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.     

Regulation and possible physiological role of the Ca2-dependent K+ channel of cortical collecting ducts of the rat

In the luminal membrane of rat cortical collecting duct (CCD) a big Ca²⁺-dependent and a small Ca²⁺-independent K⁺ channel have been described. Whereas the latter most likely is responsible for the K⁺ secretion in this nephron segment, the function of the large-conductance K⁺ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K⁺ channel was strongly voltage- and calcium-dependent. At 3 mol/l cytosolic Ca²⁺ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 mol/l Ca²⁺ activity the open probability (P _o) of this channel was pH-dependent. At pH 7.0 P _o was decreased to 4±2% (n=9) and at pH 8.5 it was increased to 425±52% (n=9) of the control. At this low Ca²⁺ activity the P _o of the channel was reduced by 1 mmol/l ATP to 8±4% (n=6). Cell swelling activated the large-conductance K⁺ channel (n=14) and hyperpolarized the membrane potential of the cells by 9±1 mV (n=23). Intracellular Ca²⁺ activity increased after hypotonic stress. This increase depended on the extracellular Ca²⁺ activity. A possible physiological function of the large-conductance K⁺ channel in rat CCD cells may be the reduction of the intracellular K⁺ concentration after cell swelling. Once this channel is activated by increases in the cytosolic Ca²⁺ activity it can be regulated by changes in cellular pH and ATP.Supported by DFG Schl 277/2-3     

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.     

Effect of depolarizing and hyperpolarizing agents on the membrane potential difference of primary cultures of rabbit aorta vascular smooth muscle cells

Vascular smooth muscle cells of rabbit aorta were enzymatically dispersed, kept in primary culture, and studied between days 1 and 7 in a bath rinsed with Ringer-like solution at 37°C. The electrical membrane potential difference (PD) was measured with microelectrodes. The mean value of PD was –50±0.4 mV (n=53). Cromakalim (BRL 34915), 1 mol/l and 10 mol/l, hyperpolarized the membrane potential by 9±1 mV (n=11) and 15±1 mV (n=53) respectively. Glibenclamide (10 mol/l) abolished the hyperpolarizing effect of cromakalim (n=6). Simultaneous addition of cromakalim and glibenclamide (both 10 mol/l, n=11) and glibenclamide itself (10 mol/l, n=7) had no effect on PD. In patch-clamp experiments in outside-out-oriented Ca²⁺-sensitive K⁺ channels, cromakalim increased the open probability (P _o) only slightly and only with a cytosolic Ca²⁺ activity of 1 mol/l. In all other series cromakalim had no effect on the P _o of these channels. Forskolin (10 mol/l) hyperpolarized PD by 6±1 mV (n=13). The nucleotides UTP, ATP and ITP (10 mol/l) depolarized PD by 12±1 mV (n=7), 8±1 mV (n=65) and 5±1 mV (n=6) respectively. GTP, [,-methylene]ATP and adenosine had no significant effect. Mn²⁺ (1 mmol/l, n=18), Ni²⁺ (1 mmol/l, n=13), Co²⁺ (1 mmol/l, n=11), Zn²⁺ (1 mmol/l, n=6) and the Ca²⁺-channel blockers verapamil and nifedipine (both 0.1 mmol/l, n=6) did not attenuate the depolarization induced by 10 mol/l ATP. Fetal calf serum (100 ml/l, n=7) depolarized PD by 11±2 mV. This effect was not abolished by nifedipine or by replacing NaCl by choline chloride. The data indicate that PD of vascular smooth muscle cells is depolarized by P₂ agonists and hyperpolarized by the K⁺-channel opener cromakalim. The effect of cromakalim is antagonized by glibenclamide. The effect of cromakalim is probably not mediated by the K⁺ channel identified in excised patches.Supported by DFG Gr 480/10     

Toxin pharmacology of the large-conductance Ca2+-activated K+ channel in the apical membrane of rabbit proximal convoluted tubule in primary culture

The patch-clamp technique was used to study the toxin pharmacology of the large-conductance Ca²⁺-activated K⁺ channel (BKCa) present in the apical membrane of rabbit proximal convoluted tubules (PCT) in primary culture. Experiments were performed with the inside-out configuration. This channel was very selective for K⁺ against Na⁺ and had a conductance of 180 pS with 140 mmol/l in the pipette and the bath. The action of toxins was studied on the extracellular side of the channel by using the pipette perfusion technique. Experimental conditions were 140 mmol/l KCl in the pipette and 140 mmol/l Nad in the bath. Pipette potential was maintained at 0 mV. Perfusion of crude venom from Leiurus quinquestriatus hebraeus inhibited reversibly the open probability (P _o) in a concentration-dependent fashion (IC₅₀=0.8 mg/l; n=3). The following synthetic or purified toxins were tested: synthetic charybdotoxin (ChTX) IC₅₀=7.3×10^–9 M (n=5); iberiotoxin (IbTX) IC₅₀=5.5×10^–7 mol/l (n=3); and kaliotoxin (KTX) IC₅₀=4.8×10^–7 mol/l (n=3). The suppression of the six first N-terminal amino-acids slightly reduced the affinity of ChTX (IC₅₀=1.2×10^–8 mol/l, n=4). Neither Dendroaspis polylepis venom nor purified dendrotoxin modified P _o even at high concentrations (20 mg/l and 10^–6 mol/l respectively). Apamin, which blocked the small-conductance K⁺ channel in cultured PCT, did not act on BKCa. These results indicate that ChTX is the most efficient known toxin against the epithelial BKCa in primary cultures of PCT. In spite of there being considerable homology of sequence between ChTX, IbTX and KTX, ChTX was about 100 times more effective than the others. Truncated ChTX kept a high affinity for this channel and could be used to obtain a labelled probe.     

