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.     

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.     

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.     

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     

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 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.     

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     

Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane

In order to study the mechanism of pancreatic HCO ₃ ^– transport, a perfused preparation of isolated intra-and interlobular ducts (i.d. 20–40 m) of rat pancreas was developed. Responses of the epithelium to changes in the bath ionic concentration and to addition of transport inhibitors was monitored by electrophysiological techniques. In this report some properties of the basolateral membrane of pancreatic duct cells are described. The transepithelial potential difference (PD_te) in ducts bathed in HCO ₃ ^– -free and HCO ₃ ^– -containing solution was –0.8 and –2.6 mV, respectively. The equivalent short circuit current (I_sc) under similar conditions was 26 and 50 A·cm^–2. The specific transepithelial resistance (R_te) was 88 cm². In control solutions the PD across the basolateral membrane (PD_bl) was –63±1 mV (n=314). Ouabain (3 mmol/l) depolarized PD_bl by 4.8±1.1 mV (n=6) within less than 10 s. When the bath K⁺ concentration was increased from 5 to 20 mmol/l, PD_bl depolarized by 15.9±0.9 mV (n=50). The same K⁺ concentration step had no effect on PD_bl if the ducts were exposed to Ba²⁺, a K⁺ channel blocker. Application of Ba²⁺ (1 mmol/l) alone depolarized PD_bl by 26.4±1.4 mV (n=19), while another K⁺ channel blocker TEA⁺ (50 mmol/l) depolarized PD_bl only by 7.7±2.0 mV (n=9). Addition of amiloride (1 mmol/l) to the bath caused 3–4 mV depolarization of PD_bl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PD_bl. An increase in the bath HCO ₃ ^– concentration from 0 to 25 mmol/l produced fast and sustained depolarization of PD_bl by 8.5±1.0 mV (n=149). It was investigated whether the effect of HCO ₃ ^– was due to a Na⁺⁺-dependent transport mechanism on the basolateral membrane, where the ion complex transferred into the cell would be positively charged, or whether it was due to decreased K⁺ conductance caused by lowered intracellular pH. Experiments showed that the HCO ₃ ^– effect was present even when the bath Na⁺ concentration was reduced to a nominal value of 0 mmol/l. Similarly, the HCO ₃ ^– effect remained unchanged after Ba²⁺ (5 mmol/l) was added to the bath. The results indicate that on the basolateral membrane of duct cells there is a ouabain sensitive (Na⁺+K⁺)-ATPase, a Ba²⁺ sensitive K⁺ conductance and an amiloride sensitive Na⁺/H⁺ antiport. The HCO ₃ ^– effect on PD_bl is most likely due to rheogenic anion exit across the luminal membrane.     

Functional heterogeneity in the hamster medullary thick ascending limb of Henle's loop

Cellular heterogeneity was examined in the hamster medullary thick ascending limb (MAL) perfused in vitro by electrophysiological measurements with an intracellular microelectrode. Random measurements of fractional resistance of basolateral membrane (Rf _B) revealed two cell populations, high basolateral conductance (HBC) cells havingRf _B of 0.05±0.01 (n=24) and low basolateral conductance (LBC) cells havingRf _B of 0.80±0.03 (n=32). Basolateral membrane potentials (V _B) were not different between HBC cells and LBC cells (–72.6±1.2,n=43 vs. –70.0±1.2,n=35). Addition of 2 mmol/l Ba²⁺ to the bath depolarized the basolateral membrane in the HBC cells from –70.4±3.2 to –20.9±5.9 mV (n=8) but not in the LBC cells (from –74.4±1.9 to –72.0±2.1 mV). Increasing K⁺ or decreasing Cl^– in the bathing solution caused marked positive deflection ofV _B in the HBC cells but little or no change inV _B in the LBC cells. Elimination of Cl^– from the lumen or addition of furosemide to the lumen enhanced the potential response of the HBC cells to basolateral application of Ba²⁺. Accordingly, with Ba²⁺ present in the bath, the potential response of the HBC cells to a decrease in bath Cl^– concentration was enhanced. These observations suggest that a K⁺ conductance exists in the basolateral membrane of HBC cells in paralled with a Cl^– conductance. The basolateral cell membrane of LBC cells also contains a Cl^– conductance. In these cells, but not in HBC cells, the potential response to decreasing bath Cl^– concentration increased when bath pH was decreased from 7.4 to 6.0 Apparent K⁺ transference numbers of the luminal membrane were higher in LBC cells (0.74±0.05,n=7) than in HBC cells (0.20±0.02,n=5). From these data, we conclude: (1) there are two distinct cell types in the hamster medullary thick ascending limb; (2) there is a low Cl^– conductance in basolateral membrane of LBC cells which is stimulated by low pH.     

