Basic properties and potential regulators of the apical K+ channel in macula densa cells

These studies examine the properties of an apical potassium (K+) channel in macula densa cells, a specialized group of cells involved in tubuloglomerular feedback signal transmission. To this end, individual glomeruli with thick ascending limbs (TAL) and macula densa cells were dissected from rabbit kidney and the TAL covering macula densa cells was removed. Using patch clamp techniques, we found a high density (up to 54 channels per patch) of K+ channels in the apical membrane of macula densa cells. An inward conductance of 41.1 +/- 4.8 pS was obtained in cell-attached patches (patch pipette, 140 mM K+). In inside- out patches (patch pipette, 140 mM; bath, 5 mM K+), inward currents of 1.1 +/- 0.1 pA (n = 11) were observed at 0 mV and single channel current reversed at a pipette potential of -84 mV giving a permeability ratio (PK/PNa) of over 100. In cell-attached patches, mean channel open probability (N,Po, where N is number of channels in the patch and Po is single channel open probability) was unaffected by bumetanide, but was reduced from 11.3 +/- 2.7 to 1.6 +/- 1.3 (n = 5, p < 0.02) by removal of bath sodium (Na+). Simultaneous removal of bath Na+ and calcium (Ca2+) prevented the Na(+)-induced decrease in N.Po indicating that the effect of Na+ removal on N.Po was probably mediated by stimulation of Ca2+ entry. This interpretation was supported by studies where ionomycin, which directly increases intracellular Ca2+, produced a fall in N.Po from 17.8 +/- 4.0 to 5.9 +/- 4.1 (n = 7, p < 0.02). In inside- out patches, the apical K+ channel was not sensitive to ATP but was directly blocked by 2 mM Ca2+ and by lowering bath pH from 7.4 to 6.8. These studies constitute the first single channel observations on macula densa cells and establish some of the characteristics and regulators of this apical K+ channel. This channel is likely to be involved in macula densa transepithelial Cl- transport and perhaps in the tubuloglomerular feedback signaling process.     

Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.     

Trek-like Potassium Channels in Rat Cardiac Ventricular Myocytes Are Activated by Intracellular ATP

Large (111 +/- 3.0 pS) K+ channels were recorded in membrane patches from adult rat ventricular myocytes using patch-clamp techniques. The channels were not blocked by 4-AP (5 mM), intracellular TEA (5 mM) or glybenclamide (100 mM). Applying stretch to the membrane (as pipette suction) increased channel open probability (Po) in both cell-attached and isolated patches (typically, Po approximately equals 0.005 with no pressure; approximately equals 0.328 with 90 cm H2O: Vm = 40 mV, pHi = 7.2). The channels were activated by a decrease in intracellular pH; decreasing pHi to 5.5 from 7.2 increased Po to 0.16 from approx. 0.005 (no suction, Vm held at 40 mV). These properties are consistent with those demonstrated for TREK-1, a member of the recently cloned tandem pore family. We confirmed, using RT-PCR, that TREK-1 is expressed in rat ventricle, suggesting that the channel being recorded is indeed TREK-1. However, we show also that the channels are activated by millimolar concentrations of intracellular ATP. At a pH of 6 with no ATP at the intracellular membrane face, Po was 0.048 +/-0.023, whereas Po increased to 0.22 +/- 0.1 with 1 mM ATP, and to 0.348 +/- 0.13 with 3 mM (n = 5; no membrane stretch applied). The rapid time course of the response and the fact that we see the effect in isolated patches appear to preclude phosphorylation. We conclude that intracellular ATP directly activates TREK-like channels, a property not previously described.     

Single channel currents in adrenocortical cells

Single channel currents have been recorded from cell-attached patches of tumoral adrenocortical cells. Our experiments suggest the existence of three sets of potassium channels in the surface membrane of these cells. All channel types can be recorded in a given membrane patch but some patches have only one type of single channel currents. One channel type has a unitary conductance of about 103 pS. The other two channels have smaller conductances and opposite voltage dependence. In one case channels open on depolarization and have a single channel conductance of 31.6 pS. In the other case the probability of being in the open state increases on hyperpolarization and the single channel conductance is of 21 pS. These channels seem to be similar to the delayed and anomalous rectifying potassium channels seen in other preparations. The role of membrane ionic permeability in steroid release induced by ACTH is discussed.     

