Ion channels in the plasma membrane of protoplasts from the halophytic angiosperm Zostera muelleri

Patch clamp studies show that there may be as many as seven different channel types in the plasma membrane of protoplasts derived from young leaves of the halophytic angiosperm Zostera muelleri. In whole-cell preparations, both outward and inward rectifying currents that activate in a timeand voltage-dependent manner are observed as the membrane is either depolarized or hyperpolarized. Current voltage plots of the tail currents indicate that both currents are carried by K⁺. The channels responsible for the outward currents have a unit conductance of approximately 70 pS and are five times more permeable to K⁺ than to Na⁺. In outside-out patches we have identified a stretch-activated channel with a conductance of 100 pS and a channel that inwardly rectifies with a conductance of 6 pS. The reversal potentials of these channels indicate a significant permeability to K⁺. In addition, the plasma membrane contains a much larger K⁺ channel with a conductance of 300 pS. Single channel recordings also indicate the existence of two Cl^– channels, with conductances of 20 and 80 pS with distinct substates. The membrane potential difference of perfused protoplasts showed rapid action potentials of up to 50 mV from the resting level. The frequency of these action potentials increased as the external osmolarity was decreased. The action potentials disappeared with the addition of Gd³⁺, an effect that is reversible upon washout.We would like to thank K. Morris and D. McKenzie for technical assistance and the Australian Research Council for financial support.     

Voltage-dependent large conductance channels in visceral smooth muscle.

The ionic conductances that underlie the resting membrane potential of visceral smooth muscle are not fully understood. Using the patch-clamp technique in the whole-cell configuration, single large conductance channels (LCCs) with unitary conductances of up to 400 pS were recorded in isolated smooth muscle cells of the opossum esophagus. These channels were active at physiological potentials (-100 to -40 mV) and opened with increasing frequency as the membrane potential was hyperpolarized. This voltage dependence gave rise to an inwardly rectifying macroscopic current which was half-maximally activated at -65 mV. The current through LCCs was carried by cations because reduction of external [NaCl] shifted the reversal potential of the LCC current towards the predicted Nernst potential for a nonselective cation current. These results suggest that LCCs may contribute to resting membrane potential in the circular muscle of the opossum esophagus.     

Ion channels in human endothelial cells.

Ion channels were studied in human endothelial cells from umbilical cord by the patch clamp technique in the cell attached mode. Four different types of ion channels were recorded: i) potassium channel current that rectifies at positive potentials in symmetrical potassium solutions (inward rectifier); ii) low-conductance non-selective cation channel with a permeability ratio K:Na:Ca = 1:0.9:0.2; iii) high-conductance cation-selective channel that is about 100 times more permeable for calcium than for sodium or potassium; iv) high-conductance potassium channel with a permeability ratio K:Na = 1:0.05. The extrapolated reversal potential of the inwardly rectifying current was near to the potassium equilibrium potential. The slope conductance decreased from 27 pS in isotonic KCl solution to 7 pS with 5.4 mmol/l KCl and 140 mmol/l NaCl in the pipette but 140 mmol/l KCl in the bath. The low-conductance non-selective cation channel showed a single-channel conductance of 26 pS with 140 mmol/l Na outside, 28 pS with 140 mmol/l K outside, and rectified in inward direction in the presence of Ca (60 mmol/l Ca, 70 mmol/l Na, 2.7 mmol/l K in the pipette) at negative potentials. The current could be observed with either chloride or aspartate as anion. The high-conductance non-selective channel did not discriminate between Na and K. The single-channel conductance was about 50 pS. The extrapolated reversal potential was more positive than +40 mV (140 K or 140 Na with 5 Ca outside). Both the 26 and 50 pS channel showed a run-down, and they rapidly disappeared in excised patches. The high-conductance potassium channel with a single-channel conductance of 170 pS was observed only rarely. It reversed near the expected potassium equilibrium potential. The 26 pS channel could be stimulated with histamine and thrombin from outside in the cell-attached mode. Both the 26 pS as well as the 50 pS channel can mediate calcium flux into the endothelial cell.     

Nonselective cation channels in intact human T lymphocytes.

