than -10 mV, had complex kinetics, and was highly voltage dependent. In an attempt to identify the membrane currents that were modulated by 5-HT, we compared the pharmacologic sensitivity of I5-HT to that of previously described K+ currents. 3. The two components of I5-HT had different sensitivities to agents that block K+ currents. The relatively voltage-independent component of I5-HT was not blocked by 2 mM 4-aminopyridine (4-AP) and was relatively insensitive to tetraethylammonium (TEA) (estimated Kd of 92 mM). In contrast, the voltage-dependent component of I5-HT was blocked by 4-AP (2 mM) and moderate concentrations of TEA (estimated Kd of 5 mM). 4. The K+ current blockers that were used to examine I5-HT were also used to examine voltage-activated membrane currents. Externally applied TEA blocked the delayed or voltage-dependent K+ current (IK.V) with an estimated dissociation constant (Kd) of 8 mM and a membrane current similar to the Ca2+-activated K+ current (IK.Ca) with an estimated Kd of 0.4 mM. In addition, externally applied 4-AP (2 mM) blocked IK.V. Thus TEA and 4-AP were equipotent in blocking both IK.V and the voltage-dependent component of I5-HT. 5. The suggestion that I5-HT contained multiple components was supported further by examining the modulatory effects of adenosine 3',5'-cyclic monophosphate (cAMP) that mediates some actions of 5-HT on membrane currents in these cells. cAMP difference currents (IcAMP) were similar to the relatively voltage-independent component of I5-HT. The subsequent addition of 5-HT to solutions already containing cAMP resulted in 5-HT difference currents similar to the voltage-dependent component of I5-HT.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three types of depolarization-activated potassium currents in acutely isolated mouse vestibular neurons

The nature and electrophysiological properties of Ca(2+)-independent depolarization-activated potassium currents were investigated in vestibular primary neurons acutely isolated from postnatal mice using the whole cell configuration of the patch-clamp technique. Three types of currents were identified. The first current, sensitive to TEA (I(TEA)) and insensitive to 4-aminopyridine (4-AP), activated at -40 mV and exhibited slow activation (tau(ac), 38.4 +/- 7.8 ms at -30 mV, mean +/- SD). I(TEA) had a half activation potential [V(ac(1/2))] of -14.5 +/- 2.6 mV and was inactivated by up to 84.5 +/- 5.7% by 10-s conditioning prepulses with a half inactivation potential [V(inac(1/2))] of -62.4 +/- 0.2 mV. The second current, sensitive to 4-AP (maximum block around 0.5 mM) and to alpha-dendrotoxin (I(DTX)) appeared at -60 mV. Complete block of I(DTX) was achieved using either 20 nM alpha-DTX or 50 nM margatoxin. This current activated 10 times faster than I(TEA) (tau(ac), 3.5 +/- 0.8 ms at -50 mV) with V(ac(1/2)) of -51.2 +/- 0.6 mV, and inactivated only slightly compared with I(TEA) (maximum inactivation, 19.7 +/- 3.2%). The third current, also sensitive to 4-AP (maximum block at 2 mM), was selectively blocked by application of blood depressing substance (BDS-I; maximum block at 250 nM). The BDS-I-sensitive current (I(BDS-I)) activated around -60 mV. It displayed fast activation (tau(ac), 2.3 +/- 0.4 ms at -50 mV) and fast and complete voltage-dependent inactivation. I(BDS-I) had a V(ac(1/2)) of -31.3 +/- 0.4 mV and V(inac(1/2)) of -65.8 +/- 0.3 mV. It displayed faster time-dependent inactivation and recovery from inactivation than I(TEA). The three types of current were found in all the neurons investigated. Although I(TEA) was the major current, the proportion of I(DTX) and I(BDS-I) varied considerably between neurons. The ratio of the density of I(BDS-I) to that of I(DTX) ranged from 0.02 to 2.90 without correlation with the cell capacitances. In conclusion, vestibular primary neurons differ by the proportion rather than the type of the depolarization-activated potassium currents they express.     