Patch recordings from the electrocytes of electrophorus. Na channel gating currents

Gating currents were recorded at 11 degrees C in cell-attached and inside-out patches from the innervated membrane of Electrophorus main organ electrocytes. With pipette tip diameters of 3-8 microns, maximal charge measured in patches ranged from 0.74 to 7.19 fC. The general features of the gating currents are similar to those from the squid giant axon. The steady-state voltage dependence of the ON gating charge was characterized by an effective valence of 1.3 +/- 0.4 and a midpoint voltage of -56 +/- 7 mV. The charge vs. voltage relation lies approximately 30 mV negative to the channel open probability curve. The ratio of the time constants of the OFF gating current and the Na current was 2.3 at -120 mV and equal at -80 mV. Charge immobilization and Na current inactivation develop with comparable time courses and have very similar voltage dependences. Between 60 and 80% of the charge is temporarily immobilized by inactivation.     

Kinetic properties of single sodium channels in rat heart and rat brain

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.     

Conductance and gating of epithelial Na channels from rat cortical collecting tubule. Effects of luminal Na and Li

The behavior of individual Na channels in the apical membrane of the rat cortical collecting tubule (CCT) was studied at different concentrations of the permeant ions Na and Li. Tubules were opened to expose their luminal surfaces and bathed in K-gluconate medium to minimize tubule-to-tubule variation in cell membrane potential and intracellular Na concentration. The patch-clamp technique was used to resolve currents through individual channels. The patch-clamp pipette was filled with solutions containing variable concentrations of either NaCl or LiCl. In one series of experiments, the concentrations were changed without substitutions. In another series, the ionic strength and Cl concentration were maintained constant by partial substitution of Li with N-methyl-D-glucamine (NMDG). In cell-attached patches, both the single-channel conductance (g) and the single-channel current (i) saturated as functions of the Na or Li activity in the pipette. Without NMDG, the saturation of i was well described by Michaelis-Menten kinetics with an apparent Km of approximately 20 mM activity for Na and approximately 50 mM activity for Li. Km was independent of voltage for both ions. With substitution for Li by NMDG, the apparent Km value for Li transport through the channels increased. The values of the probability of a channel's being open (Po) varied from patch to patch, but no effect of pipette ion activity on Po could be demonstrated. A weak dependence of Po on membrane voltage was observed, with hyperpolarization increasing Po by an average of 2.3%/mV.     

CFTR displays voltage dependence and two gating modes during stimulation

The patch-clamp technique in conjunction with current noise analysis was employed to clarify the events underlying the regulation of the CFTR (cystic fibrosis transmembrane conductance regulator) during cAMP- dependent stimulation. 3T3 fibroblast cells expressing the CFTR were stimulated in cell-attached mode with forskolin. The number (N) of activated channels per patch ranged from 1 to approximately 100. In true single-channel recordings, CFTR's gating was best described by two open states (approximately 5 and approximately 100 ms) and three closed states (< or = 5, approximately 100, and approximately 1,000 ms). Current noise analysis resulted in spectra containing two distinct Lorentzian noise components with corner frequencies of 1.3 Hz and approximately 50 Hz, respectively. Single-channel time constants were dependent on voltage. The fastest closed state increased its contribution from 48% at +100 mV to 87% at -100 mV, and the medium open state reduced its length to one half, resulting in gating dominated by fast events. Similarly, the fast Lorentzian increased its amplitude, and its corner frequency increased from 44 Hz at +100 mV to 91 Hz at - 100 mV, while the slow Lorentzian was voltage independent. In multi- channel recordings N.Po (i.e., N times open probability) increased significantly, on average by 52% between -90 and +90 mV. Stimulation with forskolin increased Po of CFTR to approximately 0.5, which resulted from a decrease of the longest closed state while the faster open and closed states were unaffected. Neither corner frequency was affected during stimulation. Recordings from multichannel patches revealed in addition, unique, very long channel openings (high Po mode, average 13 s). Channels exhibiting high Po (i.e., Po approximately 1.0) or low Po (i.e., Po approximately 0.5) gating modes were both present in multichannel recordings, and CFTRs switched modes during stimulation. In addition, the switch to the high Po mode appeared to be a cooperative event for channel pairs. High forskolin concentration (i.e., 10 microM) favored transition into the high Po mode, suggesting a cellularly mediated regulation of model switching due to a fundamental change in configuration of the CFTR. Thus, during stimulation the CFTR increased its activity through two distinct effects: the reduction of the long closed state and modal switching to the high Po mode.     

