A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.     

Plasmalemmal voltage-activated K(+) currents in protoplasts from tobacco BY-2 cells: possible regulation by actin microfilaments?

Plasmalemmal ionic currents from enzymatically isolated protoplasts of suspension-cultured tobacco 'Bright Yellow-2' cells were investigated by whole-cell patch-clamp techniques. In all protoplasts, delayed rectifier outward K(+) currents having sigmoidal activation kinetics, no inactivation, and very slow deactivation kinetics were activated by step depolarization. Tail current reversal potentials were close to equilibrium potential E(K) when external [K(+)] was either 6 or 60 mM. Several channel blockers, including external Ba(2+), niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, inhibited this outward K(+) current. Among the monovalent cations tested (NH(4)(+), Rb(+), Li(+), Na(+)), only Rb(+) had appreciable permeation (P(Rb)/P(K) (=) 0.7). In addition, in 60 mM K(+) solutions, a hyperpolarization-activated, time-dependent, inwardly rectifying K(+) current was observed in most protoplasts. This inward current activated very slowly, did not inactivate, and deactivated quickly upon repolarization. The tail current reversal potential was very close to E(K), and other monovalent cations (NH(4)(+), Rb(+), Li(+), Na(+)) were not permeant. The inward current was blocked by external Ba(2+) and niflumic acid. External Cs(+) reversibly blocked the inward current without affecting the outward current. The amplitude of the inward rectifier K(+) current was generally small compared to the amplitude of the outward K(+) current in the same cell, although this was highly variable. Similar amplitudes for both currents occurred in only 4% of the protoplasts in control conditions. Microfilament-depolymerizing drugs shifted this proportion to about 12%, suggesting that microfilaments participate in the regulation of K(+) currents in tobacco 'Bright Yellow-2' cells.     

Sodium ions as blocking agents and charge carriers in the potassium channel of the squid giant axon

Instantaneous K channel current-voltage (I-V) relations were determined by using internally perfused squid axons. When K was the only internal cation, the I-V relation was linear for outward currents at membrane potentials up to +240 mV inside. With 25-200 mM Na plus 300 mM K in the internal solution, an N-shaped I-V curve was seen. Voltage-dependent blocking of the K channels by Na produces a region of negative slope in the I-V plot (F. Bezanilla and C. M. Armstrong. 1972. J. Gen Physiol, 60: 588). At higher voltages (greater than or equal to 160 mV) we observed a second region of increasing current and a decrease in the fraction of the K conductance blocked by Na. Internal tetraethylammonium (TEA) ions blocked currents over the whole voltage range. In a second series of experiments with K-free, Na-containing internal solutions, the I-V curve turned sharply upward about +160 mV. The current at high voltages increased with increasing internal Na concentration was largely blocked by internal TEA. These data suggest that the K channel becomes substantially more permeable to Na at high voltages. This change is apparently responsible for the relief, at high transmembrane voltages, of the blocking effect seen in axons perfused with Na plus K mixtures. Each time a Na ion passed through, vacating the blocking site, the channel would transiently allow K ions to pass through freely.     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ionic currents and ion channels of lobster olfactory receptor neurons

The role of the soma of spiny lobster olfactory receptor cells in generating odor-evoked electrical signals was investigated by studying the ion channels and macroscopic currents of the soma. Four ionic currents; a tetrodotoxin-sensitive Na+ current, a Ca++ current, a Ca(++)-activated K+ current, and a delayed rectifier K+ current, were isolated by application of specific blocking agents. The Na+ and Ca++ currents began to activate at -40 to -30 mV, while the K+ currents began to activate at -30 to -20 mV. The size of the Na+ current was related to the presence of a remnant of a neurite, presumably an axon, and not to the size of the soma. No voltage-dependent inward currents were observed at potentials below those activating the Na+ current, suggesting that receptor potentials spread passively through the soma to generate action potentials in the axon of this cell. Steady-state inactivation of the Na+ current was half-maximal at -40 mV. Recovery from inactivation was a single exponential function that was half-maximal at 1.7 ms at room temperature. The K+ currents were much larger than the inward currents and probably underlie the outward rectification observed in this cell. The delayed rectifier K+ current was reduced by GTP-gamma-S and AIF-4, agents which activate GTP-binding proteins. The channels described were a 215-pS Ca(++)-activated K+ channel, a 9.7-pS delayed rectifier K+ channel, and a 35-pS voltage-independent Cl- channel. The Cl- channel provides a constant leak conductance that may be important in stabilizing the membrane potential of the cell.     

