Properties of single K+ channels in the basolateral membrane of rabbit proximal straight tubules

The basolateral membrane of rabbit straight proximal tubules, which were cannulated and perfused on one side, was investigated with the patch clamp technique. Properties of inward and outward directed single K⁺ channel currents were studied in cell-attached and insideout oriented cell-excised membrane patches. In cell-attached patches with NaCl Ringer solution both in pipette and bath, outward K⁺ currents could be detected after depolarization of the membrane patch by about 20–30 mV. The current-voltage (i/V) relationship could be fitted by the Goldman-Hodgkin-Katz (GHK) current equation, with the assumption that these channels were mainly permeable for K⁺ ions. A permeability coefficientP _K of (0.17±0.04) · 10^–12 cm³/s was obtained, the single channel slope conductance at infinite positive potentialg(V ) was 50±12 pS and the single channel conductance at the membrane resting potentialg(V _bl) was 12±3 pS (n=4). In cell-excised patches, with NaCl in the pipette and KCl in the bath, the data could also be fitted to the GHK equation and yieldedP _K = (0.1 ±0.01) ·10^–12 cm³/s,g(V ) = 40 ± 4 pS andg(V _bl) = 7 ± 1 pS (n=8). In cell-attached patches with KCl in the pipette and NaCl in the bath, inward K⁺ channels occurred at clamp potentials 60 mV, whereas outward K⁺ channel current was detected at more positive voltages. The current-voltage curves showed slight inward rectification. The single channel conductance, obtained from the linear part of the i/V curve by linear regression, was 46±3 pS and the reversal potential was 59±6 mV (n=9). In cell-excised patches with KCl in the pipette and NaCl in the bath, inward directed K⁺ channel currents could again be described by the GHK equation. The single channel parameters were similar to those recorded for outward K⁺ currents (see above). In inside-out oriented cell-excised patches with NaCl in the pipette and KCl in the bath, reducing bath (i.e. cytosolic) Ca²⁺ concentration from 10^–6 mol/l to less than 10^–9 mol/l did not affect the open state probability of single channel currents. These results demonstrate that the observed channels are permeable for K⁺ ions in both directions and that these basolateral K⁺ channels in rabbit proximal straight tubule are not directly dependent on Ca²⁺ ions.     

Potassium channels in the basolateral membrane of the rectal gland of the dogfish (Squalus acanthias)

Previous studies in isolated, in vitro perfused rectal gland tubules (RGT) have revealed that the basolateral membrane possesses a K⁺ conductive pathway. In the present study, we have utilized the patch clamp technique in RGT segments to characterize this pathway. The basolateral membrane was approached with patch pipettes at the open end of in vitro perfused segments [5]. Recordings were obtained in cell-attached as well as in excised inside-out patches. In cell-attached patches with the pipette filled with a KCl solution (274 mmol/l) and the bath containing NaCl shark Ringer (275 mmol/l), inward K⁺ currents (from pipette into cell) with a mean slope conductance of 123±26 pS (n=3) were observed. We were unable to generate outward K⁺ currents at high depolarizing (cell more positive) clamp voltages. This indicates inward rectification of this channel. To examine the rectification properties further, excised (inside out) patches were exposed to K⁺ concentration gradients, directed out of, as well as into the pipette. With NaCl in the pipette and KCl in the bath, K⁺ outward currents were observed. The current-voltage (IV) relation revealed Goldman-type rectification, with a mean single channel conductance of 185±28 pS (n=7) at high positive voltages (linear range of the IV curve). The single-channel permeability coefficient for K⁺ was 0.26±0.04 ·10^–12 cm³/s (n=7). In the reversed experiment (pipette KCl, bath NaCl), inward currents of similar kinetics and amplitude were obtained. The single channel conductance was 146±21 pS (n=7) at high negative voltages (linear range of the IV curve). The single channel permeability coefficient for K⁺ was 0.21±0.03·10^–12 cm³/s (n=7). We were not able to reverse the currents in any of these experiments, indicating that this channel is highly selective for K⁺ over Na⁺. In all three series of experiments, the kinetic appearance of the channels was similar. Bursts of activity were followed by interburst pauses. The open state was described by a single time constant of 3.0±0.2 ms, whereas the closed state was described by two time constants of 0.7±0.2 ms and 2.8±0.5 ms (n=8). It can be concluded that these channels permit K⁺ inward and outward currents. They are probably the equivalent of the basolateral K⁺ conductance as observed in a previous study [12]. Under physiological conditions a single channel conductance of some 20 pS is predicted from the present data. In cell-attached patches, with a high K⁺ concentration in the pipette, the channel behaves as an inward rectifier.Supported by Deutsche Forschungsgemeinschaft Gr 4808 and by NSF and NIH grants to the MDIBL. Parts of this study have been published in the Mount Desert Island Biol. Bulletin 1984, 1985.     