Voltage-dependent gating of the cystic fibrosis transmembrane conductance regulator Cl- channel

When excised inside-out membrane patches are bathed in symmetrical Cl--rich solutions, the current-voltage (I-V) relationship of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents inwardly rectifies at large positive voltages. To investigate the mechanism of inward rectification, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human and murine CFTR using voltage-ramp and -step protocols. Using a voltage-ramp protocol, the magnitude of human CFTR Cl- current at +100 mV was 74 +/- 2% (n = 10) of that at -100 mV. This rectification of macroscopic CFTR Cl- current was reproduced in full by ensemble currents generated by averaging single-channel currents elicited by an identical voltage-ramp protocol. However, using a voltage-step protocol the single-channel current amplitude (i) of human CFTR at +100 mV was 88 +/- 2% (n = 10) of that at -100 mV. Based on these data, we hypothesized that voltage might alter the gating behavior of human CFTR. Using linear three-state kinetic schemes, we demonstrated that voltage has marked effects on channel gating. Membrane depolarization decreased both the duration of bursts and the interburst interval, but increased the duration of gaps within bursts. However, because the voltage dependencies of the different rate constants were in opposite directions, voltage was without large effect on the open probability (Po) of human CFTR. In contrast, the Po of murine CFTR was decreased markedly at positive voltages, suggesting that the rectification of murine CFTR is stronger than that of human CFTR. We conclude that inward rectification of CFTR is caused by a reduction in i and changes in gating kinetics. We suggest that inward rectification is an intrinsic property of the CFTR Cl- channel and not the result of pore block.     

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

Modal gating behavior of cardiac sodium channels in cell-free membrane patches.

Single voltage-activated Na+ channel currents were obtained from membrane patches of isolated ventricular cells of guinea pig hearts. The currents were compared when measured from cell-attached patches and from the same patch but at least 20 minutes after manual excision. The averaged currents showed a distinctly delayed decay in the excised patches due to the appearance of long lasting openings or bursts of openings. In contrast to control patches, the open time distribution in excised patches requires at least two exponentials. A short mean open time was voltage independent for cell-attached patches (0.38 ms +/- 0.07 ms between -60 and -20 mV, 6 cell-attached patches; and 0.41 +/- 0.1 ms, 7 excised patches). The long mean open time found in excised patches was clearly voltage dependent and increased from 0.48 +/- 0.14 ms (-80 mV) to 2.87 +/- 0.35 ms (-20 mV, regression coefficient +0.88, 7 patches). Sweeps with long openings appeared in clusters. The clustering of records with long openings, short openings, or without openings (nulls) was quantified by a runs analysis which showed a highly significant nonrandom ordering. The results show that in excised patches inactivation is temporally hibernating.     

Modulation of single cardiac sodium channels by DPI 201-106

Single sodium channel currents were analysed in cell attached patches from single ventricular cells of guinea pig hearts in the presence of a novel cardiotonic compound DPI 201-106. The mean single channel conductance of DPI-treated Na channels was not changed by DPI (20.8 +/- 4 pS, control, 3 patches; 21.3 +/- 1 pS with DPI, 5 mumol/1,3 patches). DPI voltage-dependently prolongs the cardiac sodium channel openings by removal of inactivation at potentials positive to -40 mV. At potentials negative to -40 mV a clustering of short openings at the very beginning of the depolarizing voltage steps can be observed causing a transient time course of the averaged currents. Long openings induced an extremely slow inactivation. Short openings, long openings and nulls appeared in groups referring to a modal gating behaviour of DPI-treated sodium channels. DPI-modified Na channels showed a monotonously prolonged mean open time with increased depolarizing voltage steps, e.g. the open state probability within a sweep was increased. However, the number of non-empty sweeps was decreased with the magnitude of the depolarizing steps, e.g. the probability of the channel being open as calculated from the averaged currents was voltage-dependently decreased by DPI (50% decrease at -50.7 +/- 9 9 mV, 3 patches). Short and long openings of DPI-modified channels could be separated by variation of the holding potential. The occurrence of long Na channel openings was much more suppressed by reducing the holding potential (half maximum inactivation at -112 +/- 8 mV, 4 patches) than that of short openings (half maximum inactivation at -88 +/- 8 mV, 4 patches). Otherwise, short living openings completely disappeared at potentials positive to -40 mV where the occurrence of long openings was favoured. The differential voltage dependence of blocking and activating effects of DPI on cardiac Na channels as well as the differential voltage dependence of the appearance of short and long openings refers to a modal gating behaviour of cardiac Na channels.     