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)     

Differential inhibition of glial K(+) currents by 4-AP

Spinal cord astrocytes express four biophysically and pharmacologically distinct voltage-activated potassium (K(+)) channel types. The K(+) channel blocker 4-aminopyridine (4-AP) exhibited differential and concentration-dependent block of all of these currents. Specifically, 100 microM 4-AP selectively inhibited a slowly inactivating outward current (K(SI)) that was insensitive to dendrototoxin (< or = 10 microM) and that activated at -50 mV. At 2 mM, 4-AP inhibited fast-inactivating, low-threshold (-70 mV) A-type currents (K(A)) and sustained, TEA-sensitive noninactivating delayed-rectifier-type currents (K(DR)). At an even higher concentration (8 mM), 4-AP additionally blocked inwardly rectifying, Cs(+)- and Ba(2+)-sensitive K(+) currents (K(IR)). Current injection into current-clamped astrocytes in culture or in acute spinal cord slices induced an overshooting voltage response reminiscent of slow neuronal action potentials. Increasing concentrations of 4-AP selectively modulated different phases in the repolarization of these glial spikes, suggesting that all four K(+) currents serve different roles in stabilization and repolarization of the astrocytic membrane potential. Our data suggest that 4-AP is an useful, dose-dependent inhibitor of all four astrocytic K(+) channels. We show that the slowly inactivating astrocytic K(+) currents, which had not been described as separate current entities in astrocytes, contribute to the resting K(+) conductance and may thus be involved in K(+) homeostatic functions of astrocytes. The high sensitivity of these currents to micromolar 4-AP suggests that application of 4-AP to inhibit neuronal A-currents or to induce epileptiform discharges in brain slices also may influence astrocytic K(+) buffering.     

A long lasting Ca2+ -activated outward current in guinea-pig atrial myocytes

Among other characteristics, the steady-state current-voltage relationship of patch-clamped single atrial myocytes from guinea-pig hearts is defined by an outward current hump in the potential region -15 to +40 mV. This hump was reversibly suppressed by Co2+ (3 mM) or nitrendipine (5 microM) and enhanced by Bay K 8644 (5 microM). The maintained outward current component suppressed by Co2+ extended between -15.2 +/- 1.9 mV and +39.5 +/- 1.7 mV (mean +/- SEM of 14 cells) and has an amplitude of 95.7 +/- 9.4 pA at +10 mV. In isochronal I-V curves, the hump was already visible at 400 ms with essentially the same amplitude as at 1500 ms. The Co2+-sensitive outward current underlying the hump was poorly time-dependent during 1.5 s voltage pulses but slowly relaxed upon repolarization. Tail currents reversed near the K+ equilibrium potential under our experimental conditions. The current hump of the steady-state I-V curve was also abolished by caffeine (10 mM) or ryanodine (3 microM), both drugs that interfere with sarcoplasmic reticulum function. Apamin (1 microM) or quinine (100 microM) but not TEA (5-50 mM) markedly reduced its amplitude. However, at similar concentrations as required to inhibit the hump, both apamin and quinine appeared to be poorly specific for Ca2+-activated K+ currents in heart cells since they also inhibited the L-Type Ca2+ current. It is concluded that a long lasting Ca2+-activated outward current, probably mainly carried by K+ ions but not sensitive to TEA, exists in atrial myocytes which is responsible for the current hump of the background I-V curve.     