K+ channels in the basolateral membrane of rat cortical collecting duct

Impalement studies in isolated perfused cortical collecting ducts (CCD) of rats have shown that the basolateral membrane possesses a K⁺ conductive pathway. In the present study this pathway was investigated at the single-channel level using the patch-clamp technique. Patch-clamp recordings were obtained from enzymatically isolated CCD segments and freshly isolated CCD cells with the conventional cell-free, cell-attached and the cell-attached nystatin method. Two K⁺ channels were found which were highly active on the cell with a conductance of 67±5 pS (n=18) and 148±4 pS (n=21) with 145 mmol/l K⁺ in the pipette. In excised patches the first channel had a conductance of 28±2 pS (n=15), whereas the second one had a conductance of 85±1 pS (n=53) at 0 mV clamp voltage with 145 mmol/l K⁺ on one side and 3.6 mmol/l K⁺ on the other side of the membrane. So far it has not been possible to characterize the smaller channel further. Excised, and with symmetrical K⁺ concentrations of 145 mmol/l, the intermediate channel had a linear conductance of 198±19 pS (n=5). After excision in the inside-out configuration the open probability (P _o) of this channel was low (0.18±0.05, n=13) whereas in the outside-out configuration this channel had a threefold higher P _o (0.57±0.04, n=12). Several inhibitors were tested in excised membranes. Ba²⁺ (1 mmol/l), tetraethylammonium (TEA⁺, 10 mmol/l) and verapamil (0.1 mmol/l) all blocked this channel reversibly. Furthermore P _o was reversibly reduced by 10 nmol/l charybdotoxin (outside-out). This K⁺ channel of the basolateral membrane was regulated by cellular pH. P _o was reduced to 26±3% at pH 6.5 (n=6) and increased to 216±18% at pH 8.5 (n=7) compared to pH 7.4. Half-maximal inhibition was reached at pH 7.0. The channel had its highest P _o at a Ca²⁺ activity of less than 10^–8 mol/l (n=13). Increasing the Ca²⁺ activity to 1 mmol/l on the cytosolic side of the membrane resulted in a reduction of P _o to 13±3% (n=11). Half-maximal inhibition was reached at a Ca²⁺ activity of 10^–5 mol/l. The high activity of both K⁺ channels of the basolateral membrane on the cell indicates that they may serve for K⁺ recirculation across the basolateral membrane.     

ATP-sensitive K+ channels regulated by intracellular Ca2+ and phosphorylation in normal (T84) and cystic fibrosis (CFPAC-1) epithelial cells

The elementary K⁺ conductance activated by the cAMP or the Ca²⁺ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl^–-free conditions, an inwardly rectifying whole-cell K⁺ current was evoked by either forskolin 10 (mol/l) or acetylcholine 1 (mol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8±2.5 pS (n=26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K⁺ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mol/l free Ca²⁺ at the inner side of the membrane. This channel was blocked by Ba²⁺ (5 mol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca²⁺ with and EC₅₀ of 0.25 mol/l and strongly reduced by intracellular MgATP with an IC₅₀ of 600 mol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K⁺ channel was also recorded, with similar characterictics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K⁺ channel regulated by intracellular Ca²⁺ and phosphorylation supports the main K⁺ current activated by secretory agonists in normal cystic fibrosis cell lines. This conductance possibly represents the major pathway for K⁺ recycling at the basolateral membrane during transepithelial fluid secretion.     

Anion and cation channels in the basolateral membrane of rabbit parietal cells

Ion channels in the basolateral membrane of rabbit parietal cells in isolated gastric glands were studied by the patch clamp technique. Whole-cell current-clamp recordings showed that the membrane potential (E _m ) changed systematically as a function of the chloride concentrations of the basolateral bathing solution ([Cl^–]₀), and of the pipette (intracellular) solution. The relationship betweenE _m and [Cl^–]₀ was not affected by additions of histamine, dibutyryl-cAMP, 4-acetoamido-4-isothiocyanostilbene-2,2-disulfonic acid and diphenylamine-2-carboxylate. The whole-cell Cl^– conductance was insensitive to voltage. In cell-attached and cell-free patch membranes, however, single Cl^– channel opening events could not be observed. The value ofE _m depended little on the basolateral K⁺ concentration, but inward-rectifier K⁺ currents were observed in the whole-cell configuration, activated by hyperpolarizing pulses and inhibited by extracellular Ba²⁺. In cell-attached and cell-free patches, openings of single inward-rectifier K⁺ channels and non-selective cation channels were infrequently recorded. Neither cAMP nor Ca²⁺ activated these cation channels. The single K⁺ channel conductance was about 230 pS under the symmetrical high K⁺ conditions and was inhibited by intracellular tetraethylammonium ions (TEA). The non-selective cation channel had a voltage-independent single conductance of 22 pS and was not inhibited by TEA.     

