Na+ selective channels in the apical membrane of rabbit late proximal tubules (pars recta)

Using the patch-clamp technique, Na⁺ selective channels were observed in the luminal membrane of rabbit straight proximal tubule segments. In the cell-attached configuration (NaCl-Ringers in pipette and bath) influx of Na⁺ ions from the pipette into the cell through fluctuating channels was observed was observed. The current-voltage curve of these Na⁺ channels yielded a zero-current potential of 84.3±30.9 mV (n=10), reflecting the electrochemical driving force for Na⁺ influx under resting conditions. The single channel conductance was 12.0±2.1 pS (n=13). In inside-out oriented cell-excised patches the single channel conductance was not significantly different with NaCl-Ringers on both sides. At clamp potentials ranging from +50 mV to –50 mV the single channel current was ohmic and channel kinetics were independent of the voltage. With KCl-Ringers on the bath side (corresponding to cell interior), the zero current potential was 62±19 mV (n=4), indicating a high selectivity of Na⁺ over K⁺ ions. Addition of 10^–5 mol/l amiloride to the bathing solution decreased the mean channel open time slightly. This effect was more pronounced with 10^–4 mol/l amiloride, whereas the single channel conductance was unaffected by the diuretic. 10^–3 mol/l amiloride caused a complete block of the channel. It is concluded that amiloride sensitive Na⁺ channels, with similar properties to those observed in tight epithelia, contribute to Na⁺ reabsorbtion in the straight portion of proximal tubules.     

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     

Effect of bradykinin and histamine on the membrane voltage,ion conductances and ion channels of human glomerular epithelial cells (hGEC) in culture

The effects of bradykinin (BK) and histamine (Hist) on the membrane voltage (V _m), ion conductances and ion channels of cultured human glomerular epithelial cells (hGEC) were examined with the nystatin patch clamp technique. Cells were studied between passage 3 and 20 in a bath rinsed with Ringer-like solution at 37°C. The mean value of V _m was –41±0.5 mV (n=189). BK (10^–6 mol/l, n=29) and Hist (10^–5 mol/l, n= 55) induced a rapid transient hyperpolarization by 15±1 mV and 18±1 mV, respectively. The hyperpolarization was followed by a long lasting depolarization by 6±1 mV (BK 10^–6 mol/l) and 7±1 mV (Hist 10^–5 mol/l). The ED₅₀ was about 5×10^–8 mol/l for BK and 5×10^–7 mol/l for Hist. In the presence of both agonists, increases of outward and inward currents were observed. A change in the extracellular K⁺ concentration from 3.6 to 30 mmol/l depolarized V _m by 8±1 mV and completely inhibited the hyperpolarizing effect of both agents (n=11). Reduction of extracellular Cl^– concentration from 145 to 30 mmol/l led to a depolarization by 2 ±1 mV (n=25). In 30 mmol/l Cl^– the depolarizations induced by BK (10^–7 mol/l) and Hist (10^–6 mol/l) were augmented to 9±2 mV (n=14) and to 10±2 mV (n=11), respectively. Ba²⁺ (5 mmol/l) depolarized V _m by 19±5 mV (n=6) and completely inhibited the hyperpolarization induced by BK (10^–6 mol/l, n=3) and reduced that of Hist (10^–5 mol/l) markedly (n=3). Preincubation with the K⁺ channel blocker charybdotoxin (1–10 nmol/l) for 3 min had no significant effect on V _m, but reduced markedly the BK(10^–6 mol/l, n=11) and Hist-(10^–5 mol/l, n=6) induced hyperpolarizations. In 10 out of 31 experiments in the cell attached nystatin patch configuration big K⁺ channels with a conductance of 247±17 pS were found. The open probability of these K⁺ channels was increased 3- to 5-fold during the hyperpolarization induced by BK (10^–7 mol/l) or Hist (10^–5 mol/l, both n= 4). In excised inside/out patches this K⁺ channel had a mean conductance of 136±8.5 pS (n=10, clamp voltage 0 mV). The channel was outwardly rectifying and its open probability was increased when Ca²⁺ on the cytosolic side was greater than 0.1 mol/l. The data indicate that BK and Hist activate a and a in hGEC. The hyperpolarization is induced by the activation of a Ca²⁺-dependent maxi K⁺ channel.     