IA current compared to low threshold calcium current in cranial sensory neurons

Whole-cell recordings of cranial sensory neurons were obtained with CsCl- and tetraethylammonium (TEA)-loaded cells as used for ICa studies. When replacing calcium (Ca) with potassium (K) in the external medium, a transient inward current developed at low threshold, if the holding potential was below -100 mV. Its activation started above -60 mV and reached its maximum at -20 mV. Its reversal potential followed the predicted value of the K equilibrium potential (EK) characteristic of a K current. Pharmacologically, it resembles the classical outward A current since it is insensitive to TEA up to 20 mM and blocked by 4-aminopyridine (4-AP) and 3,4-AP in the millimolar range. This inward A current rises rapidly in less than 5 ms and decays monoexponentially with a time constant of about 60 ms at all potentials studied. Its steady-state inactivation occurred between -100 and -60 mV with a half-inactivation at -86 mV. All these characteristics hold for a voltage-dependent A current devoid of any Ca-dependent component. The activation and inactivation of this A inward current are compared to those of ICa,t, the other low threshold current encountered on those cells. The contribution of the two currents in the control of discharge frequency is discussed.     

Inhibitory effects of potassium channel blockers on carbachol-induced contraction in rat detrusor muscle

We present accidental findings that potassium channel blockers, such as tetraethyl-ammonium (TEA) or 4-aminopyridine (4-AP), inhibit the sustained tonic contraction induced by carbachol in rat detrusor muscle strips. The relatively lower concentrations (<2 mM) of TEA and 4-AP inhibited phasic and tonic contractions induced by 5 micro M carbachol, whilst the relatively higher concentrations of TEA and 4-AP (>5 mM) potentiated phasic contractions. The potentiation of phasic contraction was not observed in nicardipine pretreated condition. In nicardipine pretreated condition, the concentration-response curves for the negative inotropic effect of potassium channel blockers were shifted to the right by the increasing concentration of carbachol from 0.5 microM to 5 microM. IC50 was changed significantly from 0.19 to 0.64 mM (TEA) and from 0.21 to 0.96 (4-AP). Such inhibitory effects were also observed in Ca2+ depleted condition, where 0.1 mM EGTA and 1 microM thapsigargin were added into Ca2+ free solution. In conclusion, inhibitory effects of potassium channel blockers on carbachol-induced contraction may be ascribed to the direct inhibition of receptor-agonist binding.     

Calcium-dependent potassium currents in neurons from cat sensorimotor cortex.

1. Ca(2+)-dependent K+ currents were studied in large pyramidal neurons (Betz cells) from layer V of cat sensorimotor cortex by use of an in vitro brain slice and single microelectrode voltage clamp. The Ca(2+)-dependent outward current was taken as the difference current obtained before and after blockade of Ca2+ influx. During step depolarizations in the presence of tetrodotoxin (TTX), this current exhibited a fast onset of variable amplitude and a prominent slowly developing component. 2. The Ca(2+)-dependent outward current first appeared when membrane potential was stepped positive to -40 mV. Downsteps from a holding potential of -40 mV revealed little or no time-, voltage-, or Ca(2+)-dependent current. When membrane potential was stepped positive to -40 mV, a prolonged Ca(2+)-dependent outward tail current followed repolarization. The decay of this tail current at -40 mV was best described by a single exponential function having a time constant of 275 +/- 75 (SD) ms. The tail current reversed at 96 +/- 5 mV in 3 mM extracellular K+ concentration ([K+]o) and at more positive potentials when [K+]o was raised, suggesting that it was carried predominantly by K+. 3. The Ca(2+)-dependent K+ current consisted of two pharmacologically separable components. The slowly developing current was insensitive to 1 mM tetraethylammonium (TEA), but a substantial portion was reduced by 100 nM apamin. Most of the remaining current was blocked by the addition of isoproterenol (20-50 microM) or muscarine (10-20 microM). 4. The time courses of the apamin- and transmitter-sensitive components were similar when activated by step depolarizations in voltage clamp, but they were quite different when activated by a train of action potentials. Applying the voltage clamp at the end of a train of 90 spikes (evoked at 100-200 Hz) resulted in an Ca(2+)-dependent K+ current with a prominent rapidly decaying portion (time constant approximately 50 ms at -64 mV) and a smaller slowly decaying portion (time constant approximately 500 ms at -64 mV). The rapidly decaying portion was blocked by apamin (50-200 nM), and the slowly decaying portion was blocked by isoproterenol (20-50 microM). 5. When recorded with microelectrodes containing 2 mM dimethyl-bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (dimethyl-BAPTA), which causes prolonged afterhyperpolarizations, the Ca(2+)-dependent K+ current evoked by step depolarizations had an extremely slow onset and decay. The current recorded after a train of evoked spikes had a similar slow decay.(ABSTRACT TRUNCATED AT 400 WORDS)     