Small and maxi K+ channels in the basolateral membrane of isolated crypts from rat distal colon: single-channel and slow whole-cell recordings

The patch-clamp technique was used to characterize K⁺ channel activity in the basolateral membrane of isolated crypts from rat distal colon. In cell-attached patches with KCl in the pipette, channels with conductances ranging from 6 pS to 80 pS appeared. With NaCl in the pipette and KCl in the bath in excised inside-out membrane patches a small-conductance channel with a mean conductance of 12±6 pS (n=18) was observed. The channel has been identified as K⁺ channel by its selectivity for K⁺ over Na⁺ and by its sensitivity to conventional K⁺ channel blockers, Ba²⁺ and tetraethylammonium (TEA⁺). Changes of cytosolic pH did not attenuate channel activity. Activity of the 12-pS channel was increased by membrane depolarization and elevated cytosolic Ca²⁺ concentration. In addition, a maxi K⁺ channel with a mean conductance of 187±15 pS (n=4) in symmetrical KCl solutions was only occasionally recorded. The maxi K⁺ channel could be blocked by Ba²⁺ (5 mmol/l) on the cytosolic side. Using the slow-whole cell recording technique under control conditions, a cell membrane potential of –70±10mV (n=18) was measured. By application of various K⁺ channel blockers such as glibenclamide, charybdotoxin, apamin, risotilide, Ba²⁺ and TEA⁺ in the bath, only Ba²⁺ and TEA⁺ depolarized the cell membrane. The present data suggest that the small K⁺ channel (12 pS) is involved in the maintenance of the cell membrane resting potential.     

The luminal K+ channel of the thick ascending limb of Henle's loop

In vitro perfused rat thick ascending limbs of Henle's loop (TAL) were used (n=260) to analyse the conductance properties of the luminal membrane applying the patch-clamp technique. Medullary (mTAL) and cortical (cTAL) tubule segments were dissected and perfused in vitro. The free end of the tubule was held and immobilized at one edge by a holding pipette kept under continuous suction. A micropositioner was used to insert a patch pipette into the lumen, and a gigaohm seal with the luminal membrane was achieved in 455 instances out of considerably more trials. In approximately 20% of all gigaohm seals recordings of single ionic channels were obtained. We have identified only one single type of K⁺ channel in these cell-attached and cell-excised recordings. In the cell-attached configuration with KCl or NaCl in the pipette, the channel had a conductance of 60±6 pS (n=24) and 31±7 pS (n=4) respectively. In cell-free patches with KCl either in the patch pipette or in the bath and with a Ringer-type solution (NaCl) on the opposite side the conductance was 72±4 pS (n=37) at a clamp voltage of 0 mV. The permeability was 0.33±0.02 · 10^±12 cm³/s. The selectivity sequence für this channel was: K⁺=Rb⁺=NH ₄ ⁺ =Cs⁺>Li⁺Na⁺=0; the conductance sequence was K⁺Li⁺Rb⁺=Cs⁺= NH ₄ ⁺ =Na⁺=0. In excised patches Rb⁺, Cs⁺ and NH ₄ ⁺ when present in the bath at 145 mmol/l all inhibited K⁺ currents out of the pipette. The channel kinetics were described by one open (9.5±1.5 ms, n=18) and by two closed (1.4±0.1 and 14±2 ms) time constants. The open probability of this channel was increased by depolarization. The channel open probability was reduced voltage dependently by Ba²⁺ (half maximal inhibition at 0 mV: 0.07 mmol/l) from the cytosolic side. Verapamil, diltiazem, quinine and quinidine inhibited at approximately 1 mol/l ±0.1 mmol/l from either side. Similarly, the amino cations lidocaine, tetraethylammonium and choline inhibited at 10–100 mmol/l. The channel was downregulated in its open probability by cytosolic Ca²⁺ activities > 10^±7 mol/l and by adenosine triphosphate 10^±4 mol/l. The open probability was downregulated by decreasing cytosolic pH (2-fold by a decrease in pH by 0.2 units). The described channel differs in several properties from the K⁺ channels of other epithelia and of renal cells and TAL cells in culture. It appears to be responsible for K⁺ recycling in the TAL segment.Preliminary reports of the present study have been given at the following conferences: Tagung der Deutschen Physiologischen Gesellschaft, Würzburg, October 1988; Membranforum, Frankfurt, April 1989; 3rd Int. Conf. Diur., Mexico City, April 1989; 3rd Nephrology Forefront Symposium, Arrola, July, 1989; IUPS meeting, Helsinki, July 1989. This study has been supported by Deutsche Forschungsgemeinschaft Grant No. Gr 480/9     

Potassium conductance of smooth muscle cells from rabbit aorta in primary culture