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.     

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.     

Characteristics of apical chloride channels in human colon cells (HT29)

Recent studies have demonstrated that active chloride secretion in mammalian colon and other epithelia, is dependent on the induction of an increase of apical chloride conductance. Since the physical characteristics of apical chloride channels in man have not been elucidated, patch clamp analysis of human colon cells (HT₂₉), in culture, was performed, after stimulation with db-cAMP 10^–4 mol/l. In excised inside out patches of apical membranes two types of channels were found. The smaller and less frequent channel had a mean conductance of 15±1 pS (n=9). This type of channel showed identical I/V curves in NaCl and KCl solutions. It was inhibited by a chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The more frequently observed larger conductance channel was selective for anions and was impermeable to Na⁺ and K⁺. Regarding anion selectivity, the channel was similarly permeable to Cl^–, Br^–, I^–, and NO ₃ ^– , but was impermeable to gluconate. The channel was completely inhibited by the potent Cl^– channel blocker NPPB (10^–6 mol/l). This channel exhibited rectification: The conductance was 50±4 pS at positive clamp potentials (sign referred to bath with respect to pipette interior) and 32±3 (n=33) pS at negative voltages. Moreover, the open state probability was doubled when the clamp potential was increased from –20 to +20 mV. These results demonstrate the existence of chloride channels in the apical membrane of db-cAMP treated colonic carcinoma cells.     

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     

Flufenamate and Gd3+ inhibit stimulated Ca2+ influx in the epithelial cell line CFPAC-1

The relevant influx pathway for stimulated Ca²⁺ entry into epithelial cells is largely unknown. Using flufenamate (Flu) and Gd³⁺, both known pharmacological blockers of non-selective cation currents in other epithelial preparations, we tested whether the stimulated Ca²⁺ entry in CFPAC-1 cells was inhibited by these agents. Transmembraneous Ca²⁺ influx into CFPAC-1 cells was stimulated by either ATP (10^–4 and 10^–5 mol/l), carbachol (CCH, 10^–4 mol/l) or thapsigargin (TG, 10^–8 mol/l). Three different experimental approaches were used. (1) Because the plateau phase of an agonist-induced [Ca²⁺]_i transient reflects Ca²⁺ influx into these cells, we investigated the influence of Flu and Gd³⁺ on the level of the stimulated [Ca²⁺]_i plateau. (2) The fura-2 Mn²⁺-quenching technique was used to visualise divalent cation entry and monitor its inhibition. (3) During the refilling period after agonist-induced discharge of the intracellular pools the putative influx inhibitors Flu and Gd³⁺ were given and subsequently the filling state of the agonist-sensitive intracellular stores tested. The results from the first experimental approach showed that both Flu and Gd³⁺ were potent inhibitors of the stimulated Ca²⁺ entry in CFPAC-1 cells. Flu reversibly decreased the ATP-induced [Ca²⁺]_i plateau in a concentration dependent manner, with an IC₅₀ value of 33 mol/l (n = 6). Similar results were obtained for the CCH-(n = 5) and the TG-induced (n = 5) [Ca²⁺]_i plateau. Gd³⁺ concentration dependently inhibited the stimulated Ca²⁺ plateau. A complete block of the ATP-induced [Ca²⁺]_i plateau was seen at 0.5 mol/l (ATP 10^–5 mol/l, n = 8). The second approach showed that Flu (10^–4 mol/l) completely inhibited the ATP- (10^–5 mol/l, n = 3), CCH-(10^–4 mol/l, n = 4) and TG-(10^–8 mol/l, n = 3)-induced fura-2 Mn²⁺ quench. Gd³⁺ also inhibited the fura-2 Mn²⁺-quenching rate (n = 9). The third approach showed that Flu (n = 6) and Gd³⁺ (n = 8) inhibited the refilling of the ATP-sensitive intracellular Ca²⁺ store. These results show that inhibitors of non-selective cation currents in other epithelial preparations are potent inhibitors of stimulated Ca²⁺ influx in CFPAC-1 cells. Whether this inhibitory effect concerns a non-selective cation channel remains to be established.     