Action potentials and membrane currents of isolated single smooth muscle cells of cat and rabbit colon

Membrane potentials, action potentials and macroscopic currents in enzymatically dispersed, single smooth muscle cells of the circular layer of cat and rabbit colon were investigated. The cells did not exhibit spontaneous depolarizations and repolarizations (slow waves) or spontaneous action potentials. Single action potentials of smooth muscle cells were evoked by depolarizing current pulses of 5 ms to 3 s duration. A repetitive action potential discharge and an increase in the duration of the action potential was observed in cells during long depolarizing current pulses by superfusion with tetraethylammonium (TEA) or 4-aminopyridine (4-AP). Tetrodotoxin (TTX) did not alter the configuration of the action potential. Voltage-clamp experiments revealed two major outward macroscopic currents: a quasi-instantaneous (time-independent) and a time-dependent outward current. Both currents were identified as potassium (K) currents due to their pharmacological sensitivity to K antagonists [TEA, 4-AP and cesium (Cs)] and due to the reversal potential of outward tail currents. Barium selectively blocked the time-independent current. A time-dependent outward K current in colon cells was observed which appeared to be dependent upon entry of calcium ions (Ca²⁺) through voltage-dependent Ca-channels, since it was blocked by cadmium and low concentrations of nifedipine. The majority of cells did not exhibit transient outward currents. Inward currents were exposed in some of the cells when the K currents were blocked by external TEA and by replacement of K by Cs and TEA in the recording pipette. Inward currents were presumably carried by Ca²⁺, since they were not altered by TTX, were sensitive to external Ca concentrations and were abolished by the Ca channel antagonist, nifedipine. Carbachol augmented the amplitude of the inward Ca current.     

Voltage-dependent currents in isolated cells of the turtle retinal pigment epithelium

The electrophysiological properties of isolated turtle retinal pigment epithelial cells (RPE cells) were investigated using the whole-cell patch-clamp technique. Most RPE cells exhibited a voltage-dependent outward current activated by depolarization beyond about –43 mV that inactivated during a 500-ms voltage step. Tail current measurements indicated that the conductance underlying this current was potassium selective. This current inactivated with prolonged depolarization and was abolished or reduced by extracellular quinidine, barium, tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Steady-state inactivation of the voltage-dependent outward current revealed a time-independent outwardly rectifying current/voltage relationship in many cells. In addition, many cells had an outward current that activated slowly upon depolarization beyond about +40 mV and appeared to reverse near 0 mV in both 3 mM KCl and 30 mM KCl external solutions. This current was often observed in the presence of potassium channel blockers. Hyperpolarizing pulses commonly evoked inward currents that activated slowly and did not inactivate. These currents were commonly observed when fluoride was absent from the pipette, and only occasionally when fluoride was the major pipette anion. Tail current measurements indicated that this current was somewhat anion selective.These currents may play important roles in the homeostatic and phagocytic functions of RPE cells in their interactions with the neural retina.     

Functional properties of a slowly inactivating potassium current in guinea pig dorsal lateral geniculate relay neurons.