Vascular smooth muscle cells were obtained from rabbit aorta and were studied in primary culture on days 1–7 after seeding with electrophysiological techniques. In impalement experiments a mean membrane potential difference (PD) of –50±0.3 mV (n=387) was obtained with Ringer-type solution in the bath. PD was depolarized by 6±0.3 mV (n=45) and 16±2 mV (n= 5) when the bath K⁺ concentration was increased from the control value of 3.6 mmol/l to 13.6 and 23.6 mmol/l, respectively. Ba²⁺ (0.1–1 mmol/l) depolarized PD. Tetraethylammonium (TEA, 10 mmol/l) depolarized PD only slightly but significantly. Verapamil (0.1 mmol/l) and charybdotoxin (10 nmol/l) had no effect on PD. The conductance properties of these cells were further examined with the patch-clamp technique. K⁺ channels were spontaneously present in cell-attached patches. When the pipette was filled with 145 mmol/l KCl, a mean conductance (g _K) of 209.6±4.6 mV (n=17) was read from the current/voltage curves at a clamp voltage (V _c) of 0 mV. After excision K⁺ channels were found in 129 patches with inside-out and in 50 with outside-out configuration. With KCl on one and NaCl on the other side the mean g _K at a V _c of 0 mV was 134.6±3.9 pS (n=179). The mean permeability was 0.89±0.03×10^–12 cm³/s. With symmetrical KCl solution the mean g _K was 227±6 pS (n=17). The conductance sequence was g _K g _Rb= g _Cs=g _Na=0. TEA blocked dose-dependently only from the outside.(1–10 mmol/l). Lidocaine (5 mmol/l) quinidine (0.01–1 mmol/l) and quinine (0.01–1 mmol/l) blocked from both sides. Charybdotoxin (0.5–5 nmol/l) blocked only from the extracellular side. Ba²⁺ blocked from the cytosolic side and the inhibition was increased by depolarization and reduced by hyperpolarization. At a V _c of 0 mV a half-maximal inhibition (IC₅₀) of 2 mol/l was obtained. Verapamil and diltiazem blocked from both sides, verapamil with an IC₅₀ of 2 mol/l and diltiazem with an IC₅₀ of 10 mol/l. The open probability of this channel was increased by Ca²⁺ on the cytosolic side at activities > 0.1 mol/l. Half-maximal activation occurred at Ca²⁺ activities exceeding 1 mol/l. The present data indicate that the vascular smooth muscle cells of rabbit aorta in primary culture possess a K⁺ conductance. In excised patches only a maxi K⁺ channel was detected. This channel has properties different from the macroscopic K⁺ conductance. Hence, it is likely that the K⁺ conductance of the intact cell is dominated by yet another and thus far not detected K⁺ channel.Supported by DFG Gr 480/10     

K+ and Cl− currents in freshly isolated rat osteoclasts

Membrane electrical properties of freshly isolated rat osteoclasts were studied using patch-clamp recording methods. Characterization of the passive membrane properties indicated that the osteoclast cell membrane behaved as an isopotential surface. The specific membrane capacitance was 1.2±0.3 F/cm² (mean ±SD), with no difference between cells plated on glass and those adhering to a permeable collagen substrate. The current/voltage (I/V) relationship of all cells showed inward rectification and I/V curves shifted 51 mV positive per tenfold increase of [K⁺]_out, indicating an inwardly rectifying K⁺ conductance. The voltage dependence of the K⁺ chord conductance (g _K) also shifted positive along the voltage axis, and the maximum conductance increased, with elevation of [K⁺]_out. g _K for cells bathed in 4.7 mM [K⁺]_out increased e-fold per 12mV hyperpolarization, and half-maximal activation was at –89 mV. Approximately 18% (50 pS/pF) of the maximum g _K was active at –70 mV. Inward single-channel currents were recorded in cell-attached patches at hyperpolarizing potentials. With symmetrical K⁺, channel conductance was 25±3 pS and reversal was close to the K⁺ equilibrium potential, consistent with this K⁺ channel underlying the whole-cell K⁺ currents. With both conventional whole-cell and perforated-patch recording, no voltage-activated Ca²⁺ current was detected. In approximately 30% of osteoclasts studied, an outwardly rectifying current was observed, which was reversibly blocked by 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene2,2-disulphonic acid (SITS). This DIDS- and SITS-sensitive current reversed direction at the chloride equilibrium potential. We conclude that an inwardly rectifying K⁺ current is present in all rat osteoclasts and that some osteoclasts also exhibit an outwardly rectifying Cl^– current. Both these membrane conductances may play an important physiological role by dissipating the potential that arises from the electrogenic transport of H⁺ across the ruffled membrane of the osteoclast.     

cAMP-dependent activation of small-conductance Cl− channels in HT29 colon carcinoma cells

