Single-channel recordings from cultured human retinal pigment epithelial cells

We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single-channel slope conductances near 100, 45, and 25 pS as measured in on-cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane.     

Potassium inward rectifier and acetylcholine receptor channels in embryonic Xenopus muscle cells in culture

Embryonic muscle cells of the frog Xenopus laevis were isolated and grown in culture and single-channel recordings of potassium inward rectifier and acetylcholine (ACh) receptor currents were obtained from cell-attached membrane patches. Two classes of inward rectifier channels, which differed in conductance, were apparent. With 140 mM potassium chloride in the electrode, one channel class had a conductance of 28.8 ± 3.4 pS (n = 21), and, much more infrequently, a smaller channel class with a conductance of 8.6 ± 3.6 pS (n = 7) was recorded. Both channel classes had relatively long mean channel open times, which decreased with membrane hyperpolarization. The probability of finding a patch of membrane with an inward rectifier channel was high (66%) and many membrane patches contained more than one inward rectifier channel. The open state probability (with no applied potential) was high for both inward rectifier channel classes so that 70% of the time there was a channel open. Seventy-three percent of the membrane patches with ACh receptor channels (n = 11) also had at least one inward rectifier channel present when the patch electrode contained 0.1 μM ACh. Inward rectifier channels were also found at 71% of the sites of high ACh receptor density (n = 14), which were identified with rhodamine-conjugated α-bungarotoxin. The results indicate that the density of inward rectifier channels in this embryonic skeletal muscle membrane was relatively high and includes sites of membrane that have synaptic specializations. © 1996 John Wiley & Sons, Inc.     

Potassium conductance of the squid giant axon. Single-channel studies

The patch-clamp technique was implemented in the cut-open squid giant axon and used to record single K channels. We present evidence for the existence of three distinct types of channel activities. In patches that contained three to eight channels, ensemble fluctuation analysis was performed to obtain an estimate of 17.4 pS for the single-channel conductance. Averaged currents obtained from these multichannel patches had a time course of activation similar to that of macroscopic K currents recorded from perfused squid giant axons. In patches where single events could be recorded, it was possible to find channels with conductances of 10, 20, and 40 pS. The channel most frequently encountered was the 20-pS channel; for a pulse to 50 mV, this channel had a probability of being open of 0.9. In other single-channel patches, a channel with a conductance of 40 pS was present. The activity of this channel varied from patch to patch. In some patches, it showed a very low probability of being open (0.16 for a pulse to 50 mV) and had a pronounced lag in its activation time course. In other patches, the 40-pS channel had a much higher probability of being open (0.75 at a holding potential of 50 mV). The 40-pS channel was found to be quite selective for K over Na. In some experiments, the cut-open axon was exposed to a solution containing no K for several minutes. A channel with a conductance of 10 pS was more frequently observed after this treatment. Our study shows that the macroscopic K conductance is a composite of several K channel types, but the relative contribution of each type is not yet clear. The time course of activation of the 20-pS channel and the ability to render it refractory to activation only by holding the membrane potential at a positive potential for several seconds makes it likely that it is the predominant channel contributing to the delayed rectifier conductance.     

GABA increases both the conductance and mean open time of recombinant GABAA channels co-expressed with GABARAP

The single channel properties of recombinant gamma-aminobutyric acid type A (GABA(A))alphabetagamma receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing alphabetagamma receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA(A) receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (> or =40 pS), and GABA (> or =10 microM) was able to increase their conductance, up to a maximum of 60 pS. The mean open time of GABA-activated channels in control cells expressing alphabetagamma receptors alone was 2.3 +/- 0.1 ms for the main 30-pS channel and shorter for the subconductance state (20 pS, 0.8 +/- 0.1 ms). Similar values were measured for the 30- and 20-pS channels active in patches from cells co-expressing GABARAP. However higher conductance channels (> or =40 pS) remained open longer, irrespective of whether GABA or GABA plus diazepam activated them. Plotting mean open times against mean conductances revealed a linear relationship between these two parameters. Since high GABA concentrations increase both the maximum single channel conductance and mean open time of GABA(A) channels co-expressed with GABARAP, trafficking processes must influence ion channel properties. This suggests that the organization of extrasynaptic GABA(A) receptors may provide a range of distinct inhibitory currents in the brain and, further, provide differential drug responses.     