Characterization and localization of two ion-binding sites within the pore of cardiac L-type calcium channels

L-type Ca channels from porcine cardiac sarcolemma were incorporated into planar lipid bilayers. We characterized interactions of permeant and blocking ions with the channel's pore by (a) studying the current-voltage relationships for Ca2+ and Na+ when equal concentrations of the ions were present in both internal and external solutions, (b) testing the dose-dependent block of Ba2+ currents through the channels by internally applied cadmium, and (c) examining the dose and voltage dependence of the block of Na+ currents through the channels by internally and externally applied Ca2+. We found that the I-V relationship for Na+ appears symmetrical through the origin when equal concentrations of Na+ are present on both sides of the channel (gamma = 90 pS in 200 mM NaCl). The conductance for outward Ca2+ currents with 100 mM Ca2+ on both sides of the channel is approximately 8 pS, a value identical to that observed for inward currents when 100 mM Ca2+ was present outside only. This provides evidence that ions pass through the channel equally well regardless of the direction of net flux. In addition, we find that internal Cd2+ is as effective as external Cd2+ in blocking Ba2+ currents through the channels, again suggesting identical interactions of ions with each end of the pore. Finally, we find that micromolar Ca2+, either in the internal or in the external solution, blocks Na+ currents through the channels. The affinity for internally applied Ca2+ appears the same as that for externally applied Ca2+. The voltage dependence of the Ca(2+)-block suggests that the sites to which Ca2+ binds are located approximately 15% and approximately 85% of the electric field into the pore. Taken together, these data provide direct experimental evidence for the existence of at least two ion binding sites with high affinity for Ca2+, and support the idea that the sites are symmetrically located within the electric field across L-type Ca channels.     

Modulation of calcium channels by norepinephrine in internally dialyzed avian sensory neurons

Modulation of voltage-dependent Ca channels by norepinephrine (NE) was studied in chick dorsal root ganglion cells using the whole-cell configuration of the patch-clamp technique. Cells dialyzed with K+ and 2-10 mM EGTA exhibited Ca action potentials that were reversibly decreased in duration and amplitude by NE. Ca channel currents were isolated from other channel contributions by using: (a) tetrodotoxin (TTX) to block gNa, (b) internal K channel impermeant ions (Cs or Na/N-methylglucamine mixtures) as K substitutes, (c) external tetraethylammonium (TEA) to block K channels, (d) internal EGTA to reduce possible current contribution from Ca-activated channels. A marked decline (rundown) of Ca conductance was observed during continual dialysis, which obscured reversible NE effects. The addition of 2-5 mM MgATP to the intracellular solutions greatly retarded Ca channel rundown and permitted a clear assessment of modulatory drug effects. The inclusion of an intracellular creatine phosphate/creatine phosphokinase nucleotide regeneration system further stabilized Ca channels, which permitted recording of Ca currents for up to 3 h. NE reversibly decreased both steady state Ca currents and Ca tail currents in Cs/EGTA/MgATP-dialyzed cells. A possible role of several putative intracellular second messengers in NE receptor-Ca channel coupling was investigated. Cyclic AMP or cyclic GMP added to the intracellular solutions at concentrations several orders of magnitude higher than the Kd for activation of cyclic nucleotide-dependent protein kinases did not block or mask the expression of the NE-mediated decrease in gCa. Addition of internal EGTA to a final concentration of 10 mM also did not affect the expression of the NE response. These results suggest that neither cyclic AMP nor cyclic GMP nor Ca is acting as a second messenger coupling the NE receptor to the down-modulated Ca channel population.     