Nonselective cation channels were found in single channel recordings from cell-attached patches on human T lymphocytes. These channels were active under conditions that should lead to cell swelling (hypotonic bath solutions with NaCl or KCl); however, a definite dependence of activity on cell swelling has not been proven. Under these conditions similar channels were found in 20 of 23 patches from 11 different blood donors. The current-voltage relation was approximately linear for outward current (11-14 pS) and inwardly rectifying (to 23 pS) when the intact cells were depolarized with high KCl in the bath. The voltage dependence of channel activity is consistent with closing at hyperpolarized membrane potentials (Vm less than or equal to -50 mV) and block of open channels at strongly depolarized membrane potentials (Vm greater than 0 mV). Reversal potentials under all ionic gradients tested are consistent with a channel that is poorly selective between Na+ and K+ ions. Active channels in cell-attached patches were rapidly blocked by bath addition of the membrane-permeant inhibitor quinine. Channels that were active in cell-attached became quiescent after patch excision; however, two patches remained active long enough to obtain current-voltage relations. These were linear with a slope conductance for outward current of 8-11 pS. Because of the clustering of single-channel openings, detailed voltage dependence of kinetics and probability of opening were not studied.     

A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus

Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

An inwardly rectifying potassium channel in the basolateral membrane of sheep parotid secretory cells

Summary Using whole-cell patch-clamp techniques, we demonstrate that sheep parotid secretory cells have both inwardly and outwardly rectifying currents. The outwardly rectifying current, which is blocked by 10 mmol/liter tetraethylammonium (TEA) applied extracellularly, is probably carried by the 250 pS Ca²⁺-and voltage-activated K⁺ (BK) channel which has been described in previous studies. In contrast, the inwardly rectifying current, which is also carried by K⁺ ions, is not sensitive to TEA. It is similar to the inwardly rectifying currents observed in many excitable tissues in that (i) its conductance is dependent on the square root of the extracellular K⁺, (ii) the voltage range over which it is activated is influenced by the extracellular K⁺ concentration and (iii) it is blocked by the addition of Cs⁺ ions (670 µmol/liter) to the bathing solution. Our previously published cell-attached patch studies have shown that the channel type most commonly observed in the basolateral membrane of unstimulated sheep parotid secretory cells is a K⁺ channel with a conductance of 30 pS and, in this study, we find that its conductance also depends on the square root of the extracellular K⁺ concentration. It thus seems likely that it carries the inwardly rectifying K⁺ current seen in the whole-cell studies.     

Pharmacological and biophysical properties of swelling-activated chloride channel in mouse cardiac myocytes

The present study was designed to observe the properties of swelling-activated chloride channel (ICl.swell) in mouse cardiac myocytes using patch clamp techniques. In whole-cell recordings, hypotonic solution activated a chloride current that exhibited outward rectification, weak voltage-dependent inactivation, and anion selectivity with permeability sequence of I- > Br- > Cl-. The current was sensitive to Cl- channel blockers tamoxifen, NPPB and DIDS. In single-channel recordings, cell swelling activated a single channel current which showed outward rectification with open probability of 0.76 +/- 0.08 and conductance of 38.1 +/- 2.5 pS at +100 mV under [Cl-] symmetrical condition. I-V relation revealed the reversal potential as expected for a Cl(-)-selective channel. These results suggested that in mouse cardiac myocytes, swelling-activated, outward rectifying chloride channel with a single channel conductance of 38.1 +/- 2.5 pS (at +100 mV under [Cl-] symmetrical condition) underlies the volume regulatory Cl- channel.     

Voltage-activated ionic channels and conductances in embryonic chick osteoblast cultures