A calcium-activated nonselective cationic channel in the basolateral membrane of outer hair cells of the guinea-pig cochlea

The patch-clamp technique was used to investigate ion channels in the basolateral perilymph-facing membrane of freshly isolated outer hair cells (OHCs) from the guinea-pig cochlea. These sensory cells probably determine, via their motile activity, the fine tuning of sound frequencies and the high sensitivity of the inner ear. A Ca²⁺-activated nonselective cationic channel was found in excised inside-out membrane patches. The current/voltage relationship was linear with a unit conductance of 26.3±0.3 pS (n=15) under symmetrical inger conditions. The channel excluded anions (P _Na/P _Cl=18 whereP _Na/P _Cl denotes the relative permeability of Na to Cl); it was equally permeant to the Na⁺ and K⁺ ions and exhibited a low permeability toN-methyl-D-glucamine and Ba²⁺ or Ca²⁺. Channel opening required a free Ca²⁺ concentration of about 10^–6 mol/l on the internal side of the membrane and the open probability (P _o) was maximal at 10^–3 mol/l (P _o=0.72±0.06,n=12). Adenosine 5mono-, tri- and di-phosphate reducedP _o to 29±14 (n=5), 42±10 (n=8) and 51±12 (n=5) % of controlP _o, respectively, when they were added at a concentration of 10^–3 mol/l to the internal side. The channel was partially blocked by flufenamic acid (10^–4 mol/l) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10^–5 mol/l). This type of channel, together with Ca²⁺-activated K⁺ channels, might participate in the control of membrane potential and modulate the motility of OHCs.     

Ba2+ and amiloride uncover or induce a pH-sensitive and a Na+ or non-selective cation conductance in transitional cells of the inner ear

The membrane potential V _m the cytosolic pH (pH_i), the transference numbers (t) for K⁺, Cl^– and Na⁺/ non-selective cation (NSC) and the pH-sensitivity of V _m were investigated in transitional cells from the vestibular labyrinth of the gerbil. V _m, pH_i, , and the pH_i sensitivity of V _m were under control conditions were –92±1 mV (n=89 cells), pH_i 7.13±0.07 (n=11 epithelia), 0.87±0.02 (n=22), 0.02±0.01 (n=19), 0.01±0.01 (n=24) and –5 mV/pH unit (n=13 cells/n=11 epithelia), respectively. In the presence of 100 mol/l Ba²⁺ the corresponding values were: –70±1 mV (n=32), pH_i 7.16±0.08 (n=6), 0.31±0.05 (n=4), 0.06±0.01 (n=6), 0.20±0.03 (n=10) and -16 mV/pH-unit (n=15/n=6). In the presence of 500 mol/l amiloride the corresponding values were: –72±2mV (n=34), pH_i 7.00±0.07 (n=5), 0.50±0.04 (n=6), 0.04±0.01 (n=11), 0.28±0.04 (n=9) and –26 mV/pH-unit (n=20/n=5). In the presence of 20 mmol/l propionate plus amiloride the corresponding values were: –61±2 mV (n=27), pH_i 6.72±0.06 (n=5), 0.30±0.02 (n=6), 0.06±0.01 (n=5) and 0.40±0.02 (n=8), respectively. V _m was depolarized and and pH_i decreased due to (a) addition of 1 mmol/l amiloride in 150 mmol/l Na⁺ by 38±1 mV (n=8), from 0.82±0.02 to 0.17±0.02 (n=8) and by 0.13±0.01 pH unit (n=6), respectively; (b) reduction of [Na⁺] from 150 to 1.5 mmol/l by 3.3±0.5 mV (n=30), from 0.83±0.02 to 0.75±0.04 (n=9) and by 0.33±0.07 pH unit (n=4), respectively and (c) addition of 1 mmol/l amiloride in 1.5 mmol/l Na⁺ by 20±1 mV (n=11) and from 0.83±0.03 to 0.53±0.02 (n=5), respectively. These data suggest that the K⁺ conductance is directly inhibited by amiloride and Ba²⁺ and that Ba²⁺ and amiloride uncover or induce a pH-sensitive and a Na⁺/NSC conductance which may or may not be the same entity.Some of the data have been presented at various meetings and appear in abstract form in [31, 35, 37]     