Atrioventricular block in low potassium and K dependence of the nodal resting potential

A progressive conduction block leading to atrioventricular dissociation develops in perfused rabbit hearts within 20-30 min of exposure to Krebs containing 0.5 mM potassium (low K). A decrease in potassium permeability resulting in membrane depolarization (as seen in Purkinje fibers) could be responsible for the loss of excitability in nodal cells. We investigated the K dependence of the resting potential and the long-term effects of low K perfusion on the resting and action potentials of nodal cells in rabbit hearts. The resting potential of atrial, atrionodal, and nodal cells varied by 52, 41, and 34 mV per decade of change in Ko within the range of 5-50 mM K. Hyperpolarization of the resting membrane, a progressive decline in action potential amplitude, and a decrease in maximum rate of rise were observed in nodal fibers when exposed to low K. Loss of propagated activity occurred in the middle node within 20-30 min while the cells remained hyperpolarized. There was no evidence of electrogenic Na extrusion and it seems that the low nodal resting potential results from a high resting PNa/PK permeability ratio. The early decrease in rate of rise in low K probably reflects an increase in K-dependent outward currents, whereas the progressive deterioration and final loss of conducted electrical activity may result from an accumulation of internal Na and Ca overload produced by low K inhibition of the Na pump.     

Ion channels in rabbit cultured fibroblasts

Large outward currents are recorded with the whole-cell patch-clamp technique on depolarization of rabbit cultured fibroblasts. Our findings suggest that these outward currents consist of two voltage-dependent components, one of which also depends on cytoplasmic calcium concentration. Total replacement of external Cl- by the large anion ascorbate does not affect the amplitude of the currents, indicating that both components must be carried by K+. Consistent with these findings with whole-cell currents, in single channel recordings from fibroblasts we found that most patches contain high-conductance potassium-selective channels whose activation depends on both membrane potential and the calcium concentration at the cytoplasmic surface of the membrane. In a smaller number of patches, a second population of high-conductance calcium-independent potassium channels is observed having different voltage-dependence. The calcium- and voltage-dependence suggest that these two channels correspond with the two components of outward current seen in the whole-cell recordings. The single channel conductance of both channels in symmetrical KCl (150 mM) is 260-270 pS. Both channels are highly selective for K+ over both Na+ and Cl-. The conductance of the channels when outward current is carried by Rb+ is considerably smaller than when it is carried by K+. Some evidence is adduced to support the hypothesis that these potassium channel populations may be involved in the control of cell proliferation.     

A Fast Transient Outward Monovalent Current in Rat Saphenous Myocytes Passing Through Ca2+ Channels

Transient outward currents in rat saphenous arterial myocytes were studied using the perforated configuration of the patch-clamp method. When myocytes were bathed in a Na-gluconate solution containing TEA to block large-conductance Ca2+-activated K+ (BK) currents, depolarizing pulses positive to +20 mV from a holding potential of -100 mV induced fast transient outward currents. The activation and inactivation time constants of the current were voltage dependent, and at +40 mV were 3.6 +/- 0.8 ms and 23.9 +/- 6.4 ms (n = 4), respectively. The steady-state inactivation of the transient outward current was steeply voltage dependent (z = 1.7), with 50% of the current inactivated at -55 mV. The current was insensitive to the A-type K+ channel blocker 4-AP (1-5 mM), and was modulated by external Ca, decreasing to approximately 0.85 of control values upon raising Ca2+ from 1 to 10 mM, and increasing approximately 3-fold upon lowering it to 0.1 mM. Transient outward currents were also recorded following replacement of internal K+ with either Na+ or Cs+, raising the possibility that the current was carried by monovalent ions passing through voltage-gated Ca2+ channels. This hypothesis was supported by the finding that the transient outward current had the same inactivation rate as the inward Ba2+ current, and that both currents were effectively blocked by the L-type Ca2+ channel blocker, nifedipine and enhanced by the agonist BAYK8644.     