Patch-clamp measurements were made on osteoblast-like cells isolated from embryonic chick calvaria. Cell-attached-patch measurements revealed two types of high conductance (100-250 pS) channels, which rapidly activated upon 50-100 mV depolarization. One type showed sustained and the other transient activation over a 10-sec period of depolarization. The single-channel conductances of these channel types were about 100 or 250 pS, depending on whether the pipettes were filled with a low K+ (3 mM) or high K+ (143 mM) saline, respectively. The different reversal potentials under these conditions were consistent with at least K+ conduction. Whole-cell measurements revealed the existence of two types of outward rectifying conductances. The first type conducts K+ ions and activates within 20-200 msec (depending on the stimulus) upon depolarizing voltage steps from less than -60 mV to greater than -30 mV. It inactivates almost completely with a time constant of 2-3 sec. Recovery from inactivation is biphasic with an initial rapid phase (1-2 sec) followed by a slow phase (greater than 20 sec). The second whole-cell conductance activates at positive membrane potentials of greater than +50 mV. It also rapidly turns on upon depolarizing voltage steps. Activation may partly disappear at the higher voltages. Its single channels of 140 pS conductance were identified in the whole cell and did conduct K+ ions but were not highly Cl- or Na+ selective. The results show that osteoblasts may express various types of voltage controlled ionic channels. We predict a role for such channels in mineral metabolism of bone tissue and its control by osteoblasts.     

Inwardly rectifying single-channel and whole cell K+ currents in rat ventricular myocytes

Summary The voltage-dependent properties of inwardly rectifying potassium channels were studied in adult and neonatal rat ventricular myocytes using patch voltage-clamp techniques. Inward rectification was pronounced in the single-channel currentvoltage relation and outward currents were not detected at potentials positive to the calculated reversal potential for potassium (E _k). Single-channel currents having at least three different conductances were observed and the middle one was predominant. Its single-channel conductance was nonlinear ranging from 20 to 40 pS. Its open-time distribution was fit by a single exponential and the time constants decreased markedly with hyperpolarization fromE _k. The distribution of the closed times required at least two exponentials for fitting, and their taus were related to the bursting behavior displayed at negative potentials. The steady-state probability of being open (P _o) for this channel was determined from the single-channel records; in symmetrical isotonic K solutionsP _o was 0.73 at –60 mV, but fell to 0.18 at –100 mV. The smaller conductance was about one-half the usual value and the open times were greatly prolonged. The large conductance was about 50 percent greater than the usual value and the open times were very brief. TheP _o(V) relation, the kinetics and the conductance of the predominant channel account for most of the whole cell inwardly rectifying current. The kinetics suggest that an intrinsic K⁺-dependent mechanism may control the gating, and the conductance of this channel. In the steady state, the opening and closing probabilities for the two smaller channels were not independent of each other, suggesting the possibility of a sub-conductance state or cooperativity between different channels.     

Single potassium channels of human glioma cells.

Single potassium channels in the membrane of human malignant glioma cells U-118MG were studied using the technique of patch clamp in cell-attached and inside-out configurations. Three types of potassium channels were found which differed from each other under conditions close to physiological in their conductance and gating characteristics. The lowest-conductance channel (20 pS near the reversal potential) showed a mild outward rectification up to 45 pS at positive voltages and spontaneous modes of high and low activity. At extreme values of potentials its activity was generally low. The intermediate conductance channel had an S-shaped I-V curve, giving a conductance of 63 pS at reversal, and a low and voltage independent opening probability. The high-conductance (215 pS) channel was found to be activated by both membrane potential and Ca2+ ions and blocked by internal sodium at high voltages. The current-voltage curves of all three channel types displayed saturation.     

The inwardly rectifying potassium current of embryonic chick hepatocytes

The single channel and whole-cell properties of an inward, rectifying potassium current in cultured embryonic chick hepatocytes were studied at 20°C. In cell-attached patches, channels open upon membrane hyperpolarization and are present in about 90% of cellattached patches. With 145 mm potassium in the pipette, inward current has a slope conductance of 80 pS. The conductance is not a linear function of the external potassium concentration. Current saturates at high external potassium and has a Michaelis-Menten affinity constant of 275 mm potassium. Substitution of gluconate for chloride in the external solution has no significant effect on conductance, and the reversal potential shifts approximately 18 mV with a change in external potassium from 72.5 to 145 mm indicating potassium selectivity. Channel openings are characterized by multiple brief closures during a burst. The channel is inhibited by external cesium in a concentration-dependent manner. Block is characterized by an increased frequency of transient closures. Whole-cell dialysis with 145 mm CsCl of cells bathed in 145 mm KCl reveals time-independent inward currents that reverse at 0 mV in response to 200 msecvoltage steps. Although voltage ramps evoke currents that are 75% potassium dependent and cesium sensitive, the mean chord conductance (425 pS) indicates that less than five channels are open at any instant. We suggest that the inwardly rectifying potassium channel is partially inactivated in the dialysed hepatocyte.We thank K. Paula S. Hettiaratchi and Eunice Y. Wang for expert cell isolation and culture technique, and the Natural Sciences and Engineering Research Council of Canada for supporting this work.     