Electrophysiological characterization of rabbit distal convoluted tubule cell

The distal convoluted tubule (DCT) from rabbit kidney were perfused in vitro to study the conductive properties of the cell membranes by using electrophysiological methods. When the lumen and the bath were perfused with a biearbonate free solution buffered with HEPES, the transepithelial voltage (V _T) averaged –2.8±0.6 mV (n=20), lumen negative. The basolateral membrane voltage (V _B) averaged –77.8±1.1 mV (n=33) obtained by intracellular impalement of microelectrodes. Cable analysis performed by injecting a current from perfusion pipette revealed that the transepithelial resistance was 21.8±1.7 ·cm² and the fractional resistance of the luminal membrane was 0.78±0.03 (n=8), indicating the existence of ionic conductances in the luminal membrane. Addition of amiloride (10^–5 mol/l) to the luminal perfusate or Na⁺ removal from the lumen abolished the lumen negativeV _T and hyperpolarized the apical membrane. An increase in luminal K⁺ concentration from 5 to 50 mmol/l reduced the apical membrane potential (V _A) by 37.5±2.6 mV (n=7), whereas a reduction of Cl^– in the luminal perfusate did not changeV _A significantly (0.5±0.5 mV,n=4). Addition of Ba²⁺ to the lumen reducedV _A by 42.6±1.0 mV (n=4). When the bathing fluid was perfused with 50 mmol/l K⁺ solution, the basolateral membrane voltage (V _B) fell from –76.8±1.5 to –31.0±1.3 mV (n=18), and addition of Ba²⁺ to the bath reducedV _B by 18.3±4.8 mV (n=7). Although a reduction of Cl^– in the bathing fluid from 143 to 5 mmol/l did not cause any significant fast initial depolarization (1.8±1.7 mV,n=8), a spike like depolarization (14.0±2.5 mV,n=4) was observed, upon Cl^– reduction in the presence of Ba²⁺ in the bath. From these results, we conclude that the apical membrane of DCT has both K⁺ and Na⁺ conductances and the basolateral membrane has a K⁺ conductance and a small Cl^– conductance.     

Ion conductances of isolated cortical collecting duct cells

The study of ion conductances in the intact cortical collecting duct (CCD) with the patch-clamp method is rather difficult. An optimized method to isolate CCD cells from rat kidneys using an in vivo followed by an in vitro enzyme digestion is described. Individual CCD segments were collected after this digestion and incubated in EGTA-buffered medium. This procedure resulted in single cells or cell clusters. These freshly isolated CCD cells were studied with different modifications of the patch-clamp method. Membrane voltages measured in the cell-attached-nystatin configuration were –74 ±1mV (n=13) and –68±3 mV (n=22) in cells isolated from normal and mineralocorticoid-treated rats respectively. These values and those measured with the nystatin-perforated slow-whole-cell configuration (–79 ±1mV, n=23) are comparable to those measured in principal cells of isolated CCD segments. The cells hyperpolarized after the addition of amiloride and depolarized with the addition of adiuretin to the bath. The amiloride effect was enhanced when cells were isolated from deoxycorticosterone-acetate-treated rats. The cells were strongly depolarized upon elevation of the extracellular K⁺-concentration and did not demonstrate a measurable Cl^– conductance. A large-conductance K⁺ channel (174 pS, n=5, cell-attached, 145 mmol/l K⁺ in the pipette; 140 pS, n=12, cell-free, 3.6 mmol/l K⁺ in the bath) was seen. It had a very low activity on the cell, but a high open probability when excised into a solution with 1 mmol/l Ca²⁺ on the cytosolic side. More often a small-conductance K⁺ channel (36–52 pS, n=19, cell-attached; 30 pS, n=5, cell-free) with a high open probability was found on the cell. These freshly isolated cells seem to be a powerful preparation to study the properties and regulation of ion conductances of rat CCD with several electrophysiological methods. These freshly isolated CCD cells maintain the conductance properties known from principal cells of the intact CCD.     

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     

A stretch-activated K+ channel in the basolateral membrane ofXenopus kidney proximal tubule cells

The present study examined whether a basolateral potassium ion (K⁺) channel is activated by membrane-stretching in the cell-attached patch. A K⁺ channel of conductance of 27.5 pS was most commonly observed in the basolateral membrane ofXenopus kidney proximal tubule cells. Channel activity increased with hyperpolarizing membrane potentials [at more positive pipette potentials (V _p)]. Open probability (P _o) was 0.03, 0.13, and 0.21 atV _p values of 0, 40, and 80 mV, respectively. Barium (0.1 mM) in the pipette reducedP _o by 79% at aV _p of 40 mV. Application of negative hydraulic pressure (−16 to −32 cm H₂O) to the pipette markedly activated outward currents (fromP _o=0.01 to 0.75) at aV _p of −80 mV, but not inward currents at aV _p of 80 mV. The size of the activated outward currents (from cell to pipette) did not change by replacing chloride with gluconate in the pipette. These results indicate that a stretch-activated K⁺ channel exists in the basolateral membrane of proximal tubule cells. It may play an important role as a K⁺ exit pathway when the cell membrane is stretched (for example, by cell swelling).     