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells

The present study examines the properties of Cl^–channels in cultured respiratory cells of cystic fibrosis (CF) patients and normal (N) individuals. In excised membrane patches the conductances for CF and N Cl^– channels were larger at positive as compared to negative clamp voltages (V _c): 74±2.6 (V _c > 0) and 47±2.0 pS (V _c < 0) for CF (n= 57) and 69±3.6 (V _c > 0) and 45±2.3 pS (V _c < 0) for N (n=35). The open probability (P _o) of the channel increased markedly with depolarization. Both the voltage dependence of the conductance and of P _o contribute to the outward rectification of the channel. The time histogram analysis reveals two open and two closed time constants. The selectivity of the channel was Cl^–=Br^– =I^– > NO ₃ ^– gluconate. The channel was inhibited reversibly by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) at 10^–7 mol/l to 10^–5 mol/l. While Cl^– channels were present in cell attached patches of N cells, they were absent in those of CF cells. The mean conductance for cell attached (N) Cl^– channels was 76±3.2 pS for positive clamp voltages (V _c) and 46±3.9 pS for negative V _c (n=8). When the membrane patches were excised from CF cells Cl^– currents appeared spontaneously (n=19). The immediate appearance (within 1 s) of Cl^– channels after excision was observed at positive (n=6) as well as at negative clamp voltage (n=13). Excision activation of CF Cl^– channels was observed at low (< 10^–9 mol/l) or high (10^–3 mol/l) calcium activities on the cytosolic side of the excised patch. Variation of the Ca⁺ activity (< 10^–9–10^–3 mol/l) or pH (6.5–8.5) on the cytosolic side exerted no effects on these Cl^– channels. These results suggest that Cl^– channels are present in the apical membrane of CF and N respiratory cells but they seem to be inhibited in intact CF cells. Excision of the patch and hence removal of the cytosolic inhibitor leads to an activation of Cl^– channels. The Cl^– channels in excised patches of N and CF cells have identical properties.     

A 23-pS Ca2+-activated K+ channel in MCF-7 human breast carcinoma cells: an apparent correlation of channel incidence with the rate of cell proliferation

In studies on the apical membranes of cultured MCF-7 human breast carcinoma cells, we found two conspicuous K⁺ channel types with conductances of 23 and 70 pS, respectively. Of these, the 23-pS K⁺ channel was most conspicuous. In cell-attached patches with KCl in the pipette, it had a linear current/voltage (I/V) relation and was activated by depolarisation and in excised insideout patches it was highly selective for K⁺ over Na⁺ (permeability ratio of Na⁺ to K⁺, P _Na/P _K=0.02). Rubidium (Rb⁺) had a similar permeability to K⁺, although it was only conducted at 20% of the rate of K⁺, and cesium (Cs⁺) had a permeability less than 30% that of K⁺ and was not conducted at all. Both Cs⁺ and Rb⁺ acted as partial blockers when applied internally but the channel was not blocked by external tetraethylammonium (TEA, 10 mmol/l), quinidine (200 mol/l) or apamin (50 nmol/l). It was activated by Ca^{2 +} in the range 10^–7–10^–6 mol/l. In cell-attached patches at a pipette potential of 0 mV, the open-time histogram was described by a single exponential (time constant 1.6 ms) and the closed-time histogram by two exponentials (time constants 0.5 and 1.5 ms). The incidence of the 23-pS but not the 70-pS channel depended on the rate of cell proliferation. Thus, in studies on cell-attached patches from cells in the exponential growth phase, the 23-pS channel was observed in 78% of patches. However, when the proliferation rate was decreased, whether as a result of allowing the monolayer to reach confluence, or of cell treatment with an anti-oestrogen (tamoxifen, 10 mol/l), or a phorbol ester [phorbol 12-myristate 13-acetate (TPA), 2.6 nmol/l], the channel incidence was reduced to 42%, 60% and 42%, respectively. The activity of the 23-pS channel is not obligatory for cell division, however, since the rate of cell proliferation remained the same in MCF-7 cultures in which the channel was not expressed.     