1. The time- and voltage-dependent properties of a slowly inactivating and depolarization-activated potassium current and the functional consequences of its activation was investigated with current and single-electrode voltage-clamp techniques applied to guinea pig dorsal lateral geniculate neurons maintained as a slice in vitro. 2. In current clamp, application of a step depolarization to near firing threshold resulted in a slowly rising membrane potential that took up to 10 s to reach steady state and firing threshold. In voltage clamp, step depolarization of the membrane potential to values positive to approximately -65 mV resulted in the rapid activation followed by slow inactivation of an outward current. In both cases the sudden depolarization was associated with a large increase in membrane conductance, which gradually lessened in parallel with the slow depolarization in current clamp or with the decrease in outward current in voltage clamp. 3. The time course of inactivation of the outward current, which we refer to as IAs, was well fitted by a two-exponential function with time constants of 96 and 2,255 ms, suggesting the presence of a fast and slow phase of inactivation. The activation threshold for IAs was about -65 to -60 mV, whereas inactivation was incomplete even at -50 mV, suggesting the presence of a substantial "window" current. The time course of removal of inactivation of IAs at -85 to -100 mV was well fitted by a single exponential function with time constant of 91 ms. 4. IAs appears to be mediated by K+. Increasing [K+]o from 2.5 to 10 mM resulted in a reduction in amplitude of IAs, whereas changing from 10 to 2.5 mM [K+]o enhanced this current. Intracellular injection of Cs+ resulted in an abolition of IAs, whereas extracellular application of Ba2+ resulted in a large decrease in the apparent input conductance but relatively little reduction of IAs. 5. Both phases of inactivation of the transient outward current were completely blocked by low doses (100 microM) of 4-aminopyridine (4-AP), but not by extracellular application of Cs+, tetraethylammonium (TEA), tetrodotoxin (TTX), or after block of transmembrane Ca2+ currents. Local application of 4-AP to neurons depolarized to near firing threshold resulted in depolarization associated with a decrease in apparent input conductance, thereby confirming the presence of a window current.4+ this bias against depolarizing inputs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of 4-AP and TEA sensitivities in mammalian myelinated nerve fibers

1. The sensitivities of mammalian myelinated axons to potassium channel blockers was studied over the course of development using in vitro sucrose gap and intra-axonal recording techniques. 2. Application of 4-aminopyridine (4-AP; 1.0 mM) to young nerves led to a delay in return to base line of the sciatic nerve compound action potential and to a postspike positivity (indicative of hyperpolarization) lasting for tens of milliseconds. These effects were very much attenuated during the course of maturation. 3. Tetraethylammonium chloride (TEA; 10 mM) application alone had little effect on the waveform of the compound action potential at any age. However, the 4-AP-induced postspike positivity was blocked by TEA, Ba/+, and Cs+. This block was observed in Ca2+-free electrolyte solutions containing EGTA (1.0 mM). 4. Immature sciatic nerves (approximately 3 wk postnatal) were incubated in a potassium-free electrolyte solution containing 120 mM CsCl for up to 1 h in an attempt to replace internal potassium with cesium. When the nerves were tested in the sucrose gap chamber using solutions containing 3.0 mM CsCl substituted for KCl, the compound action potential was broadened and a prolonged depolarization appeared, but there was no postspike positivity; the CsCl effect was similar to the combined effects of 4-AP and TEA. 5. Intra-axonal recordings were obtained to study the effects of 4-AP and TEA on individual axons. In the presence of 4-AP a single stimulus led to a burst of action potentials followed by a pronounced afterhyperpolarization (AHP) in sensory fibers. The AHP was blocked by TEA. In motor fibers 4-AP application resulted in action potential broadening with no AHP. 6. Repetitive stimulation (200-500 Hz; 100 ms) was followed by a pronounced AHP in both sensory and motor fibers at all ages studied. This activity-elicited AHP was sensitive to TEA at all ages. 7. The results indicate that 4-AP and TEA sensitivity change over the course of development in rat sciatic nerve. The effects of 4-AP are much more pronounced in immature nerves than in mature nerves, suggesting that 4-AP-sensitive channels become masked as they are covered by myelin during maturation. However, the TEA-sensitive channels, demonstrable after repetitive firing, remain accessible to TEA after myelination. These channels therefore may have a nodal representation.