Ion channels activated by L-glutamate and GABA in cultured cerebellar neurons of the rat

Glutamate and GABA-receptor channels were investigated in explants of rat cerebellum grown in cell culture. The patch-clamp technique was used to examine neurons under whole cell clamp and the properties of channels were derived by analysis of glutamate and GABA-evoked current noise. In addition, single channel currents activated by glutamate were recorded from isolated outside-out patches of membrane. We found evidence for at least two types of glutamate receptor-channels in cerebellar cells. Some neurons exhibited a channel of 50 pS conductance with a Lorentzian noise spectrum of 5.9 ms time constant. Single channels were readily resolved both in whole cell clamp and excised patches. Other neurons possessed low conductance channels which produced two component spectra. Estimates of the single channel conductance gave a value of about 140 fS. GABA channel noise obtained from these cells was also fitted by two component spectra which gave single channel conductance of 16 pS.     

Calcium- and voltage-activated potassium channels in human macrophages.

Single calcium-activated potassium channel currents were recorded in intact and excised membrane patches from cultured human macrophages. Channel conductance was 240 pS in symmetrical 145 mM K+ and 130 pS in 5 mM external K+. Lower conductance current fluctuations (40% of the larger channels) with the same reversal potential as the higher conductance channels were noted in some patches. Ion substitution experiments indicated that the channel is permeable to potassium and relatively impermeable to sodium. The frequency of channel opening increased with depolarization and intracellular calcium concentration. At 10(-7) M (Ca++)i, channel activity was evident only at potentials of +40 mV or more depolarized, while at 10(-5) M, channels were open at all voltages tested (-40 to +60 mV). In intact patches, channels were seen at depolarized patch potentials of +50 mV or greater, indicating that the ionized calcium concentration in the macrophage is probably less than 10(-7) M.     

Calcium channels of amphibian stomach and mammalian aorta smooth muscle cells.

Whole-cell and single-channel calcium currents were studied using single smooth muscle cells enzymatically-isolated from stomach of Amphiuma tridactylum and from guinea-pig aorta. These cells have a high specific resistance and can sustain calcium action potentials after suppression of potassium currents. Dialyzed Amphiuma smooth muscle cells had calcium currents which were stable for several hours whereas the calcium currents of aortic cells ran down quickly. Single channel calcium currents in cell-attached patches behaved similarly for the two cell types. Calcium channel conductance in 110 mM barium was 12 pS and the mean open time was 1.4 ms at a nominal membrane potential of +10 mV. Exposure of both cell types to BAY K8644 resulted in a dramatic prolongation of the calcium channel open times and a shift in the probability of opening to more negative potentials. Low-threshold calcium channels were not identified in the extensively studied amphibian cells. High-threshold calcium channels therefore appear to be the primary pathway for the calcium influx that produces contraction in these smooth muscle cells.     

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.     

Spontaneous and GABA-induced single channel currents in cultured murine spinal cord neurons

Intracellular and patch clamp recordings were made from embryonic mouse spinal cord neurons growing in primary cell culture. Outside-out membrane patches obtained from these cells usually showed spontaneous single channel currents when studied at the resting potential (-56 +/- 1.5 mV). In 18 out of 30 patches tested, spontaneous single channel activity was abolished by making Tris+ the major cation on both sides of the membrane. The remaining patches continued to display spontaneous single channel currents under these conditions. These events reversed polarity at a patch potential of 0 mV and displayed a mean single channel conductance of 24 +/- 1.2 pS. Application of the putative inhibitory transmitter gamma-aminobutyric acid (0.5-10 microM) to outside-out patches of spinal cord cell membrane induced single channel currents in 10 out of 15 patches tested. These channels had a primary conductance of 29 +/- 2.8 pS in symmetrical 145 mM Cl- solutions. Frequency distributions for the open times of these channels were well fit by the sum of a fast exponential term ("of") with a time constant tau of = 4 +/- 1.3 ms and a slow exponential term ("os") with a time constant tau os = 24 +/- 8.1 ms. Frequency distributions for channel closed times were also well fit by a double exponential equation, with time constants tau cf = 2 +/- 0.2 ms and tau cs = 62 +/- 20.9 ms.     