Inhibition of Ca2+-activated K+ channels in pig pancreatic acinar cells by Ba2+, Ca2+, quinine and quinidine

Patch-clamp whole-cell and single-channel current recordings were made from pig pancreatic acinar cells to test the effects of quinine, quinidine, Ba2+ and Ca2+. Voltage-clamp current recordings from single isolated cells showed that high external concentrations of Ba2+ or Ca2+ (88 mM) abolished the outward K+ currents normally associated with depolarizing voltage steps. Lower concentrations of Ca2+ only had small inhibitory effects whereas 11 mM Ba2+ almost blocked the K+ current. 5.5 mM Ba2+ reduced the outward K+ current to less than 30% of the control value. Both external quinine and quinidine (200-500 microM) markedly reduced whole-cell outward K+ currents. In single-channel current studies it was shown that external Ba2+ (1-5 mM) markedly reduced the probability of opening of high-conductance Ca2+ and voltage-activated K+ channels whereas internal Ba2+ (6 X 10(-6) to 3 X 10(-5) M) caused activation at negative membrane potentials and inhibition at positive potentials. Quinidine (200-400 microM) evoked rapid chopping of single K+ channel openings acting both from the outside and inside of the membrane and in this way markedly reduced the total current passing through the channels.     

Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons.

Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch clamp recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current amplitude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.     

Block by calcium of ATP-activated channels in pheochromocytoma cells

We have investigated the effects of Ca2+ on Na+ influx through ATP- activated channels in pheochromocytoma PC12 cells using single channel current recordings. Under cell-attached patch-clamp conditions with 150 mM Na+ and 2 mM Ca2+ in the pipette, the unitary current activity showed an open level of about -4.3 pA at -150 mV. The channel opening was interrupted by flickery noise as well as occasional transition to a subconducting state of about -1.7 pA at -150 mV. The open level was decreased with increased external Ca2+, suggesting that external Ca2+ blocks Na+ permeation. We assessed the block by Ca2+ as the mean amplitude obtained with heavy filtration according to Pietrobon et al. (Pietrobon, D., B. Prod'hom, and P. Hess, 1989. J. Gen. Physiol. 94:1- 21). The block was concentration dependent with a Hill coefficient of 1 and a half-maximal concentration of approximately 6 mM. A similar block was observed with other divalent cations, and the order of potency was Cd2+ > Mn2+ > Mg2+ not equal to Ca2+ > Ba2+. High Ca2+, Mg2+ and Ba2+ did not block completely, probably because they can carry current in the channel. The block by external Ca2+ did not exhibit voltage dependence between -100 and -210 mV. In the inside-out patch-clamp configuration, the amplitude of inward channel current obtained with 150 mM external Na+ was reduced by increased internal Ca2+. The reduction was observed at lower concentrations than that by external Ca2+. Internal Ba2+ and Cd2+ induced similar reduction in current amplitude. This inhibitory effect of internal Ca2+ was voltage dependent; the inhibition was relieved with hyperpolarization. The results suggest that both external and internal Ca2+ can block Na+ influx through the ATP-activated channel. A simple one-binding site model with symmetric energy barriers is not sufficient to explain the Ca2+ block from both sides.     

Open sodium channel properties of single canine cardiac Purkinje cells.

Open channel properties of canine cardiac Purkinje cell Na+ channels were studied with single channel cell-attached recording and with whole cell macroscopic current recording in internally perfused cells. Single channel currents and membrane currents increased with an increase in Na+ concentration, but showed evidence of saturation. Assuming first-order binding, the Km for Na+ was 370 mM. PCs/PNa was 0.020 and PK/PNa was 0.094. The current-voltage relationship for single channels showed prominent flattening in the hyperpolarizing direction. This flattening was accentuated by 10 mM Ca2+ and was greatly reduced in O mM Ca2+, indicating that the rectification was a consequence of Ca2+ block of the Na+ channels. A similar instantaneous current-voltage relationship was seen for the whole cell membrane currents. These results demonstrate that the cardiac channel shows substantial Ca2+ block, although it is relatively insensitive to tetrodotoxin. The Na+ and Ca2+ binding properties could be modeled by the four-barrier Eyring rate theory model, with similar values to those reported for the neuroblastoma Na+ channel (Yamamoto, D.,J.Z. Yeh, and T. Narahashi, 1984, Biophys J., 45:337-344).     

Calcium currents in squid giant axon.