The present study was performed to examine the conductance properties in the colon carcinoma cell line HT₂₉ and the activation of Cl^– channels by cAMP. A modified cell-attached nystatin patch-clamp technique was used, allowing for the simultaneous recording of the cell membrane potential (PD) and the conductance properties of the cell-attached membrane. In resting cells, PD was –56±0.4 mV (n=294). Changing the respective ion concentrations in the bath indicate that these cells possess a dominating K⁺ conductance and a smaller Cl^– conductance. A significant non-selective cation conductance, which could not be inhibited by amiloride, was only observed in cells examined early after plating. The K⁺ conductance was reversibly inhibited by 1–5 mmol/l Ba²⁺. Stimulation of the cells by the secretagogues isoproterenol and vasointestinal polypeptide (VIP) depolarized PD and induced a Cl^– conductance. Similar results were obtained with compounds increasing cytosolic cAMP: forskolin, 3-isobutyl-1-methylxanthine, cholera toxin and 8-bromoadenosine cyclic 3,5-monophosphate (8-Br-cAMP). VIP (1 nmol/l, n=10) and isoproterenol (1 umol/l, n=12) depolarized the cells dose-dependently and reversibly by 12±2 mV and 13±2 mV. The maximal depolarization was reached after some 20 s. The depolarization was due to increases in the fractional Cl^– conductance. Simultaneously the conductance of the cellattached membrane increased from 155±31 pS to 253±40 pS (VIP, n=4) and from 170±43 pS to 268±56 pS (isoproterenol, n=11), reflecting the gating of Cl^– channels in the cell-attached membrane. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) was without significant effects in resting and in forskolin-stimulated HT₂₉ cells. The agonist-induced conductance increase of the cell-attached nystatin patches was not paralleled by the appearance of detectable single-channel events in these membranes. These data suggest activation of small, non-resolvable Cl^– channels by cAMP.Supported by DFG Gr 480/10 and BMFT 01 GA 88/6     

Ca2+-activated K+ channels in airway smooth muscle are inhibited by cytoplasmic adenosine triphosphate

Large-conductance Ca²⁺-activated K⁺ channels were studied in membranes of cultured rabbit airway smooth muscle cells, using the patch-clamp technique. In cell-attached recordings, channel openings were rare and occurred only at very positive potentials. Bradykinin (10 M), an agonist which releases Ca²⁺ from the sarcoplasmic reticulum, transiently increased channel activity. The metabolic blocker 2,4-dinitrophenol (20 M), which lowers cellular adenosine triphosphate (ATP) levels, induced a sustained increase of channel activity in cell-attached patches. In excised patches, these channels had a slope conductance of 155 pS at 0 mV, were activated by depolarization and by increasing the Ca²⁺ concentration at the cytoplasmic side above 10^–7 mol/l. ATP, applied to the cytoplasmic side of the patches, dose-dependently decreased the channel's open-state probability. An inhibition constant (K _i) of 0.2 mmol/l was found for the ATP-induced inhibition. ATP reduced the Ca²⁺ sensitivity of the channel, shifting the Ca²⁺ activation curve to the right and additionally reducing its steepness. Our results demonstrate that cytoplasmic ATP inhibits a large-conductance Ca²⁺-activated K⁺ channel in airway smooth muscle. This ATP modulation of Ca²⁺-activated K⁺ channels might serve as an important mechanism linking energy status and the contractile state of the cells.     

G-protein-mediated regulation of a Ca2+-dependent K+ channel in cultured vascular endothelial cells

The purpose of the present study was to determine the mechanism by which bradykinin activates the small conductance, inwardly rectifying, Ca²⁺-activated K⁺ channel (K_Ca) found in cultured bovine aortic endothelial cells. Channel activity was studied using the patch-clamp technique in whole-cell, cell-attached, inside-out and outside-out configurations. Channel conductance at potentials positive to 0 mV was 10±2 pS and at potentials negative to 0 mV 30±3 pS (n=7) when examined in symmetrical K⁺ (150 mmol/l) solutions. The channel open probability (P _o) was only weakly voltage dependent changing approximately 0.2 units over 160 mV. In contrast, raising the intracellular Ca²⁺ concentration from 100 nmol/l to 10 mol/l at –60 mV produced a graded increase in channel P _o from 0.15 to 0.96; the concentration required for half-maximum response (apparent K_0.5) was 719 nmol/l. At a constant Ca²⁺ concentration, application of guanosine triphosphate (GTP) to the cytoplasmic surface of the patch increased channel P _o. This effect was dependent upon the simultaneous presence of both GTP and Mg²⁺, and was reversed by the subsequent application of the guanosine diphosphate (GDP) analogue, guanosine-5-O-(2-thiodiphosphate) (GDPS). The hydrolysis-resistant GTP analogue, guanosine-5-O-(3-thiotriphosphate) (GTPS), induced a long-lasting increase in channel P _o. In the presence of Mg²⁺-GTP, the apparent K_0.5 for Ca²⁺ decreased from a control value of 722 nmol/l to 231 nmol/l. Addition of bradykinin to outside-out patches previously exposed to intracellular Mg²⁺-GTP further enhanced K_Ca activity, shifting the apparent K_0.5 for Ca²⁺ from 228 nmol/l to 107 nmol/l. This activation by bradykinin was not observed in patches following prior exposure to GDPS. These results suggest that bradykinin can activate the K_Ca channel of vascular endothelial cells via a G-protein-mediated change in the sensitivity of the channel for Ca²⁺. We postulate that vasoactive agonists may use this mechanism to maintain an elevated K⁺ permeability as the intracellular Ca²⁺ concentration returns towards normal resting levels.     