Muscarinic acetylcholine receptor produced in recombinant baculovirus infected Sf9 insect cells couples with endogenous G-proteins to activate ion channels.

Following the infection of insect ovarian cells (Sf9) with recombinant bearing the cDNA coding for the rat muscarinic acetylcholine (ACh) receptor subtype m3, ionic flux across the membrane in response to the application of ACh was examined electrophysiologically. We show that ACh activates potassium currents. The response is abolished when cells are treated with pertussis toxin. No ACh-induced currents are observed from uninfected cells or cells infected with virus which do not contain the cDNA coding for ACh receptors in its genome. The characteristics of single channel currents show time-dependent changes following the application of ACh. Initially, ACh activates brief channel currents with a conductance of about 5 pS. The conductance level of channels gradually increases in steps to 10 pS and then to 20 pS and 40 pS. At the same time, channel open probability also increases. Thereafter, additional channels appear, opening and closing independently of, or at times in synchrony with, the original channel.     

Conductance of recombinant GABA (A) channels is increased in cells co-expressing GABA(A) receptor-associated protein

High conductance gamma-aminobutyric acid type A (GABA(A)) channels (>40 picosiemens (pS)) have been reported in some studies on GABA(A) channels in situ but not in others, whereas recombinant GABA(A) channels do not appear to display conductances above 40 pS. Furthermore, the conductance of some native GABA(A) channels can be increased by diazepam or pentobarbital, which are effects not reported for expressed GABA(A) channels. GABARAP, a protein associated with native GABA(A) channels, has been reported to cause clustering of GABA(A) receptors and changes in channel kinetics. We have recorded single channel currents activated by GABA in L929 cells expressing alpha(1), beta(1), and gamma(2S) subunits of human GABA(A) receptors. Channel conductance was never higher than 40 pS and was not significantly increased by diazepam or pentobarbital, although open probability was increased. In contrast, in cells expressing the same three subunits together with GABARAP, channel conductance could be significantly higher than 40 pS, and channel conductance was increased by diazepam and pentobarbital. GABARAP caused clustering of receptors in L929 cells, and we suggest that there may be interactions between subunits of clustered GABA(A) receptors that make them open co-operatively to give high conductance "channels." Recombinant channels may require the influence of GABARAP and perhaps other intracellular proteins to adopt a fuller repertoire of properties of native channels.     

Single potassium channel of anomalous (inward) rectification in mollusk neurons

Currents passing through individual potassium channels with anomalous (inward) rectification were recorded at the neuronal membrane ofPlanorbarius corneus using the patch clamp technique. These currents could be detected, whether in "right side out" or "inside out" configurations in the presence of 50 mM potassium ions or one of the potassium channel blockers: tetraethylammonium (TEA), barium, or cesium (2–20 mM) on the external side of the membrane. Inward currents were observed in individual channels at potentials more negative than level of potassium equilibrium potential (E_k); conductance of these measured 81±12 pS (n=11). At more positive potentials than E_k, conductance fell to zero. Potassium channels with anomalous (inward) rectification inPlanorbarius corneus resemble equivalent channels in other cells in their kinetics: time scale of the open state may be described by a single exponential function. This would imply that the ionic channel has a single open state. Time scale of the closed state was biexponential, thus indicating the possible existence of two kinetically different nonconducting states of the potassium channel with anomalous (inward) rectification at the neuronal membrane ofPlanorbarius corneus.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 31–38, January–February, 1989.     

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.     

