Characterization of Vacuolar Malate and K Channels under Physiological Conditions

Patch-clamp techniques were employed to study the electrical properties of vacuoles from sugar beet (Beta vulgaris) cell suspensions at physiological concentrations of cytoplasmic Ca²⁺. Vacuoles exposed to K⁺ malate revealed the activation of instantaneous and time-dependent outward currents by positive membrane potentials. Negative potentials induced only instantaneous inward currents. The time-dependent outward currents were 10 times more selective for malate than for K⁺ and were completely blocked by zinc. Vacuoles exposed to KCl developed instantaneous currents when polarized to positive or negative membrane potentials. The time-dependent outward channels could serve as the route for the movement of malate into the vacuole, whereas K⁺ could move through the time-independent inward and outward channels.     

Properties of Single K and Cl Channels in Asclepias tuberosa Protoplasts

Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K⁺ and Cl⁻, respectively. Whole cell K⁺ currents activated exponentially during step depolarizations, while voltage-dependent Cl⁻ channels were activated by hyperpolarizations. Single K⁺ channel conductance was 40 ± 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs⁺ and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 ± 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn²⁺ and ethacrynic acid. Whole-cell Cl⁻ currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development.     

Characterization of potassium-dependent currents in protoplasts of corn suspension cells

Protoplasts obtained from corn (Zea mays) suspension cells were studied using the whole cell patch-clamp technique. One time-independent current, as well as two time-dependent currents were identified. All three currents were reduced by tetraethylammonium (9 millimolar), a K⁺ channel blocker. The time-independent current had a nearly linear current-voltage relationship and its reversal potential, defined as the voltage at which there is zero current, was highly dependent on the extracellular potassium concentration. One of the two time-dependent currents was activated, with rapid kinetics, by membrane hyperpolarization to potentials more negative than −100 millivolts. The second time-dependent current was activated with a sigmoidal time course by membrane depolarization to potentials more positive than −60 millivolts. It exhibited no inactivation and was carried primarily by potassium ions. These characteristics suggest that this latter current is caused by the voltage-dependent opening of delayed-rectifier K⁺ channels. These three currents, which are not generated by the plasmalemma H⁺-ATPase, are likely to assist in the regulation of the cellular K⁺ fluxes and membrane potential.     

K Channels Are Responsible for an Inwardly Rectifying Current in the Plasma Membrane of Mesophyll Protoplasts of Avena sativa

In whole-cell recording, the conductance of the plasma membrane of protoplasts isolated from mesophyll cells of leaves of oat (Avena sativa) was greater for inward than outward current. The inward current in both the whole-cell mode and with isolated patches was dependent on [K⁺]_o. When the membrane voltage was more positive than −50 millivolts, the membrane conductance in the whole-cell mode was low, and K⁺ channels in cell-attached or outside-out patches had a low probability of being open. At a membrane voltage more negative than −50 millivolts, the membrane conductance increased by sevenfold in the whole-cell mode, and the probability of the channels being open increased. The inward current was highly selective for K⁺ compared with Cs⁺, Na⁺, choline or Cl⁻. Low concentrations of [Cs⁺]_o or [Na⁺]_o blocked the inward current in a strongly voltage-dependent fashion. Comparison of single-channel with the macroscopic current yields an estimate of about 200 inwardly rectifying K⁺ channels per cell at a density of 0.035 per square micrometer. At physiological membrane voltages and [K⁺]_o about 10 millimolar, the influx through these channels is sufficient to increase the internal [K⁺] by 2 millimolar per minute. These K⁺ channels are activated by membrane voltages in the normal physiological range and could contribute to K⁺ uptake whenever the membrane is more negative than the K⁺ equilibrium potential.     

Time-dependent Outward Currents through the Inward Rectifier Potassium Channel IRK1 : The Role of Weak Blocking Molecules