Tetraethylammonium ion sensitivity of a 35-pS Ca2+-activated K+ channel in GH3 cells that is activated by thyrotropin-releasing hormone

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. We have previously demonstrated the coexistence of large-conductance and small-conductance (280 pS and 11 pS in symmetrical 150 mM K⁺, respectively) Ca²⁺-activated K⁺ channels in this cell line (Lang and Ritchie 1987). Here we report the existence of a third type of Ca²⁺-activated K⁺ channel that has a conductance of about 35 pS under similar conditions. In excised inside-out patches, this channel can be activated by elevations of the internal free Ca²⁺ concentration, and the open probability increases as the membrane potential is made more positive. In excised patches, the sensitivity of this 35-pS channel to internal Ca²⁺ is low; at positive membrane potentials, this channel requires Ca²⁺ concentrations greater than 10 M for activation. However, 35-pS channels have a much higher sensitivity to Ca²⁺ in the first minute after excision (activated by 1 M Ca²⁺ at –50 mV). Therefore, it is possible that the Ca²⁺ sensitivity of this channel is stabilized by intracellular factors. In cell-attached patches, this intermediate conductance channel can be activated (at negative membrane potentials) by thyrotropin-releasing hormone-induced elevations of the intracellular Ca²⁺ concentration and by Ca²⁺ influx during action potentials. The intermediate conductance channel is inhibited by high concentrations of external tetraethylammonium ions (K _d=17 mM) and is relatively resistant to inhibition by apamin.     

Regulation of ion channels in rat hepatocytes

 Patch-clamp studies have been performed to elucidate single ion channels in rat hepatocytes. In rat hepatocytes two types of ion channel have been identified: an inwardly rectifying K⁺ channel with a mean inward conductance of 55 ± 6.5 pS (n = 20) and a mean outward conductance of 25 ± 3.2 pS (n = 20) in the inside-out configuration with 145 mmol/l KCl on either side of the patch as well as an outwardly rectifying Cl^– channel with a mean outward conductance of 30 ± 4.5 pS (n = 8) and a mean inward conductance of 10 ± 2.3 pS (n = 6) in the inside-out configuration with symmetrical 145 mmol/l KCl. The open probability of these channels is virtually insensitive to Ca²⁺ activity on the intracellular side. Accordingly, the Ca²⁺ ionophore ionomycin had no effect on cell membrane potential. Dibutyryl-cAMP (db-cAMP) hyperpolarizes the cell membrane and increases the activity of the 55-pS inwardly rectifying K⁺ channel by reducing the duration of closure between bursts. Forskolin similarly hyperpolarizes the cell membrane. The inwardly rectifying K⁺ channel is inhibited by progesterone, while the outwardly rectifying Cl^– channel is insensitive to progesterone. Received: 21 May 1997 / Received after revision: 7 August 1997 / Accepted: 19 August 1997     

Amiloride-blockable Ca2+-activated Na+-permeant channels in the fetal distal lung epithelium

The Na⁺ transport function of alveolar epithelium represents an important mechanism for clearance of fluid in air space at birth. I observed the activity of two types of amiloride-blockable Na⁺-permeant cation channels in the apical membrane of fetal distal lung epithelium cultured on permeable filters for 2 days after harvesting of the cells from Wistar rats of 20 days' gestation (term = 22 days). One type was a nonselective cation (NSC) channel and had a linear current/voltage (I/V) relationship with a single-channel conductance of 26.9 ± 0.8 pS (n = 5). The other type was highly Na⁺ selective (i.e. Na⁺ channel) and had an inwardly rectifyingI/V relationship with a single-channel conductance of 11.8 ± 0.2 pS (n = 5) around resting membrane potential. The NSC channel was more frequently observed (1.37 ± 0.15 per patch membrane;n = 73) than the Na⁺ channel (0.15 ± 0.40 per patch membrane;n = 73). However, the open probability of the NSC channel was smaller than that of the Na⁺ channel. Both types of the channels were activated by cytosolic Ca²⁺, however the sensitivity to cytosolic Ca²⁺ was much higher in the Na⁺ channel than in the NSC channel. Furthermore, both types of the channels were blocked by amiloride or benzamil. The half-maximal inhibitory concentration (IC₅₀) of amiloride or benzamil of the Na⁺ channel was 1–2 M, while that of NSC channel was less than 1 M. Both channels were activated by insulin.     

A ubiquitous non-selective cation channel in the mouse renal tubule with variable sensitivity to calcium