Cation channel blocked by extracellular Ca2+ in the apical membrane of the chick embryonic ectoderm

In the chick embryo (20 h incubation, gastrula stage), the apical membrane of the ectodermal cells shows a high density of a non-selective cation channel which is blocked by very low extracellular Ca²⁺ concentrations. Properties of this channel were studied at the single-channel level using the patch-clamp technique in the cell-attached mode.With 1 mmol/l Ca²⁺ in the pipette, only outward current was present and the channel conductance measured at +120 mV was 25.5 pS. In the absence of Ca²⁺, also inward current through the channel was observed. The conductances measured at –50 mV were 49.5 pS with Na⁺ as the charge carrier, 72.5 pS with K⁺, 49.1 pS with Cs⁺, and 18.5 pS with Li⁺. The conductance measured at +80 mV was around 23 pS in all four cases. The reversal potential was similar (around 25 mV) for all four ions, which indicates a poor selectivity of the channel. In the absence of Ca²⁺ and the presence of 1 mmol/l ethylene-bis(oxonitrilo) tetraacetate (EGTA), the kinetics of the channel were characterized by bursts of the order of seconds. During a burst, the channel flickered between one open and one closed level. The open time was constant between –30 mV and –80 mV, while the closed time decreased with hyperpolarization. The open time varied according to the permeant ion (K⁺+=Cs⁺+).Extracellular Ca²⁺ blocked the inward current in a voltage-dependent manner. The K _d values, 1 mol/l at –30 mV and 3.2 mol/l at –80 mV, indicate that Ca²⁺ ions exit the channel toward the intracellular side. A weak voltage dependency of the association rate constant suggests that the Ca²⁺-binding site is close to the outside mouth. Extracellular Ca²⁺ was much less efficient at blocking the outward current (K _d about 1 mmol/l at 80 mV). Tetracaine, but not uraniumdioxide, decreased the opening probability of the channel. The embryonic channel shows similarities with the Ca²⁺-blockable, poorly selective channel described in the epithelium of toad urinary bladder.     

Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells

 We have previously shown that a new type of K⁺ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl^- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K⁺ channel of 16 ± 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0.1 mmol/l, n = 26). Reduction of extracellular Ca²⁺ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SK_Ca). It was also inactivated by forskolin (5 μmol/l, n = 38), whilst the K⁺ channel noise caused by the very small K⁺ channel increased. Activity of non-selective cation channels (NS_cat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NS_cat, with a mean conductance of 49 ± 1.0 pS (n = 96), was Ca²⁺ activated and required >10 μmol/l Ca²⁺ (cytosolic side = cs). It was reversibly inhibited by ATP (<1 mmol/l, n = 13) and by 3′,5-dichloro-diphenylamine-2-carboxylate (10–100 μmol/l, n = 5). SK_Ca was also Ca²⁺ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 μmol/l (cs) and then fell sharply to almost zero at 0.1 μmol/l Ca²⁺ (cs, n = 12). SK_Ca was inhibited by Ba²⁺ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca²⁺ activity ([Ca²⁺]_i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K⁺ concentration to 30 mmol/l, reduced [Ca²⁺]_i markedly (n = 8–10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca²⁺-sensitive K⁺ channel. This channel is activated promptly by very small increments in [Ca²⁺]_i and is inactivated by a fall in [Ca²⁺]_i induced by forskolin. Received: 15 April 1996 / Received after revision and accepted: 17 June 1996     