Biophysical properties and metabolic regulation of a TASK-like potassium channel in rat carotid body type 1 cells

The single channel properties of TASK-like oxygen-sensitive potassium channels were studied in rat carotid body type 1 cells. We observed channels with rapid bursting kinetics, active at resting membrane potentials. These channels were highly potassium selective with a slope conductance of 14-16 pS, values similar to those reported for TASK-1. In the absence of extracellular divalent cations, however, single channel conductance increased to 28 pS in a manner similar to that reported for TASK-3. After patch excision, channel activity ran down rapidly. Channel activity in inside-out patches was markedly increased by 2 and 5 mM ATP and by 2 mM ADP but not by 100 microM ADP or 1 mM AMP. In cell-attached patches, both cyanide and 2,4-dinitrophenol strongly inhibited channel activity. We conclude that 1) whilst the properties of this channel are consistent with it being a TASK-like potassium channel they do not precisely conform to those of either TASK-1 or TASK-3, 2) channel activity is highly dependent on cytosolic factors including ATP, and 3) changes in energy metabolism may play a role in regulating the activity of these background K+ channels.     

Voltage dependent calcium channels in cerebellar granule cell primary cultures

Voltage activated calcium channels were studied in rat cerebellar granule cells in primary culture. Macroscopic currents, carried by 20mM Ba2+, were measured in the whole-cell configuration. Slowly inactivating macroscopic currents, with a maximum value at a membrane potential around 5 mV, were recorded between the 1st and the 4th day in culture. These currents were completely blocked by 5mM Co2+, partially blocked by 10 microM nifedipine, and increased by 2 to 5 microM BAY K-8644. Two types of channels, in the presence of 80 mM Ba2+, were identified by single channel recording in cell-attached patches. The first type, which was dihydropyridine agonist sensitive, had a conductance of 18 pS, a half activation potential of more than 10 mV and did not inactivate. This type of channel was the only type found during the first four days in culture, although it was also present up to the 11th day. The second type of channel was dihydropyridine insensitive, had a conductance of 10 pS, a half activation potential less than -15 mV, and displayed voltage dependent inactivation. This second type of channel was found in cells for more than four days in culture.     

GABA-induced potassium channels in cultured neurons

When gamma-aminobutyric acid (GABA) or baclofen were applied to cultured rat hippocampal neurons, single-channel potassium currents appeared after a delay of 30 s or more in patches of membrane on the cell surface isolated from the agonists by the recording pipette. The appearance of currents in patches not exposed to agonist, the delay in their appearance and the suppression of currents in cells pre-incubated with pertussis toxin indicate the involvement of an intracellular second messenger system. The channels were associated with a GABAB receptor rather than a GABAA receptor as they were blocked by baclofen, a GABAB antagonist, but were not affected by bicuculline, a GABAA antagonist. A feature of the single channel currents was their variable amplitude: they had a maximum conductance of ca. 70 pS and displayed many lower conductance states that were integral multiples of 5-6 pS. In several cells exposed to GABA or baclofen, first small currents and then progressively larger currents appeared: current amplitude was a multiple of an elementary current. It is suggested that binding of GABA to GABAB receptors activates a second messenger system causing opening of oligomeric potassium channels.     

Ion channels in mammalian proximal renal tubules.