Basolateral membranes of microdissected collagenase-treated fragments of renal tubules from the mouse were examined using the cell-attached and the cell-free variants of the patch-clamp technique. With a K⁺-rich solution in the pipette, a highly active, inwardly rectifying K⁺ channel was observed on intact cells of the cortical collecting tubule (CCT). The mean inward and outward conductances were 38.5±3.1 pS and 17.3±1.8 pS, respectively (n=4). In contrast, cell-attached patches were usually inactive when a Na⁺-rich solution filled the patch pipette. However, another type of channel with a conductance of 20–30 pS exhibited a sparse activity in 4/20 CCT. In excised, inside-out patches, the most frequent channel in CCT had an ohmic unit conductance of 27.1±1.2 pS (n=17), excluded anions (P _Cl /P _Na=0.09), discriminated little between NH₄ ⁺, K⁺ and Na⁺ (P _NH4 /P _Na=1.5;P _K /P _Na=0.9), and was much less permeable to Ca²⁺ and Ba²⁺ than to Na⁺ (P _Ca /P _Na=0.09;P _Ba /P _Na≈0). The cation channel was moderately voltagedependent, showing a decreased open probability (P _o) at negative voltages. It was activated by internal calcium (threshold: 1 μmol/l–0.1 mmol/l calcium), and inhibited by the adenine nucleotides ATP, ADP and AMP with half-maximal inhibition ofP _o at 1.2 umol/l AMP. As in other cell models, 3′,5′-dichlorodiphenylamine-2-carboxylic acid blocked channel activity when added to the internal surface of the membrane patch. Extending our study to other parts of the renal tubule, we found that the basolateral membranes of the proximal (pars recta), distal convoluted, connecting and outer medullary collecting tubules, the thin descending limb and the medullary thick ascending limb all contained a similar Ca- and ATP-sensitive cation channel. The calcium sensitivity varied from one part to another.     

Cation specificity and pharmacological properties of the Ca2+-dependent K+ channel of rat cortical collecting ducts

The luminal membrane of principal cells of rat cortical collecting duct (CCD) is dominated by a K⁺ conductance. Two different K⁺ channels are described for this membrane. K⁺ secretion probably occurs via a small-conductance Ca²⁺-independent channel. The function of the second, large-conductance Ca²⁺-dependent channel is unclear. This study examines properties of this channel to allow a comparison of this K⁺ channel with the macroscopic K⁺ conductance of the CCD and with similar K⁺ channels from other preparations. The channel is poorly active on the cell. It has a conductance of 263±11 pS (n=36, symmetrical K⁺ concentrations) and of 139±3 pS (n=91) with 145 mmol/l K⁺ on one side and 3.6 mmol/l K⁺ on the other side of the membrane. Its open probability is high after excision (0.71±0.03, n=85). The channel flickers rapidly between open and closed states. Its permeability in the cell-free configuration was 7.0±0.2×10^–13 cm³/s (n=85). It is inhibited by several typical blockers of K⁺ channels such as Ba²⁺, tetraethylammonium, quinine, and quinidine and high concentrations of Mg²⁺. The Ca²⁺ antagonists verapamil and diltiazem also inhibit this K⁺ channel. As is typical for the maxi K⁺ channel, it is inhibited by charybdotoxin but not by apamin. The selectivity of this large-conductance K⁺ channel demonstrates significant differences between the permeability sequence (P _K > P _Rb > P _NH4 > P _Cs=P _Li=P _Na=P _choline=0) and the conductance sequence (g _K > g _NH4 > g _Rb > g _Li=g _choline > g _Cs=g _Na=0). The only other cations that are significantly conducted by this channel besides K⁺ (g _K at V _c = is 279±8 pS, n=88) are NH ₄ ⁺ (g _NH4=127±22 pS, n=10) and Rb⁺ (g _Rb=36±5 pS, n=6). The K⁺ currents through this channel are reduced by high concentrations of choline⁺, Cs⁺, Rb⁺, and NH ₄ ⁺ . These properties and the dependence of this channel on Ca²⁺ and voltage classify it as a maxi K⁺ channel. A possible physiological function of this channel is discussed in the accompanying paper.Supported by DFG Gr 480/10, by Schl 277/2-3 and by GIF 88/II     

Effect of NH4 +/NH3 on cytosolic pH and the K+ channels of freshly isolated cells from the thick ascending limb of Henle's loop