Forskolin and PMA pretreatment of HT29 cells alters their chloride conductance induced by cAMP,Ca2+ and hypotonic cell swelling

HT₂₉ cells were preincubated with forskolin (10^–5 mol/l, FORHT) or phorbol 12-myristate 13-acetate (PMA) (10^–7 mol/l, PMAHT) for 20 h, which has been shown previously and is also shown here, to upregulate and downregulate, respectively, the expression of the cystic fibrosis transmembrane conductance regulator (CFTR). CFPAC-1 cells underwent the same protocols. HT₂₉ cells were examined by slow (SWC) and fast (FWC) whole-cell patch-clamp techniques. The results of SWC and FWC were indistinguishable and were pooled. CFPAC-1 cells were examined with FWC. The membrane voltage (V) of FORHT was -41.8±1.4 mV (n=77) and that of PMAHT was -43.6±2.4 mV (n=76). The conductance (G) of FORHT (9.4 ±0.9 nS, n=77) was significantly larger than that of PMAHT (3.7±0.4 nS, n=76). Acute application of forskolin (10^–5 mol/l, FOR) plus 0.5 mmol/l 8-(4-chlorophenylthio)-cAMP (cAMP) depolarized V by 12 (FORHT) and 8 (PMAHT) mV, respectively. The acute increase of G by FOR plus cAMP was by 7.6±1.9 nS for FORHT (n=22) and only 2.2±1 nS for PMAHT (n=13). ATP (10^–4 mol/l) depolarized V in both types of cells. It enhanced G by 16.7±4.1 nS in FORHT (n=14) and significantly less (by 5.5±1.2nS, n=14) in PMAHT. Also the G increase lasted longer in FORHT. Neurotensin (NT, 10^–8 mol/l) also had a stronger and longer lasting effect in FORHT. Exposure to hypotonic bath solution (160 mosmol/l) depolarized V in both types of cells. The increase in G was by 15±2.2 nS in FORHT (n=18) and by 11±1.3 nS in PMAHT (n=23). After being returned to the normotonic media, the decline of G to the control value was delayed in FORHT when compared to PMAHT. Ionomycin (10^–7 mol/l) increased G significantly more (to 47±8.5 nS, n=13) in FORHT when compared to PMAHT (to 28±4 nS, n=13). The present data indicate that a 20-h exposure of HT₂₉ cells to FOR versus PMA alters markedly the CFTR concentration. The cells with high CFTR (FORHT) when compared to those with low CFTR (PMAHT) differ not only in their acute G response to cAMP, but also in that to ATP, NT, hypotonic cell swelling, and ionomycin. In contrast, the same pretreatment of CFPAC-1 cells did not alter the G changes induced by ionomycin or hypotonic cell swelling. These results indicate that changes in CFTR expression correlate with the Cl^– conductances induced by cAMP, Ca²⁺ and hypotonic cell swelling.     