In the plasma membranes of mammalian proximal renal tubules single ion channels were investigated mainly in isolated tubules perfused on one side, in isolated nonperfused (collapsed) tubules and in primary cell cultures. With these techniques, the following results were obtained: in the luminal membrane of isolated one-sided perfused tubules of rabbit and mouse S3 segments, K(+)-selective channels with single-channel conductance (g) of 33 pS and 63 pS, respectively, were recorded. In primary cultures of rabbit S1 segments, a small-conductance (42 pS) as well as a large-conductance (200 pS) K+ channel were observed. The latter was Ca2(+)- and voltage-sensitive. In cultured cells a Ca2(+)-activated, nonselective cation channel with g = 25 pS was also recorded. On the other hand, an amiloride-sensitive channel with g = 12 pS, which was highly selective for Na+ over K+, was observed in the isolated perfused S3 segment. In the basolateral membrane of isolated perfused S3 segments, two types of K+ channels with g = 46 pS and 36 pS, respectively, were observed. The latter channel was not dependent on cytosolic Ca2+ in cell-excised patches. A K+ channel with g = 54 pS was recorded in isolated nonperfused S1 segments. This channel showed inward rectification and was more active at depolarizing potentials. In isolated perfused S3 segments, in addition to the K+ channels also a nonselective cation channel with g = 28 pS was observed. This channel was highly dependent on cytosolic Ca2+ in cell-free patches. It can be concluded that the K+ channels both in the luminal and contraluminal cell membrane are involved in the generation of the cell potential. Na+ channels in the luminal membrane may participate in Na+ reabsorption, whereas the function of a basolateral cation channel remains unclear. Recently, single anion-selective channels were recorded in membranes of endocytotic vesicles, isolated from rat proximal tubules. Vesicles were enlarged by the dehydration/rehydration method and investigated with the patch clamp technique. The Cl- channel had a conductance of 73 pS, the current-voltage curve was linear and the channel inactivated at high negative clamp potentials. It is suggested that this channel is responsible for charge neutrality during active H+ uptake into the endosomes.     

Calcium- and voltage-activated potassium channels in adrenocortical cell membranes

Current flowing through single Ca- and voltage-activated K channels has been recorded from cell-attached and inside-out excised membrane patches of cultured Y-1 adrenocortical cells. In intact cells, single-channel current amplitude and the time a channel stays in the open state increase with membrane depolarization. In excised patches bathed in symmetrical 130 mM K solutions, single-channel conductance is 170 pS. This value is constant in the membrane potential range of +/- 50 mV but decreases at larger hyper- and depolarizations. Channel open probability is heavily influenced by the concentration of ionic Ca at the inner surface of the membrane in the range between 0.01 and 10 microM. When internal Ca concentration is close to 0.01 microM, channels are usually closed even at large depolarizing voltages. With larger Ca concentrations, channel open probability increases and its voltage dependence is greater. These channels are uniformly distributed in the plasma membrane, since one to four channels were seen in more than 99% of the patches isolated in this study. There are previous reports suggesting a role for calcium ions in the secretory response of adrenocortical cells to ACTH. Therefore, it is possible that, as in other endocrine cells, these K channels modulate Ca influx across the plasma membrane and thus contribute to regulate steroid biosynthesis and release.     

钾通道活化剂对豚鼠气道平滑肌电压依赖性钾通道特性的影响

钾通道活化剂可激活钾离子通道并松驰支气管平滑肌,在急性分离的豚鼠支气管平滑肌细胞上,用膜片钳技术的细胞贴附式和内面向外式研究了其对电压依赖性钾通道的直接作用。结果证实：在全细胞记录条件下,卡吗克林和拉吗克林不影响静息膜电位。但在去极化时可使通道电导从７５．２±５．１ｐＳ分别增大到８５．９±１１．８ｐＳ和８２．１±５．５ｐＳ。通道动力学特性也发生了改变,通道平均开放时间的τｏ２值延长和开放概率显著增加,其中拉吗克林的作用更为强。两者均可诱发通道出现多级开放。表明这两类活化剂可使去极化时钾离子外流增加。     

Nonselective voltage-gated ionic channels in type II taste cells

In cells of different types outward voltage-gated (VG) ion currents are generally carried by potassium ions. However, in mouse type II taste cells these currents persist when K⁺-selective ion channels are inhibited. In this study, we examined the ion channels that provide a pathway for atypical VG outward currents in type II taste cells. These channels are found to be weakly selective and permeabile to large molecules such as NMDG, gluconate, and ATP. According to non-stationary fluctuation analysis, single channel conductance is about 200 pS. The data obtained suggest that the nonselective ion channels are similar to hemichannels formed by connexins, the gap-junction proteins, in the plasma membrane of vertebrate cells.