The conductance properties of the luminal membrane of cells from the thick ascending limb of Henle's loop of rat kidney (TAL) are dominated by K⁺. In excised membrane patches the luminal K⁺ channel is regulated by pH changes on the cytosolic side. To examine this pH regulation in intact cells of freshly isolated TAL segments we measured the membrane voltage (V _m) in slow-whole-cell (SWC) recordings and the open probability (P _o) of K⁺ channels in the cell-attached nystatin (CAN) configuration, where channel activity and part of V _m can be recorded. The pipette solution contained K⁺ 125 mmol/l and Cl^– 32 mmol/l. Intracellular pH was determined by 2,7 bis(2-carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) fluorescence. pH changes were induced by the addition of 10 mmol/l NH₄ ⁺/NH₃ to the bath. In the presence of NH₄ ⁺/NH₃ intracellular pH acidified by 0.53±0.11 units (n=7). Inhibition of the Na⁺2Cl^– K⁺ cotransporter by furosemide (0.1 mmol/l) reversed this effect and led to a transient alkalinisation by 0.62±0.14 units (n=7). In SWC experiments V _m of TAL cells was -72±1 mV (n=70). NH₄ ⁺/NH₃ depolarised V _m by 22±2 mV (n=25). In 11 SWC experiments furosemide (0.1 mmol/l) attenuated the depolarising effect of NH₄ ⁺ from 24±3 mV to 7±3 mV. Under control conditions the single-channel conductance of TAL K⁺ channels in CAN experiments was 66±5 pS and the reversal voltage for K⁺ currents was 70±2 mV (n=35). The P _o of K⁺ channels in CAN patches was reduced by NH₄ ⁺/NH₃ from 0.45±0.15 to 0.09±0.07 (n=7). NH₄ ⁺/NH₃ exposure depolarised the zero current voltage of the permeabilised patches by-9.7±3.6 mV (n=5). The results show that TAL K⁺ channels are regulated by cytosolic pH in the intact cell. The cytosolic pH is acidified by NH₄ ⁺/NH₃ exposure at concentrations which are physiologically relevant because Na⁺2Cl^–K⁺(NH₄ ⁺) cotransporter-mediated import of NH₄ ⁺ exceeds the rate of NH₃ diffusion into the TAL. K⁺ channels are inhibited by this acidification and the cells depolarise. In the presence of furosemide TAL cells alkalinise proving that NH₄ ⁺ uptake occurs by the Na⁺2Cl^–K⁺ cotransporter. The findings that, in the presence of NH₄ ⁺/NH₃ and furosemide, V _m is not completely repolarised and that K⁺ channels are not activated suggest that the respective K⁺ channels may in addition to their pH regulation be inhibited directly by NH₄ ⁺/NH₃.     

Small-conductance Cl− channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP

The present study demonstrates the activation of Cl^– channels in HT₂₉ cells by agonist (ATP, neurotensin, carbachol) increasing cytosolic Ca²⁺, by hypotonic cell swelling and by cGMP. Cell-attached nystatin patch-clamp (CAN) as well as slow and fast wholecell recordings were used. The cell membrane potential was depolarized in a dose-dependent manner with halfmaximal effects at 0.4 umol/l for ATP, 60 pmol/l for neurotensin and 0.8 mol/l for carbachol. The depolarization, which was caused by Cl^– conductances increases, occurred within 1 s and was accompanied by a simultaneous and reversible increase of the input conductance of the cell-attached membrane from 295±32 pS to 1180±271 pS (ATP; 10 mol/l, n=21) and 192±37 pS to 443±128 pS (neurotensin; 1 nmol/l, n=8). The effects of the agonists could be mimicked by ionomycin (0.2 umol/l), suggesting that an increase in intracellular Ca²⁺ was responsible for the activation of Cl^– channels. The depolarization was followed by a secondary hyperpolarization. Hypotonic cell swelling also depolarized the cells and induced an increase in the membrane conductance. With 120 mmol/l NaCl the depolarization was 10±0.8 mV and the cell-attached conductance increased from 228±29 pS to 410±65 (n=26) pS. NaCl at 90 mmol/l and 72.5 mmol/l had even stronger effects. Comparable conductance increases were also obtained when the different agonists or hypotonic cell swelling were examined in whole cell experiments.5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) did not prevent the effects of Ca²⁺-increasing hormones and of hypotonic solutions. An increase in Cl^– conductance was also induced by 8-Br-cGMP (1 mmol/l) but not by heat-stable Escherichia coli toxin. In contrast to their conductance-increasing effects in CAN patches, the different agonists and cell swelling did not activate resolvable single channels in these cell-attached membranes. This indicates that the Cl^– channels involved have a single-channel conductance too small ( 4 pS, 150 Hz) to be resolved by our techniques.     

Anoxia opens ATP regulated K channels in isolated heart cells of the guinea pig

We studied single channel ionic currents in cell-attached patches of guinea pig heart cells under conditions of anoxia (pO₂<0.1 torr) to identify the type of channels which contribute to the anoxia-induced time-independent K current in whole cells. In most experiments, K currents were recorded at negative potentials as inward currents with 150 mmol/l KCl in the pipette. After periods of 5–60 minutes of anoxia, opening events of one to four voltage-independent 83 pS channels developed whose open probability reached a steady state value between 0.6 and 0.95 (T=35°C). The reversal potential of the unitary currents, determined at 150 mmol/l and 10.8 mmol/l K⁺ in the pipette, showed that the channels were highly selective for K⁺ ions. Open time histograms were fitted by two or three exponentials of which the fast time constant (_o1=0.46±0.20 ms, mean±SD) was bandwidth-limited by our filter and the slow components substantially varied (_o2=1.5–19 ms: _o3=23–200 ms). Voltage ramp experiments showed that the channels were slightly rectifying in an inward direction. The unitary conductance of anoxia-induced outward currents at reduced K⁺ in the pipette was smaller (11 pS at 5.4 mmol K⁺, 25 pS at 10.8 mmol/l K⁺) than in excised patches. It is concluded that in isolated cardiocytes substrate-free anoxia causes opening of ATP regulated K channels whose conductance is reduced at physiological levels of [K⁺]_o by a fast block, most likely by intracellular Mg⁺⁺ and Na⁺.