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.     

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in HT29 colonic carcinoma cells

Cl^– secretion in HT₂₉ cells is regulated by agonists such as carbachol, neurotensin and adenosine 5-triphosphate (ATP). These agonists induce Ca²⁺ store release as well as Ca²⁺ influx from the extracellular space. The increase in cytosolic Ca²⁺ enhances the Cl^– and K⁺ conductances of these cells. Removal of extracellular Ca²⁺ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca²⁺ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=6) inhibited ATP (0.1 mmol·l^–1) induced increases in whole-cell conductance (G _m). When Cl^– and K⁺ currents were inhibited by the presence of Cs₂SO₄ in the patch pipette and gluconate in the bath, ATP (0.1 mmol·l^–1) still induced a significant increase in G _m from 1.2±0.3 nS to 4.7±1 nS (n=24). This suggests that ATP induces a cation influx with a conductance of approximately 3–4 nS. This cation influx was inhibited by flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=9). When Ba²⁺ (5 mmol·l^–1) and 4,4-diisothiocyanatostilbene-2-2-disulphonic acid (DIDS, 0.1 mmol·l^–1) were added to the KCl/K-gluconate pipette solution to inhibit K⁺ and Cl^– currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol·l^–1) reduced the membrane current (I _m) significantly from 86±14 pA to 54±11 pA (n=13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5–10 mmol·l^–1 1,2-bis-(2-aminoethoxy)ethane-N,N,N,N-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA). The zero-current membrane voltage (V _m) and I _m (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30–120 s after membrane rupture. V _m depolarised significantly from –33±2 mV to –12±1 mV, and I _m fell significantly from 17±2 pA to 8.9±1.0 pA (n=71). This negative current, representing a cation inward current, was activated when Ca²⁺ stores were emptied and was reduced significantly (I _m) when Ca²⁺ and/or Na⁺ were removed from the bathing solution: removal of Ca²⁺ in the absence of Na⁺ caused a I _m of 5.0±1.2 pA (n=12); removal of Na⁺ in the absence of Ca²⁺ caused a I _m of 12.8±3.5 pA (n=4). The cation inward current was also reduced significantly by La³⁺, Gd³⁺, and flufenamate. We conclude that store depletion induces a Ca²⁺/Na⁺ influx current in these cells. With 145 mmol·l^–1 Na⁺ and 1 mmol·l^–1 Ca²⁺, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.     

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     

Transmitter-induced changes of the membrane voltage of HT29 cells

The colonic carcinoma cell line HT₂₉ was used to examine the influence of agonists increasing cytosolic cAMP and Ca²⁺ activity on the conductances and the cell membrane voltage (V _m). HT₂₉ cells were grown on glass cover-slips. Cells were impaled by microelectrodes 4–10 days after seeding, when they had formed large plaques. In 181 impalements V _m was –51±1 mV. An increase in bath K⁺ concentration from 3.6 mmol/l to 18.6 mmol/l or 0.5 mmol/l Ba²⁺ depolarized the cells by 10±1 mV (n=49) or by 9±2 mV (n=3), respectively. A decrease of bath Cl^– concentration from 145 to 30 mmol/l depolarized the cells by 11±1 mV (n=24). Agents increasing intracellular cAMP such as isobutylmethylxanthine (0.1 mmol/l), forskolin (10 mol/l) or isoprenaline (10 mol/l) depolarized the cells by 6±1 (n=13), 15±3 (n=5) and 6±2 (n=3) mV, respectively. In hypoosmolar solutions (225 mosmol/l) cells depolarized by 9±1 mV (n=6). Purine and pyrimidine nucleotides depolarized the cells dose-dependently with the following potency sequence: UTP > ATP > ITP > GTP > TIP > CTP = 0. The depolarization by ATP was stronger than that by ADP and adenosine. The muscarinic agonist carbachol led to a sustained depolarization by 27±6 mV (n=5) at 0.1 mmol/l, and to a transient depolarization by 12±4 mV (n=5) at 10 mol/l. Neurotensin depolarized with a half-maximal effect at around 5 nmol/l. The depolarization induced by nucleotides and neurotensin was transient and followed by a hyperpolarization. We confirm that HT₂₉ cells possess Cl^–- and K⁺-conductive pathways. The Cl^– conductance is regulated by intracellular cAMP level, cytosolic Ca²⁺ activity, and cell swelling. The K⁺ conductance in HT₂₉ cells is regulated by intracellular Ca²⁺ activity.Supported by DFG Gre 480/10 and GIF Proj. no. I-86-100.10/ 88     

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.