Outward currents through the inward rectifier K⁺ channel contribute to repolarization of the cardiac action potential. The properties of the IRK1 channel expressed in murine fibroblast (L) cells closely resemble those of the native cardiac inward rectifier. In this study, we added Mg²⁺ (0.44–1.1 mM) or putrescine (∼0.4 mM) to the intracellular milieu where endogenous polyamines remained, and then examined outward IRK1 currents using the whole-cell patch-clamp method at 5.4 mM external K⁺. Without internal Mg²⁺, small outward currents flowed only at potentials between −80 (the reversal potential) and ∼−40 mV during voltage steps applied from −110 mV. The strong inward rectification was mainly caused by the closed state of the activation gating, which was recently reinterpreted as the endogenous-spermine blocked state. With internal Mg²⁺, small outward currents flowed over a wider range of potentials during the voltage steps. The outward currents at potentials between −40 and 0 mV were concurrent with the contribution of Mg²⁺ to blocking channels at these potentials, judging from instantaneous inward currents in the following hyperpolarization. Furthermore, when the membrane was repolarized to −50 mV after short depolarizing steps (>0 mV), a transient increase appeared in outward currents at −50 mV. Since the peak amplitude depended on the fraction of Mg²⁺-blocked channels in the preceding depolarization, the transient increase was attributed to the relief of Mg²⁺ block, followed by a re-block of channels by spermine. Shift in the holding potential (−110 to −80 mV), or prolongation of depolarization, increased the number of spermine-blocked channels and decreased that of Mg²⁺-blocked channels in depolarization, which in turn decreased outward currents in the subsequent repolarization. Putrescine caused the same effects as Mg²⁺. When both spermine (1 μM, an estimated free spermine level during whole-cell recordings) and putrescine (300 μM) were applied to the inside-out patch membrane, the findings in whole-cell IRK1 were reproduced. Our study indicates that blockage of IRK1 by molecules with distinct affinities, spermine and Mg²⁺ (putrescine), elicits a transient increase in the outward IRK1, which may contribute to repolarization of the cardiac action potential.     

Voltage-sensitive chloride ion channels in Anopheles gambiae Sua-1B cells

In this study, we performed electrophysiological analysis of Anopheles gambiae Sua-1B cells having “neuron-like” morphologies using the patch clamp method. The recorded cells (n = 79) had processes resembling axons/dendrites, with 63 % unipolar, 22 % bipolar, and 15 % multipolar. While no inward currents were observed following step depolarizations (holding potential = ?80 mV), a slowly activating outward current was observed in 96 % of the cells, especially at depolarized potentials. The amplitude of the current was attenuated nearly 70 % by reducing extracellular Cl^? ion concentration, or by incubating with 100 μM DIDS, a known voltage-sensitive chloride channel blocker, suggesting that the current was mediated by chloride ions. No qualitative difference was found between recordings made with Cs⁺ ions in the intracellular pipette solution (inhibits K⁺ currents) and those made with normal physiological solution, indicating a deficiency of potassium channels. Additionally, recordings made with Ca²⁺-free extracellular bath solution eliminated the slowly activating outward current. A subset of cells (n = 3) lacked this current, but had outward currents with voltage-dependent properties similar to those of volume-regulated chloride channels. Taken together, our results suggest that the voltage-sensitive currents observed in the majority of Sua-1B cells are mediated primarily by chloride channels of the calcium-dependent subtype.     

Inward Rectifying K Channels in the Plasma Membrane of Arabidopsis thaliana

Ion channels in the plasma membrane of protoplasts isolated from cultured cells of Arabidopsis thaliana were studied by means of the patch-clamp technique applied in the whole-cell configuration. In some protoplasts, depolarizing pulses and, in other protoplasts, hyperpolarizing pulses elicited time-dependent currents; both kinds of current were only rarely observed in the same protoplast. The hyperpolarization-activated inward rectifying currents, the focus of this paper, appeared to be due to the relatively slow opening of channels (activation time constant = 150 to 300 milliseconds), which closed at positive potentials. The reversal potential of this current, measured in the presence of different ion concentrations (symmetrical or asymmetrical K⁺ and Cl⁻ or gluconate), was always close to the electrochemical equilibrium potential of K⁺. The currents were inhibited by 10 millimolar tetraethylammonium, a K⁺ channel blocker. These data show that the hyperpolarization-activated currents flow through K⁺ channels, which can provide a pathway for the passive diffusion of K⁺ down its electrochemical gradient.     