Evidence for agonist-induced export of intracellular Ca2+ in epithelial cells

There is increasing evidence that some agonists not only induce intracellular Ca²⁺ increases, due to store release and transmembranous influx, but also that they stimulate Ca²⁺ efflux. We have investigated the agonist-stimulated response on the intracellular Ca²⁺ activity ([Ca²⁺]_i) in the presence of thapsigargin (10^–8 mol/l, TG) in HT₂₉ and CFPAC-1 cells. For CFPAC-1 the agonists ATP (10^–7–10^–3 mol/l, n=9), carbachol (10^–6–10^–3 mol/l, n=5) and neurotensin (10^–10–10^–7 mol/l, n=6) all induced a concentration-dependent decrease in [Ca²⁺]_i in the presence of TG. Similar results were obtained with HT₂₉ cells. This decrease of [Ca²⁺]_i could be caused by a reduced Ca²⁺ influx, either due to a reduced driving force for Ca²⁺ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca²⁺ permeability. Using the fura-2 Mn²⁺ quenching technique we demonstrated that ATP did not slow the TG-induced Mn²⁺ quench. This indicates that the agonist-induced [Ca²⁺]_i decrease in the presence of TG was not due to a reduced influx of Ca²⁺ into the cell, but rather due to stimulation of Ca²⁺ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of –49±3.6 mV (n=9). Within the first 3 min ATP was still able to induce a depolarization which could be attributed to an increase in Cl^– conductance. This was expected, since at this time after TG stimulation all Ca²⁺ agonists still liberated some [Ca²⁺]_i. When TG incubation was prolonged, agonist application led to strongly attenuated or to no electrical responses. Therefore, the agonist-stimulated [Ca²⁺]_i decrease cannot be explained by the reduction of the driving force for Ca²⁺ into the cell. In the same cells hypotonic swelling (160 mosmol/l, n=15) still induced a further [Ca²⁺]_i increase in the presence of TG and concomitantly induced Cl^– and K⁺ conductances. We conclude that the agonist-induced decrease of [Ca²⁺]_i in the presence of TG probably unmasks a stimulation of [Ca²⁺]_i export.     

Effect of vasoactive intestinal peptide,carbachol and other agonists on the membrane voltage of pancreatic duct cells

The regulation of pancreatic exocrine secretion involves hormonal, neural and neurohormonal components. Many agonists are known to be effective in pancreatic acinar cells, but less is known about the ducts. Therefore, we wanted to investigate the influence of various agonists on isolated perfused pancreatic ducts and, as a physiological response, we measured the basolateral membrane voltage of the duct cells (V _bl) with microelectrodes. Pancreatic ducts were dissected from pancreas of normal rats and bathed in a HCO₃ ^–-containing solution. Under control conditions, the average V _bl was between -50 and -70 mV. Vasoactive intestinal peptide (VIP) and carbachol (CCH) reversibly depolarized V _bl when applied to the bath. VIP (9×10^–9 mol/l) depolarized V _bl from -72±3 mV to -53±3 mV (n=20) and CCH (10^–5 mol/l) from -62±3 to -35±4mV (n=10). Furthermore, a decrease of the Cl^– concentration in the lumen led to an increase of VIP-induced depolarization of V _bl, suggesting that a luminal Cl^– conductance was increased. Cholecystokinin (CCK, 10^–10-10^–7 mol/l) and bombesin (10^–8, 10^–5 mol/l), which stimulate pancreatic exocrine secretion in acini or whole glands, showed no significant effect on V _bl of the duct cells tested in our preparation (n=7, 6). Neurotensin (10^–8 mol/l) had a marked depolarizing effect in two out of ten cases; V _bl depolarized from about -65 mV to-29 mV and the effect was reversible. Substance P (2×10^–7 mol/l), alone or in combination with secretin, had no effect on V _bl of the tested duct cells (n=11). We propose that the basolateral membrane of pancreatic duct cells possesses receptors for VIP, acetylcholine and neurotensin. CCK, bombesin and substance P had no detectable effects on V _bl of the duct cells tested, which could be due to the lack of corresponding receptors on these cells, or due to the absence of electrophysiologically detectable effects, in spite of receptor presence.Preliminary reports of the present study were presented at the 70th and 71st Meetings of the German Physiological Society, Germany, September 1991, March 1992     

Effects of arylaminobenzoate-type chloride channel blockers on equivalent short-circuit current in rabbit colon