Voltage-clamp experiments were carried out on intracellularly perfused squid giant axons in a Na-free solution of 100 mM CaCl2+sucrose. The internal solution was 25 mM CsF+sucrose or 100 mM RbF+50mM tetraethylammonium chloride+sucrose. Depolarizing voltage clamp steps produced small inward currents; at large depolarizations the inward current reversed into an outward current. Tetrodotoxin completely blocked the inward current and part of the outward current. No inward current was seen with 100 mM MgCl2+sucrose as internal solution. It is concluded that the inward current is carried by Ca ions moving through the sodium channel. The reversal potential of the tetrodotoxin-sensitive current was +54mV with 25 mM CsF+sucrose inside and +10 mV with 100 mM RbF+50 mM tetraethylammonium chloride+sucrose inside. From the reversal potentials measured with varying external and internal solutions the relative permeabilities of the sodium channel for Ca, Cs and Na were calculated by means of the constant field equations. The results of the voltage-clamp experiments are compared with measurements of the Ca entry in intact axons.     

Ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis

The ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis were examined by using conventional two-electrode voltage clamp and whole-cell patch clamping methods. Several separable currents were identified. These include: (1) a transient and (2) a steady-state voltage-activated inward current; both are tetrodotoxin (TTX) and saxitoxin (STX) insensitive, partly reduced by decreasing external Ca2+ or Na+ or by addition of 5 mM Co2+, D-600 or verapamil and are totally blocked with 5 mM Cd2+; (3) an early, transient, cation-dependent, outward K+ current (IKCa/Na); (4) a transient, voltage-activated, outward K+ current provisionally identified as IA; (5) a delayed, steady-state, voltage-activated outward K+ current (IK) and (6) a late, transient, outward K+ current which is blocked by Cd2+ and evident only during long voltage pulses. Despite their phylogenic origin, most of these currents are similar to currents identified in many vertebrate smooth and cardiac muscle preparations, and other excitable cells in higher animals.     

Steady-state current-voltage relationship of the Na/K pump in guinea pig ventricular myocytes

Whole-cell currents were recorded in guinea pig ventricular myocytes at approximately 36 degrees C before, during, and after exposure to maximally effective concentrations of strophanthidin, a cardiotonic steroid and specific inhibitor of the Na/K pump. Wide-tipped pipettes, in combination with a device for exchanging the solution inside the pipette, afforded reasonable control of the ionic composition of the intracellular solution and of the membrane potential. Internal and external solutions were designed to minimize channel currents and Na/Ca exchange current while sustaining vigorous forward Na/K transport, monitored as strophanthidin-sensitive current. 100-ms voltage pulses from the -40 mV holding potential were used to determine steady-state levels of membrane current between -140 and +60 mV. Control experiments demonstrated that if the Na/K pump cycle were first arrested, e.g., by withdrawal of external K, or of both internal and external Na, then neither strophanthidin nor its vehicle, dimethylsulfoxide, had any discernible effect on steady-state membrane current. Further controls showed that, with the Na/K pump inhibited by strophanthidin, membrane current was insensitive to changes of external [K] between 5.4 and 0 mM and was little altered by changing the pipette [Na] from 0 to 50 mM. Strophanthidin-sensitive current therefore closely approximated Na/K pump current, and was virtually free of contamination by current components altered by the changes in extracellular [K] and intracellular [Na] expected to accompany pump inhibition. The steady-state Na/K pump current-voltage (I-V) relationship, with the pump strongly activated by 5.4 mM external K and 50 mM internal Na (and 10 mM ATP), was sigmoid in shape with a steep positive slope between about 0 and -100 mV, a less steep slope at more negative potentials, and an extremely shallow slope at positive potentials; no region of negative slope was found. That shape of I-V relationship can be generated by a two-state cycle with one pair of voltage-sensitive rate constants and one pair of voltage-insensitive rate constants: such a two-state scheme is a valid steady-state representation of a multi-state cycle that includes only a single voltage-sensitive step.     