Synergistic effect of light and fusicoccin on stomatal opening : epidermal peel and patch clamp experiments

Upon incubation of epidermal peels of Commelina communis in 1 millimolar KCl, a synergistic effect of light and low fusicoccin (FC) concentrations on stomatal opening is observed. In 1 millimolar KCl, stomata remain closed even in the light. However, addition of 0.1 micromolar FC results in opening up to 12 micrometers. The same FC concentration stimulates less than 5 micrometers of opening in darkness. The synergistic effect (a) decreases with increasing FC or KCl concentrations; (b) is dark-reversible; (c) like stomatal opening in high KCl concentrations (120 millimolar) is partially inhibited by the K⁺ channel blocker, tetraethyl-ammonium⁺ (20 millimolar). In whole-cell patch-clamp experiments with guard cell protoplasts of Vicia faba, FC (1 or 10 micromolar) stimulates an increase in outward current that is essentially voltage independent between - 100 and +60 millivolts, and occurs even when the membrane potential is held at a voltage (−60 millivolts) at which K⁺ channels are inactivated. These results are indicative of FC activation of a H⁺ pump. FC effects on the magnitude of inward and outward K⁺ currents are not observed. Epidermal peel and patch clamp data are both consistent with the hypothesis that the plasma membrane H⁺ ATPase of guard cells is a primary locus for the FC effect on stomatal apertures.     

Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

Prolonged depolarization induces a slow inactivation process in some K⁺ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K⁺ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K⁺ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.     

Ion channels in Arabidopsis plasma membrane : transport characteristics and involvement in light-induced voltage changes

White light (25 watts per square meter) induced an increase in plasma membrane K⁺-channel activity and a 30- to 70-millivolt transient membrane depolarization (completed in 2-3 minutes) in Arabidopsis thaliana leaf mesophyll cells. Transport characteristics of three types of ion channels in the plasma membrane were determined using inside-out patches. With 220 millimolar K⁺ on the cytoplasmic side of the patch and 50 millimolar K⁺ in the pipette, (220/50 K), the open-channel current-voltage curves of these channels were sigmoidal and consistent with an enzyme kinetic model. Two channel types were selective for K⁺ over Na⁺ and Cl⁻. One (named PKC1) had a maximum conductance (G_max) of 44 picosiemens at a membrane voltage (V_m) of −65 mV in (220/50 K) and is stimulated by light. The other (PKC2) had G_max = 66 picosiemens at V_m = 60 millivolts in (220/50 K). The third channel type (PCC1) transported K⁺ and Na⁺ about equally well but not Cl⁻. It had G_max = 109 picosiemens at V_m = 55 millivolts in (250/50 K) with 10 millimolar Ca²⁺ on the cytoplasmic side. Reducing Ca²⁺ to 0.1 millimolar increased PCC1 open-channel currents by approximately 50% in a voltage-independent manner. Averaged over time, PKC2 and PCC1 currents strongly outward rectified and PKC1 currents did so weakly. Reductants (1 millimolar dithiothreitol or 10 millimolar β-mercaptoethanol) added to the cytoplasmic side of an excised patch increased the open probability of all three channel types.     

Inward and outward currents of native and cloned K(ATP) channels (Kir6.2/SUR1) share single-channel kinetic properties

BackgroundThe ATP-sensitive K⁺ (K(ATP)) channel is found in a variety of tissues extending from the heart and vascular smooth muscles to the endocrine pancreas and brain. Common to all K(ATP) channels is the pore-forming subunit Kir6.x, a member of the family of small inwardly rectifying K⁺ channels, and the regulatory subunit sulfonylurea receptor (SURx). In insulin secreting β-cells in the endocrine part of the pancreas, where the channel is best studied, the K(ATP) channel consists of Kir6.2 and SUR1. Under physiological conditions, the K(ATP) channel current flow is outward at membrane potentials more positive than the K⁺ equilibrium potential around ?80 mV. However, K(ATP) channel kinetics have been extensively investigated for inward currents and the single-channel kinetic model is based on this type of recording, whereas only a limited amount of work has focused on outward current kinetics.MethodsWe have estimated the kinetic properties of both native and cloned K(ATP) channels under varying ionic gradients and membrane potentials using the patch-clamp technique.ResultsAnalyses of outward currents in K(ATP) and cloned Kir6.2ΔC26 channels, alone or co-expressed with SUR1, show openings that are not grouped in bursts as seen for inward currents. Burst duration for inward current corresponds well to open time for outward current.ConclusionsOutward K(ATP) channel currents are not grouped in bursts regardless of membrane potential, and channel open time for outward currents corresponds to burst duration for inward currents.     