Structural determinants of gating in inward-rectifier K+ channels

The gating characteristics of two ion channels in the inward-rectifier K+ channel superfamily were compared at the single-channel level. The strong inward rectifier IRK1 (Kir 2.1) opened and closed with kinetics that were slow relative to those of the weakly rectifying ROMK2 (Kir 1.1b). At a membrane potential of -60 mV, both IRK and ROMK had single-exponential open-time distributions, with mean open times of 279 +/- 58 ms (n = 4) for IRK1 and 23 +/- 1 ms (n = 7) for ROMK. At -60 mV (and no EDTA) ROMK2 had two closed times: 1.3 +/- 0.1 and 36 +/- 3 ms (n = 7). Under the same conditions, IRK1 exhibited four discrete closed states with mean closed times of 0.8 +/- 0.1 ms, 14 +/- 0.6 ms, 99 +/- 19 ms, and 2744 +/- 640 ms (n = 4). Both the open and the three shortest closed-time constants of IRK1 decreased monotonically with membrane hyperpolarization. IRK1 open probability (Po) decreased sharply with hyperpolarization due to an increase in the frequency of long closed events that were attributable to divalent-cation blockade. Chelation of divalent cations with EDTA eliminated the slowest closed-time distribution of IRK1 and blunted the hyperpolarization-dependent fall in open probability. In contrast, ROMK2 had shorter open and closed times and only two closed states, and its Po was less affected by hyperpolarization. Chimeric channels were constructed to address the question of which parts of the molecules were responsible for the differences in kinetics. The property of multiple closed states was conferred by the second membrane-spanning domain (M2) of IRK. The long-lived open and closed states, including the higher sensitivity to extracellular divalent cations, correlated with the extracellular loop of IRK, including the "P-region." Channel kinetics were essentially unaffected by the N- and C-termini. The data of the present study are consistent with the idea that the locus of gating is near the outer mouth of the pore.     

Cl- channels in intact human T lymphocytes.

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.     

GTP-dependent regulation of myometrial KCa channels incorporated into lipid bilayers

The regulation of calcium-activated K (KCa) channels by a G protein-mediated mechanism was studied. KCa channels were reconstituted in planar lipid bilayers by fusion of membrane vesicles from rat or pig myometrium. The regulatory process was studied by exploring the actions of GTP and GTP gamma S on single channel activity. KCa channels had a conductance of 260 +/- 6 pS (n = 25, +/- SE, 250/50 mM KCl gradient) and were voltage dependent. The open probability (Po) vs. voltage relationships were well fit by a Boltzmann distribution. The slope factor (11 mV) was insensitive to internal Ca2+. The half activation potential (V1/2) was shifted -70 mV by raising internal Ca2+ from pCa 6.2 to pCa 4. Addition of GTP or GTP gamma S activated channel activity only in the presence of Mg2+, a characteristic typical of G protein-mediated mechanisms. The Po increased from 0.18 +/- 0.08 to 0.49 +/- 0.07 (n = 7, 0 mV, pCa 6 to 6.8). The channel was also activated (Po increased from 0.03 to 0.37) in the presence of AMP-PNP, a nonphosphorylating ATP analogue, suggesting a direct G protein gating of KCa channels. Upon nucleotide activation, mean open time increased by a factor of 2.7 +/- 0.7 and mean closed time decreased by 0.2 +/- 0.07 of their initial values (n = 6). Norepinephrine (NE) or isoproterenol potentiated the GTP-mediated activation of KCa channels (Po increased from 0.17 +/- 0.06 to 0.35 +/- 0.07, n = 10). These results suggest that myometrium possesses beta-adrenergic receptors coupled to a GTP-dependent protein that can directly gate KCa channels. Furthermore, KCa channels, beta-adrenergic receptors, and G proteins can be reconstituted in lipid bilayers as a stable, functionally coupled, molecular complex.     