Arylaminobenzoates were examined in rabbit colon mounted in an Ussing chamber. The open-circuit transepithelial voltage (V _te) and resistance (R _te) were measured and the equivalent short-circuit current (I _SC=V _te/ R _te) was calculated. After serosal (s) and mucosal (m) addition of indomethacin (1 mol/l) I _SC was –71±11 (n = 118) A/cm². Amiloride (0.1 mmol/l, m) inhibited this current and reversed the polarity to + 32±4 (n=118) A/cm². In the presence of amiloride and indomethacin, prostaglandin E₂ (1 mol/l, s), known to induce Cl^– secretion, generated an I _SC of -143 ± 8 (n = 92) A/cm². The arylaminobenzoate and Cl^– channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) reduced I _SC reversibly with a half-maximal inhibition (IC₅₀) at approximately 0.35 mmol/l and 0.2 mmol/l for mucosal and serosal application respectively. To test whether the poor effect was caused by mucus covering the luminal surface, dose/response curves of the mucosal effect were repeated after several pretreatments. Acidic pH on the mucosal side reduced IC₅₀ to approximately 0.1 mmol/l. A similar effect was observed after N-acetyl-l-cysteine (m) preincubation. Pretreatment with N-acetyl-l-cysteine (m) and carbachol (s), in order to exhaust mucus secretion, and l-homocysteine (m) were more effective and reduced IC₅₀ to approximately 50 mol/l. To test whether this effect of NPPB was caused by non-specific effects, the two enantiomers of 5-nitro-2-(+/–1-phenylethylamino)-benzoate were tested of which only the (+) form inhibited the Cl^– conductance in the thick ascending limb of the loop of Henle (TAL). In the present study the (+) enantiomer inhibited significantly more strongly than the (–) form. This suggests that the inhibitory effect of NPPB, even though it requires rather high concentrations, is probably due to Cl^– channel inhibition. For other arylaminobenzoates the sequence of potencies was different from that determined for the TAL. The present data indicate that substances that have been designed to block the Cl^– conductance of the TAL segment also inhibit reversibly but with much lower affinity the PGE₂-induced Cl^– secretion in rabbit colon.Supported by DFG Gr 480/10     

Negative inotropic effects of aldosterone antagonists in isolated human and guinea-pig ventricular heart muscle

Summary The effects of K⁺-canrenoate (Aldactone^® pro inj.) and its metabolite canrenone on isometric force of contraction were measured in isolated guinea-pig and human papillary muscle preparations driven electrically at a frequency of 1 Hz. In guinea-pig hearts both substances exerted a concentration-dependent negative inotropic effect; the IC₅₀ of K⁺-canrenoate and canrenone were 129±22 µmol l^–1 (n=5) and 85±11 µmol l^–1 (n=12), respectively. At the maximally tested concentration canrenone (250 µmol l^–1) and K⁺-canrenoate (1,000 µmol l^–1) reduced force of contraction by 68±4% (n=12) and 83±3% (n=5), respectively. The negative inotropic effects of canrenone and K⁺-canrenoate were not affected by 10 µmol l^–1 atropine. The negative inotropic effect of canrenone was also not affected by 14 µmol l^–1 aldosterone, but canrenone (10 µmol l^–1) diminished the maximal positive inotropic effect of dihydro-ouabain from 554±75% (n=4) to 269±39% (n=4) of the predrug value.In human heart muscles K⁺-canrenoate and canrenone also exerted a concentration-dependent negative inotropic effect. K⁺-canrenoate (1,000 µmol l^–1) and canrenone (250 µmol l^–1) reduced force of contraction by 57±7% (n=8) and 67±2% (n=6), respectively. A positive inotropic effect of both substances was never observed.It is concluded that the improvement of cardiac performance after application of aldosterone antagonists observed in patients cannot be explained by a direct effect on the heart. K⁺-canrenoate and canrenone are devoid of any direct cardiotonic action. Instead, K⁺-canrenoate and canrenone have direct negative inotropic effects at high concentrations.Abbreviations Aldactone^® pro inj Aldactone^® pro injection - g gramm - Hz Hertz - IC₅₀ concentration of drugs which produce 50% inhibition of force of contraction - i.v. intravenous - min minutes - mm millimeter - mm s^–1 millimeter per second - mmol l^–1 millimolar - mg milligramm - mN milli newton - ms millisecond - SC 8109 spironolactone derivative - SEM standard error of the mean - TRIS Tris(hydroxymethyl)-aminomethan - v/v volume per volume - µmol l^–1 micromolar This work was supported by the Deutsche Forschungsgemeinschaft     

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.