Membrane potential measurements of transitional cells from the crista ampullaris of the Gerbil

Transitional cells of the crista ampullaris were impaled with microelectrodes in order to record the membrane potential (PD) and to investigate membrane properties. In control solution the PD was –87±1 mV (n=103). This value is not significantly different from –83±2 mV (n=24) measured in Cl^– free solution. [Cl^–] steps from 150 to 15 mmol/l (n=24) depolarized the membrane by about 2 mV, indicating a minor Cl^– conductance. The transference number for K⁺ was 0.75±0.01 (n=79) obtained from the PD responses to K⁺ steps from 3.6 to 25 mmol/l. The cell membrane depolarized and the amplitude of PD responses to [K⁺] steps was reduced by Ba²⁺ (2·10^–6 to 10^–3 mol/l), quinidine (10^–3 mol/l), quinine (10^–3 mol/l), Rb⁺ (20 mmol/l), Cs⁺ (20 mmol/l), NH₄ ⁺ (20 mmol/l) and Tl⁺ (0.5 mmol/l), whereas tetraethylammonium (TEA, 20 mmol/l) had no effect. The dose-response curve for Ba²⁺ in the presence of 3.6 mmol/l K⁺ was shifted to the right by approximately three decades in the presence of 25 mmol/l K⁺ and by a factor of about 4 in the presence of 135 mmol/l gluconate as a substitute for Cl^–. Transitional cells were depolarized by ouabain, suggesting the presence of (Na⁺+K⁺-ATPase.This work was supported by grants from the Deafness Research Foundation to PhW and the National Institute of Health (NS 19490) to DCM     

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.     

Ion channels in isolated mouse jejunal crypts

 The patch-clamp technique was used to characterise the ion channels in cells located in the mid region of mouse jejunal crypts. Six different channels were seen. A large outwardly rectified K⁺ channel (BK) (conductance, g at 0 mV = 92 ± 6 pS), which was highly selective for K⁺ [P _K ⁺ (1) > P _Rb ⁺ (0.6) >> P _Cs ⁺ (0.09) ≈ P _Na ⁺ (0.07) > P _Li ⁺ (0.04)], had a low, voltage-independent open probability (P _o) in the on-cell (O/C) configuration and appeared in 66% of the patches. In inside-out (I/O) patches, this channel had a linear current/voltage (I/V) relationship (g = 132 ± 3 pS), P _o was voltage dependent and it was blocked by cytoplasmic Ba²⁺ (5 mmol/l). An intermediate K⁺ channel (IK) which was present in 49% of O/C patches, had a linear I/V (g = 38 ± 3 pS), ran-down in O/C patches, and was not seen in I/O patches. A number of smaller channels (SC) with conductances ranging from 5 to 20 pS were seen in 16% of O/C patches. Also present in the basolateral membrane were a Cl^– channel (ICOR) and a nonselective cation channel (NSCC). These channels were only seen in I/O patches. ICOR had an outwardly rectified conductance (g at 0 mV = 36 ± 2 pS), its P _o was independent of voltage and unaffected by variations in cytoplasmic Ca²⁺ (100 nmol/l to 1 mmol/l) or ATP (0–1 mmol/l). The NSCC had a linear conductance (20 ± 1 pS), its P _o increased with depolarisation and elevation of cytoplasmic [Ca²⁺] (≥ 10 μmol/l), but was reduced by cytoplasmic ATP. None of the basolateral channels described here were activated by cAMP-dependent secretagogues, although a Cl^– conductance was activated. This cAMP-dependent Cl^– conductance was distinct from the basolateral Cl^– channel and thus is most likely located in the apical membrane. Received: 25 June 1997 / Accepted: 14 October 1997     

Large conductance calcium-activated potassium channels in cultured retinal pericytes under normal and high-glucose conditions