Interaction of internal anions with potassium channels of the squid giant axon

The interaction of internal anions with the delayed rectifier potassium channel was studied in perfused squid axons. Changing the internal potassium salt from K+ glutamate- to KF produced a reversible decline of outward K currents and a marked slowing of the activation of K channels at all voltages. Fluoride ions exert a differential effect upon K channel gating kinetics whereby activation of IK during depolarizing steps is slowed dramatically, but the rate of closing after the step is not much altered. These effects develop with a slow time course (30-60 min) and are specific for K channels over Na channels. Both the amplitude and activation rate of IK were restored within seconds upon return to internal glutamate solutions. The fluoride effect is independent of the external K+ concentration and test membrane potential, and does not recover with repetitive application of depolarizing voltage steps. Of 11 different anions tested, all inorganic species induced similar decreases and slowing of IK, while K currents were maintained during extended perfusion with several organic anions. Anions do not alter the reversal potential or shape of the instantaneous current-voltage relation of open K channels. The effect of prolonged exposure to internal fluoride could be partially reversed by the addition of cationic K channel blocking agents such as TEA+, 4-AP+, and Cs+. The competitive antagonism between inorganic anions and internal cationic K channel blockers suggests that they may interact at a related site(s). These results indicate that inorganic anions modify part of the K channel gating mechanism (activation) at a locus near the inner channel surface.     

Effects of tetraethylammonium on potassium currents in a molluscan neurons

The effects of tetraethylammonium (TEA) on the delayed K+ current and on the Ca2+-activated K+ current of the Aplysia pacemaker neurons R-15 and L-6 were studied. The delayed outward K+ current was measured in Ca2+-free ASW containing tetrodotoxin (TTX), using brief depolarizing clamp pulses. External TEA blocks the delayed K+ current reversibly in a dose-dependent manner. The experimental results are well fitted with a Michaelis-Menten expression, assuming a one-to-one reaction between TEA and a receptor site, with an apparent dissociation constant of 6.0 mM. The block depends on membrane voltage and is reduced at positive membrane potentials. The Ca2+-activated K+ current was measured in Ca2+-free artificial seawater (ASW) containing TTX, using internal Ca2+ ion injection to directly activate the K+ conductance. External TEA and a number of other quaternary ammonium ions block the Ca2+-activated K+ current reversibly in a dose-dependent manner. TEA is the most effective blocker, with an apparent dissociation constant, for a one-to-one reaction with a receptor site, of 0.4 mM. The block decreases with depolarization. The Ca2+-activated K+ current was also measured after intracellular iontophoretic TEA injection. Internal TEA blocks the Ca2+-activated K+ current (but the block is only apparent at positive membrane potentials), is increased by depolarization, and is irreversible. The effects of external and internal TEA can be seen in measurements of the total outward K+ current at different membrane potentials in normal ASW.     

Action of quinidine on ionic currents of molluscan pacemaker neurons

The effects of quinidine on the fast, the delayed, and the Ca2+- activated K+ outward currents, as well as on Na+ and Ca2+ inward currents, were studied at the soma membrane from neurons of the marine mollusk Aplysia californica. External quinidine blocks these current components but to different degrees. Its main effect is on the voltage- dependent, delayed K+ current, and it resembles the block produced by quaternary ammonium ions (Armstrong, C. M., 1975, Membranes, Lipid Bilayers and Biological Membranes: Dynamic Properties, 3:325-358). The apparent dissociation constant is 28 microM at V = +20 mV. The blocking action is voltage and time dependent and increases during maintained depolarization. The data are consistent with the block occurring approximately 70-80% through the membrane electric field. Internal injection of quinidine has an effect similar to that obtained after external application, but its time course of action is faster. External quinidine may therefore have to pass into or through the membrane to reach a blocking site. The Ca2+-activated K+ current is blocked by external quinidine at concentrations 20-50-fold higher compared with the delayed outward K+ current. In addition, it prolongs the time course of decay of the Ca2+-activated K+ current. Na+ and Ca2+ inward currents are also blocked by external quinidine, but again at higher concentrations. The effects of quinidine on membrane currents can be seen from its effect on action potentials and the conversion of repetitive "beating" discharge activity to "bursting" pacemaker activity.