Basolateral outward rectifier chloride channel in isolated crypts of mouse colon

Single channel patch-clamp techniques were used to demonstrate the presence of outwardly rectifying chloride channels in the basolateral membrane of crypt cells from mouse distal colon. These channels were rarely observed in the cell-attached mode and, in the inside-out configuration, only became active after a delay and depolarizing voltage steps. Single channel conductance was 23.4 pS between -100 and -40 mV and increased to 90.2 pS between 40 and 100 mV. The channel permeability sequence for anions was: I(-) > SCN(-) > Br(-) > Cl(-) > NO(3)(-) > F(-)> SO(4)(2-) approximately gluconate. In inside-out patches, the channel open probability was voltage dependent but insensitive to intracellular Ca(2+) concentration. In cell-attached mode, forskolin, histamine, carbachol, A-23187, and activators of protein kinase C all failed to activate the channel, and activity could not be evoked in inside-out patches by exposure to the purified catalytic subunit of cAMP-dependent protein kinase A. The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, 9-anthracenecarboxylic acid, and DIDS. Stimulation of G proteins with guanosine 5'-O-(3-thiotriphosphate) decreased the channel open probability and conductance, whereas subsequent addition of guanosine 5'-O-(2-thiodiphosphate) reactivated the channel.     

Light-dependent K(+) channels in the mollusc Onchidium simple photoreceptors are opened by cGMP

Light-dependent K(+) channels underlying a hyperpolarizing response of one extraocular (simple) photoreceptor, Ip-2 cell, in the marine mollusc Onchidium ganglion were examined using cell-attached and inside-out patch-clamp techniques. A previous report (Gotow, T., T. Nishi, and H. Kijima. 1994. Brain Res. 662:268-272) showed that a depolarizing response of the other simple photoreceptor, A-P-1 cell, results from closing of the light-dependent K(+) channels that are activated by cGMP. In the cell-attached patch recordings of Ip-2 cells, external artificial seawater (ASW) was replaced with a modified ASW containing 150 mM K(+) and 200 mM Mg(2+) to suppress any synaptic input and to maintain the membrane potential constant. When Ip-2 cells were equilibrated with this modified ASW, the internal K(+) concentration was estimated to be 260 mM. Light-dependent single-channels in the cell-attached patch on these cells were opened by light but scarcely by voltage. After confirming the light-dependent channel activity in the cell-attached patches, an application of cGMP to the excised inside-out patches newly activated a channel that disappeared on removal of cGMP. Open and closed time distributions of this cGMP-activated channel could be described by the sum of two exponents with time constants tau(o1), tau(o2) and tau(c1), tau(c2), respectively, similar to those of the light-dependent channel. In both the channels, tau(o1) and tau(o2) in ms ranges were similar to each other, although tau(c2) over tens of millisecond ranges was different. tau(o1), tau(o2), and the mean open time tau(o) were both independent of light intensity, cGMP concentration, and voltage. In both channels, the open probability increased as the membrane was depolarized, without changing any of tau(o2) or tau(o). In both, the reversal potentials using 200- and 450-mM K(+)-filled pipettes were close to the K(+) equilibrium potentials, suggesting that both the channels are primarily K(+) selective. Both the mean values of the channel conductance were estimated to be the same at 62 and 91 pS in 200- and 450-mM K(+) pipettes at nearly 0 mV, respectively. Combining these findings with those in the above former report, it is concluded that cGMP is a second messenger which opens the light-dependent K(+) channel of Ip-2 to cause hyperpolarization, and that the channel is the same as that of A-P-1 closed by light.     

Single voltage-dependent K+ and Cl- channels in cultured rat astrocytes

The kinetic reactions of a voltage-dependent K+ channel, which constituted about 14% of all the recorded K+ channels in the membrane of cultured rat astrocytes were studied in detail. A scheme of one open and three closed states is necessary to describe the kinetic reactions of this channel. The channel contributes little to the resting membrane potential. Its steady state open probability (Po) is 0.06 at -70 mV. When the cell is depolarized to O mV, Po approaches 1. This represents a 17-fold increase. Such channels could contribute to the potassium clearance by enhancing the effect of "spatial buffering." Additionally, single anion-selective channels with very high conductances were found in inside-out patches in approximately 15% of all recorded channels in the membrane of rat astrocytes. Channel openings are characterized by more than one conductance level; the main level showed a mean conductance of 400 pS. These channels are divided into two groups. Approximately 90% of the recorded chloride channels showed a strong voltage dependency of their current fluctuations. Within a relatively small potential range (+/- 15 mV) the channels have a high probability of being in the active state. After a voltage jump to varying testing potentials in the range of +/- 20 to +/- 50 mV the channels continued to be in the active state for some time and then closed to a shut state. If the testing potential persisted, the channels were not able to leave this shut state.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 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.