Pericytes are considered to contribute to the regulation of retinal microcirculation which is impaired in diabetic retinopathy. Single, large-conductance, Ca²⁺-dependent K⁺ channels (BK) were studied in cultured bovine retinal capillary pericytes using the patch-clamp method. In excised patches with symmetrical 135-mmol/l K⁺ solutions a single channel conductance of 238±9.9 pS was measured. With a K⁺ gradient of 4/ 135 mmol/l (extracellular/intracellular) the slope conductance averaged 148±2.9 pS at 0 mV. The mean permeability was 4.2×10^–13 cm³/s. The channel was highly selective for K⁺ with a permeability ratio for K⁺ over Na⁺ of 1/0.02. The mean open time and the open probability (P_o) of the BK channel increased with depolarization and with increasing internal [Ca²⁺] showing a maximal sensitivity to Ca²⁺ between 10^–4 and 10^–5 mol/l Ca²⁺. Ba²⁺ (5 mmol/l), quinine (5 mmol/l), and verapamil (Michaelis constant 1.5×10^–5 mol/l) blocked from the intracellular side. Tetraethylammonium induced a dose-dependent block from the outside only with a halfmaximal blocking concentration of 2.5×10^–4 mol/l. Charybdotoxin (10^–8 mol/l) blocked completely from the extracellular side. The channel activity was not changed by either internal adenosine triphosphate (ATP, 10^–4 mol/l) or the putative opener of ATP-sensitive K⁺ channels Hoe 234 (10^–6 mol/l). In cell-attached patches channelP _o was less than 3%. After a 3-day incubation in culture medium containing an elevated glucose concentration (22.5 mmol/l) the channel activity in attached patches was markedly increased. These data indicate that cultured retinal pericytes possess a BK channel. The activity of the channel increases after incubation with elevated glucose concentrations, which could indicate altered regulation of the channel under these conditions. The implications of altered function of BK channels are discussed with respect to haemodynamic changes observed in diabetic retinopathy.     

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.     

Ion channels in the basolateral membrane of intralobular duct cells of mouse mandibular glands

We have used single-channel patch-clamp techniques to study the ion channels in the basolateral membranes of intralobular duct cells from the mouse mandibular gland. In 39% of cell-attached patches, we observed a K⁺ channel that had an inwardly rectifying current/voltage (I/V) relation with a maximum slope conductance of 123±9 pS (n=12) and a zero current potential of +49.4±3.4 mV (n=5) relative to the resting cell potential. The selectivity sequence of this channel, as estimated by zero current potential measurements, was: K⁺ (1) > Rb⁺ (0.38) > NH ₄ ⁺ (<0.34), Cs⁺ (<0.16) > Na⁺ (<0.028). The activity of the channel was not affected by changes in membrane potential, nor was it affected by changes in the free Ca²⁺ concentration on the cytosolic side of inside-out excised patches in the range 1 nmol/l to 1 mol/l. In 38% of cell-attached patches we observed a second K⁺ channel type with a maximum slope conductance of 62±3 pS (n=12) and an inwardly rectifyingI/V relation. The selectivity sequence of this channel was K⁺ (1) > Rb⁺ (<0.5) > NH ₄ ⁺ (<0.2) > Na⁺ (<0.09). The activity of this channel type was not affected by changes in membrane potential. In 18% of excised patches, we also observed a non-selective cation channel that was not demonstrable in cell-attached patches. It had a slope conductance of 22±2 pS (n=6) and was blocked by the non-selective cation channel blocker, flufenamate (10 mol/l). A fourth channel type, observed only in 5% of patches was a Cl^– channel with a slope conductance of 40 pS and a linearI/V relation. The K⁺ channels observed in this study seem likely to underlie the K⁺ conductance described in the basolateral membrane of extralobular ducts by in vitro perfusion studies. Our finding that they are inwardly rectifying suggests that they may not be the sole route of K⁺ transport across the basolateral membrane.     

Solubilisation and reconstitution of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel into liposomes suitable for patch clamp studies

Sarcoplasmic reticulum (SR) membrane vesicles have been prepared from rabbit skeletal muscle and solubilised using K⁺ cholate. Solubilised membrane proteins were reconstituted into small asolectin liposomes by dialysis against cholate-free solution. Large liposomes were produced by freezing and thawing at –80°C and room temperature, respectively. The liposomes were assayed for the SR K⁺ channel using the patch clamp technique. Channel density was modulated by varying protein: lipid ratios during reconstitution. Channels inserted into the membrane with a preferred orientation. The solubilised and reconstituted channel behaves ohmically over the holding potential range ±70 mV and has a conductance of 178.4±4.4 pS (mean ± SE,n=37) in 200 mM KCl. The channel has a selectivity sequence of K⁺>NH ₄ ⁺ >Rb⁺>Na⁺ and K⁺ conductance is blocked by hexamethonium and decamethonium. The opening probability of the reconstituted channel is voltage dependent. The conductance and gating characteristics displayed by the solubilised and reconstituted channel correlate well with those previously observed following the fusion of native SR membrane vesicles with planar phospholipid bilayers.