Inward and outward K+-selective currents in the plasma membrane of protoplasts from maize root cortex and stele

In an attempt to understand the processes mediating ion transport within the root, the patch clamp technique was applied to protoplasts isolated from the cortex and stele of maize roots and their plasma membrane conductances investigated. In the whole-cell configuration, membrane hyperpolarization induced a slowly activating inwardly rectifying conductance in most protoplasts isolated from the root cortex. In contrast, most protoplasts isolated from the stele contained a slowly activating outwardly rectifying conductance upon plasma membrane depolarization. The reversal potential of the inward current indicated that it was primarily due to the movement of K⁺; the outwardly rectifying conductance was comparatively less selective for K⁺. Membrane hyperpolarization beyond a threshold of about ?70 mV induced inward currents. When E_K was set negative of this threshold, inward currents activated negative of E_K and no outward currents were observed positive of E_K. Outward currents in the stelar protoplasts activated at potentials positive of ?85 mV. However, when E_K was set positive of ?85 mV a small inward current was also observed at potentials negative (and slightly positive) of the equilibrium potential for K⁺. Inwardly and outwardly rectifying K⁺ channels were observed in outside-out patches from the plasma membrane of cortical and stelar cells, respectively. Characterization of these channels showed that they were likely to be responsible for the macroscopic ‘whole-cell’ currents. Inward and outward currents were affected differently by various K⁺ channel blockers (TEA⁺, Ba²⁺ and Cs⁺). In addition, Ca²⁺ above 1 mM partially blocked the inward current in a voltage-dependent manner but had little effect on the outward current. It is suggested that the inwardly rectifying conductance identified in protoplasts isolated from the cortex probably represents an important component of the low-affinity K⁺ uptake mechanism (mechanism II) identified in intact roots. The outwardly rectifying conductance identified in protoplasts isolated from the stele could play a role in the release of cations into the xylem vessels for transport to the shoot.     

Inwardly rectifying potassium current in rabbit osteoclasts: A whole-cell and single-channel study

Summary Ionic conductances of rabbit osteoclasts were investigated using both whole-cell and cell-attached configurations of the patch-clamp recording technique. The predominant conductance found in these cells was an inwardly rectifying K⁺ conductance. Whole-cell currents showed an N-shaped current-voltage (I–13;V) relation with inward current activated at potentials negative to E_K. When external K⁺ was varied, I-V curves shifted 53 mV/10-fold change in [K⁺]_out, as predicted for a K⁺-selective channel. Inward current was blocked by Ba²⁺ and showed a time-dependent decline at negative potentials, which was reduced in Na⁺-free external solution. Inward single-channel currents were recorded in the cell-attached configuration. Single-channel currents were identified as inward-rectifier K⁺ channels based on the following observations: (i) Unitary I-V relations rectified, with only inward current resolved. (ii) Unitary conductance () was 31 pS when recorded in the cell-attached configuration with 140 mm K⁺ in the pipette and was found to be dependent on [K⁺]. (iii) Addition of Ba²⁺ to the pipette solution abolished single-channel events. We conclude that rabbit osteoclasts possess inwardly rectifying K⁺ channels which give rise to the inward current recorded at negative potentials in the whole-cell configuration. This inwardly rectifying K⁺ current may be responsible for setting the resting membrane potential and for dissipating electrical potential differences which arise from electrogenic transport of protons across the osteoclast ruffled border.This work was supported by The Arthritis Society and the Medical Research Council of Canada. M.E.M.K. was supported by a fellowship, S.J.D. a development Grant and S.M.S. a scholarship from the Medical Research Council. We thank Dr. Zu Gang Zheng for help with scanning microscopy.     