The k/na selectivity of a cation channel in the plasma membrane of root cells does not differ in salt-tolerant and salt-sensitive wheat species

The characteristics of cation outward rectifier channels were studied in protoplasts from wheat root (Triticum aestivum L. and Triticum turgidum L.) cells using the patch clamp technique. The cation outward rectifier channels were voltage-dependent with a single channel conductance of 32 ± 1 picosiemens in 100 millimolar KCl. Whole-cell currents were dominated by the activity of the cation outward rectifiers. The time- and voltage-dependence of these currents was accounted for by the summed behavior of individual channels recorded from outside-out detached patches. The K⁺/Na⁺ permeability ratio of these channels was measured in a salt-sensitive and salt-tolerant genotype of wheat that differ in rates of Na⁺ accumulation, using a voltage ramp protocol on protoplasts in the whole-cell configuration. Permeability ratios were calculated from shifts in reversal potentials following ion substitutions. There were no significant differences in the K⁺/Na⁺ permeability ratios of these channels in root cells from either of the two genotypes tested. The permeability ratio for K⁺/Cl⁻ was greater than 50:1. The K⁺/Na⁺ permeability ratio averaged 30:1, which is two to four times more selective than the same type of channel in guard cells and suspension culture cells. Lowering the Ca²⁺ concentration in the bath solution to 0.1 millimolar in the presence of 100 millimolar Na⁺ had no significant effect on the K⁺/Na⁺ permeability ratios of the channel. It seems unlikely that the mechanism of salt tolerance in wheat is based on differences in the K⁺/Na⁺ selectivity of these channels.     

Potassium Channels in Chara corallina: CONTROL AND INTERACTION WITH THE ELECTROGENIC H PUMP

Plasmalemma electrical properties were used to investigate K⁺ transport and its control in internodal cells of Chara corallina Klein ex Willd., em R.D.W. Cell exposure to solutions containing 10 mm KCl caused the potential, normally −250 millivolts (average), to depolarize in two steps. The first step was a 21 millivolt depolarization that lasted from 1 to 40 minutes. The second step started with an action potential and left the membrane potential at −91 millivolts, with a 10-fold reduction in resistance. We suggest that the second step was caused by the opening of K⁺ -channels in the membrane. This lowered the resistance and provided a current pathway that partially short-circuited the electrogenic pump. Although largely short-circuited, the electrogenic pump was still operating as indicated by: (a) the depolarized potential of −91 millivolts was more negative than Ek (=−42 millivolts in 10 mm K⁺); (b) a large net K⁺ uptake occurred while the cell was depolarized; (c) both the electrogenic pump inhibitor, diethylstilbestrol, and the sulfhydryl-reagent N-ethylmaleimide (which increased the passive membrane permeability) further depolarized the potential in 10 mm KCl.A two-phase recovery back to normal cell potentials occurred upon lowering the K⁺ concentration from 10 to 0.2 mm. The first phase was an apparent Nernst potential response to the change in external K⁺ concentration. The second phase was a sudden hyperpolarization accompanied by a large increase in membrane resistance. We attribute the second phase to the closing of K⁺ -channels and the removal of the associated short-circuiting effect on the electrogenic pump, thereby allowing the membrane to hyperpolarize. Further experiments indicated that the K⁺ -channel required Ca²⁺ for normal closure, but other ions could substitute, including: Na⁺, tetraethylammonium, and 2,4,6-triaminopyrimidine. Apparently, K⁺ -channel conductance is determined by competition between Ca²⁺ and K⁺ for a control (gating?) binding site.     

Ca2+-activated K+ currents inNecturus choroid plexus

Summary The tight-seal whole-cell recording method has been used to studyNecturus choroid plexus epithelium. A cell potential of –59±2 mV and a whole cell resistance of 56±6 M were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110mm NaCl Ringer solution and the interior of the cell contained a solution of 110mm KCl and 5nm Ca²⁺, stepping the membrane potential from a holding value of –50 to –10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca²⁺-activated K^_ channels. Based on the voltage dependence of the activation of Ca²⁺-activated K⁺ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K⁺ as their reversal potential at different K⁺ concentration gradients followed the Nernst potential for K⁺. These currents were reduced by the addition of TEA⁺ to the bath solution and were eliminated when Cs⁺ or Na⁺ replaced intracellular K⁺. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t _1/2) to the steady value. Increasing the pipette Ca²⁺ also decreased the delay and decreasedt _1/2. For instance, when pipette Ca²⁺ was increased from 5 to 500nm, the delay andt _1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K⁺ currents through Ca²⁺-activated K⁺ channels.At the resting membrane potential of –60 mV, Ca²⁺-activated K⁺ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20–30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca²⁺. Thus the Ca²⁺-activated K⁺ channels may play a specific role in maintaining intracellular K⁺ concentrations during CSF secretion.     

HERG-like K+ Channels in Microglia

A voltage-gated K⁺ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K⁺ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K⁺ conductance–voltage relation was half maximal at −59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (−14 vs. −39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795–805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs⁺ or Ba²⁺ occurred only at millimolar concentrations, La³⁺ blocked with K _i = ∼40 μM, and the HERG-selective blocker, E-4031, blocked with K _i = 37 nM. Implications of the presence of HERG-like K⁺ channels for the ontogeny of microglia are discussed.     

The Human Red Cell Voltage-regulated Cation Channel. The Interplay with the Chloride Conductance,the Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> Channel and the Ca<Superscript>2+</Superscript> Pump

Electrocytes from the electric organ of Electrophorus electricus exhibited sodium action potentials that have been proposed to be repolarized by leak currents and not by outward voltage-gated potassium currents. However, patch-clamp recordings have suggested that electrocytes may contain a very low density of voltage-gated K⁺ channels. We report here the cloning of a K⁺ channel from an eel electric organ cDNA library, which, when expressed in mammalian tissue culture cells, displayed delayed-rectifier K⁺ channel characteristics. The amino-acid sequence of the eel K⁺ channel had the highest identity to Kv1.1 potassium channels. However, different important functional regions of eel Kv1.1 had higher amino-acid identity to other Kv1 members, for example, the eel Kv1.1 S4-S5 region was identical to Kv1.5 and Kv1.6. Northern blot analysis indicated that eel Kv1.1 mRNA was expressed at appreciable levels in the electric organ but it was not detected in eel brain, muscle, or cardiac tissue. Because electrocytes do not express robust outward voltage-gated potassium currents we speculate that eel Kv1.1 channels are chronically inhibited in the electric organ and may be functionally recruited by an unknown mechanism.     

Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Phe-Met-Arg-Phe-amide Activates a Novel Voltage-dependent K+ Current through a Lipoxygenase Pathway in Molluscan Neurones

The neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) dose dependently (ED₅₀ = 23 nM) activated a K⁺ current in the peptidergic caudodorsal neurones that regulate egg laying in the mollusc Lymnaea stagnalis. Under standard conditions ([K⁺]_o = 1.7 mM), only outward current responses occurred. In high K⁺ salines ([K⁺]_o = 20 or 57 mM), current reversal occurred close to the theoretical reversal potential for K⁺. In both salines, no responses were measured below −120 mV. Between −120 mV and the K⁺ reversal potential, currents were inward with maximal amplitudes at ∼−60 mV. Thus, U-shaped current–voltage relations were obtained, implying that the response is voltage dependent. The conductance depended both on membrane potential and extracellular K⁺ concentration. The voltage sensitivity was characterized by an e-fold change in conductance per ∼14 mV at all [K⁺]_o. Since this result was also obtained in nearly symmetrical K⁺ conditions, it is concluded that channel gating is voltage dependent. In addition, outward rectification occurs in asymmetric K⁺ concentrations. Onset kinetics of the response were slow (rise time ∼650 ms at −40 mV). However, when FMRFa was applied while holding the cell at −120 mV, to prevent activation of the current but allow activation of the signal transduction pathway, a subsequent step to −40 mV revealed a much more rapid current onset. Thus, onset kinetics are largely determined by steps preceding channel activation. With FMRFa applied at −120 mV, the time constant of activation during the subsequent test pulse decreased from ∼36 ms at −60 mV to ∼13 ms at −30 mV, confirming that channel opening is voltage dependent. The current inactivated voltage dependently. The rate and degree of inactivation progressively increased from −120 to −50 mV. The current is blocked by internal tetraethylammonium and by bath- applied 4-aminopyridine, tetraethylammonium, Ba²⁺, and, partially, Cd²⁺ and Cs⁺. The response to FMRFa was affected by intracellular GTPγS. The response was inhibited by blockers of phospholipase A₂ and lipoxygenases, but not by a cyclo-oxygenase blocker. Bath-applied arachidonic acid induced a slow outward current and occluded the response to FMRFa. These results suggest that the FMRFa receptor couples via a G-protein to the lipoxygenase pathway of arachidonic acid metabolism. The biophysical and pharmacological properties of this transmitter operated, but voltage-dependent K⁺ current distinguish it from other receptor-driven K⁺ currents such as the S-current- and G-protein-dependent inward rectifiers.     

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.