Roles of G proteins in coupling of receptors to ionic channels and other effector systems

Guanine nucleotide binding (G) proteins are heterotrimers that couple a wide range of receptors to ionic channels. The coupling may be indirect, via cytoplasmic agents, or direct, as has been shown for two K+ channels and two Ca2+ channels. One example of direct G protein gating is the atrial muscarinic K+ channel K+[ACh], an inwardly rectifying K+ channel with a slope conductance of 40 pS in symmetrical isotonic K+ solutions and a mean open lifetime of 1.4 ms at potentials between -40 and -100 mV. Another is the clonal GH3 muscarinic or somatostatin K+ channel, also inwardly rectifying but with a slope conductance of 55 pS. A G protein, Gk, purified from human red blood cells (hRBC) activates K+ [ACh] channels at subpicomolar concentrations; its alpha subunit is equipotent. Except for being irreversible, their effects on gating precisely mimic physiological gating produced by muscarinic agonists. The alpha k effects are general and are similar in atria from adult guinea pig, neonatal rat, and chick embryo. The hydrophilic beta gamma from transducin has no effect while hydrophobic beta gamma from brain, hRBCs, or retina has effects at nanomolar concentrations which in our hands cannot be dissociated from detergent effects. An anti-alpha k monoclonal antibody blocks muscarinic activation, supporting the concept that the physiological mediator is the alpha subunit not the beta gamma dimer. The techniques of molecular biology are now being used to specify G protein gating. A "bacterial" alpha i-3 expressed in Escherichia coli using a pT7 expression system mimics the gating produced by hRBC alpha k.     

Two Distinct K+ Channels in Lamprey (Lampetra fluviatilis) Erythrocyte Membrane Characterized by Single Channel Patch Clamp

Two channels, distinguished by using single-channel patch-clamp, carry out potassium transport across the red cell membrane of lamprey erythrocytes. A small-conductance, inwardly rectifying K⁺-selective channel was observed in both isotonic and hypotonic solutions (osmolarity decreased by 50%). The single-channel conductance was 26 ± 3 pS in isotonic (132 mm K⁺) solutions and 24 ± 2 pS in hypotonic (63 mm K⁺) solutions. No outward conductance was found for this channel, and the channel activity was completely inhibited by barium. Cell swelling activated another inwardly rectifying K⁺ channel with a larger inward conductance of 65 pS and outward conductance of 15 pS in the on-cell configuration. In this channel, rectification was due to the block of outward currents by Mg²⁺ and Ca²⁺ ions, since when both ions were removed from the cytosolic side in inside-out patches the conductance of the channel was nearly ohmic. In contrast to the small-conductance channel, the swelling-activated channel was observed also in the presence of barium in the pipette. Neither type of channel was dependent on the presence of Ca²⁺ ions on the cytosolic side for activity. Received: 18 July 1997/Revised: 30 January 1998     

Expression of an inwardly rectifying K+ channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

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.     

Developmental regulation of a novel outwardly rectifying mechanosensitive anion channel in Caenorhabditis elegans

The nematode Caenorhabditis elegans offers unique experimental advantages for defining the molecular basis of anion channel function and regulation. However, the relative inaccessibility of somatic cells in adult animals greatly limits direct electrophysiological studies of channel activity. We developed methods to routinely isolate and patch clamp C. elegans embryo cells and oocytes and to culture and patch clamp neurons and muscle cells. Dissociated embryonic cells express a robust outwardly rectifying anion current that is activated by membrane stretch and depolarization. This current, termed I(Cl,mec), is inhibited by anion and mechanosensitive channel inhibitors. I(Cl,mec) has broad anion selectivity and the channel has a unitary conductance of 5-7 picosiemens. I(Cl,mec) is not detectable in whole-cell or isolated patch recordings from oocytes, cultured muscle cells, and cultured neurons but is expressed in single cell and later embryos. Channel density is high, and the current is observed in >80% of membrane patches. Macroscopic currents of 40-120 pA at +100 mV are typically observed in inside-out membrane patches formed using low resistance patch pipettes. Isolated membrane patches of early embryonic cells therefore contain 60-200 I(Cl,mec) channels. The apparent activation of I(Cl,mec) shortly after fertilization and its down-regulation in terminally differentiated cells suggests that the channel may play important roles in embryogenesis and/or cytokinesis.