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.     

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     

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.     

Single Ca2+-activated K+ channels in excised membrane patches of hamster oocytes

The properties of the Ca²⁺-activated K⁺ channel in unfertilized hamster oocytes were investigated at the single-channel level using inside-out excised membrane patches. The results indicate a new type of Ca²⁺-activated K⁺ channel which has the following characteristics: (1) single-channel conductance of 40–85 pS for outward currents in symmetrical K⁺ (150 mM) solutions, (2) inward currents of smaller conductance (10–50 pS) than outward currents, i.e. the channel is outwardly rectified in symmetrical K⁺ solutions, (3) channel activity dependent on the internal concentration of free Ca⁺ and the membrane potential, (4) modification of the channel activity by internal adenosine 5 diphosphate (0.1 mM) producing a high open probability regardless of membrane potential.     

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

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

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     

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).     

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.     

Properties of membrane currents in isolated smooth muscle cells from guinea-pig trachea

Tracheal smooth muscle cells were enzymatically isolated from guinea-pig trachea. These cells contracted in response to acetylcholine (0.01–10 M) in a concentration-dependent fashion. Under current-clamp conditions with 140 mM K⁺ in the pipette solution, the membrane potential oscillated spontaneously at around –30 mV. Under voltage-clamp conditions, there appeared spontaneous but steady oscillations of outward current (I _o). On depolarization from a holding potential at –40 mV, three components of outward current were elicited: transient outward current (I _T), steady-state outward current (I _s) and I _o. These three components of outward current reversed around the K⁺ equilibrium potential and were abolished by Cs⁺ in the pipette, indicating that K⁺ was the major charge carrier of these outward currents. All these three components were completely suppressed by extracellular tetraethylammonium (10 mM). Both I _T and I _o were depressed by quinidine (1 mM), 4-aminopyridine (10 mM) and nifedipine (100 nM), but I _s was not affected. I _T and I _o were suppressed by a Ca²⁺-free perfusate with less than 1 nM Ca²⁺ in the pipette, while with 10 nM Ca²⁺ in the pipette, only I _o was suppressed. In both conditions, I _s was not affected by the Ca²⁺-free perfusate. Therefore, it is suggested that I _o, I _T and I _s are separate types of K⁺ current. With Cs⁺ in the pipette, K⁺ currents were almost completely suppressed and a transient inward current was observed during depolarizing pulses. The inward current was not affected by tetrodotoxin and increased when the concentration of extracellular Ca²⁺ was raised, indicating that the current is a Ca²⁺ channel current. Even with a holding potential of –80 mV, the low-threshold inward current could not be observed. The high-threshold Ca²⁺ current was abolished by nifedipine (100 nM) and was enhanced by Bay K 8644 (100 nM). The order of permeation of divalent cations through the Ca²⁺ channel was Ba²⁺ >Sr²⁺ Ca²⁺. Cd²⁺ blocked the Ca²⁺ current more effectively than Ni²⁺. These results may indicate that the Ca²⁺ current of tracheal smooth muscle cells is mainly composed of the current through an L-type Ca²⁺ channel.     

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

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

K+ and Cl− currents in freshly isolated rat osteoclasts

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

Single calcium-activated potassium channel in cultured mammary epithelial cells

The properties of Ca²⁺-activated K⁺ channels in mouse mammary epithelial cells in primary culture were studied by the patch-clamp technique. In cell-attached patches, spontaneous channel openings were sometimes observed; the slope conductance of the currents was about served; the slope conductance of the currents was about 12 pS at negative membrane potentials with a physiological solution (152 mM Na⁺, 5.4 mM K⁺) in the pipette. External application of A23187, a calcium ionophore, activated this channel. In excised inside-out patches, the channel was activated by increasing the internal Ca²⁺ concentration (10^–7 to 10^–6 M). No voltage dependence of the channel activity was observed. Internal Na⁺ blocked the outward K⁺ current in a voltage dependent manner and this block led to the non-linear I–V relationship at positive membrane potentials. The channel was blocked by internal Ba²⁺ (0.1 mM) and tetracthylammonium (TEA⁺, 20–50 mM). Ba²⁺ reduced the open probability but not the single channel conductance, whereas TEA⁺ reduced the single channel conductance. The single channel conductance of this channel, measured from the inward current with a high-K⁺ solution (150 mM K⁺) in the pipette, was large (about 40 pS), and showed inward rectification. These results suggest that this channel is different from the usual small conductance Ca²⁺-activated K⁺ channels observed in many other cells.     

Properties of single potassium channels in hypothalamic neurons

In this work the cell-attached and the excised membrane patch configurations have been used to determine properties of voltage-dependent K⁺ channels in cultured rat hypothalamic neurons. With inside-out patches and 140 mM K⁺ in the bath and 5 mM K⁺ in the pipette step depolarizations, in excess of 20 mV, elicited channel activity with at least two independent current levels. The larger current level was studied presently and current-voltage plots showed the conductance of the channel to be 48 pS; measurements of the changes in reversal potential with 140 mM or 5 mM K⁺ in the bathing solution indicated the channel to be selective for K⁺. The 48 pS channel was blocked when the bathing solution contained 140 mM Cs⁺. A concentration of 10 mM TEA applied to the outside of the patch in the outside-out configuration caused a loss of channel activity whereas 4-AP had no obvious effect on channel conductance or kinetics. Changes in the bath concentration of Ca²⁺ had no apparent effect to alter the frequency or duration of channel openings for inside-out patches. In some instances obvious changes in channel kinetics were observed during the course of an experiment; these included long periods where no channel opening events occurred and on a few other occasions progressive increases in mean open time. In cell-attached patch experiments with no TTX in the bathing medium to block sodium channels, action potentials could be recorded through capacitive coupling between the cell and the pipette. In such cases the heights of spontaneous K⁺ channel currents were very similar to those associated with channel openings during the repolarization phase of the spike. The properties of the K⁺ channel studied here are consistent with those associated with the delayed rectifier K⁺ conductance (I _K) and would serve to control electrical excitability in hypothalamic neurons.     

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     

Patch-clamp study of rubidium and potassium conductances in single cation channels from mammalian exocrine acini

Single-channel current recordings were carried out on excised inside-out patches of baso-lateral plasma membrane from exocrine acinar cells. The mouse pancreas and submandibular gland as well as the pig pancreas were investigated.In the mouse pancreas the voltage-insensitive Ca²⁺-activated cation channel was studied. Single-channel current-voltage (i/v) relationships were studied in symmetrical Rb⁺-rich solutions and in asymmetrical Rb⁺/Na⁺ and Na⁺/Rb⁺ solutions. In all cases the i/v relations were linear and had the same slope representing a single-channel conductance of about 33 pS which is identical to that previously obtained with symmetrical Na⁺ solutions or asymmetrical Na⁺/K⁺ solutions.In the mouse submandibular gland and the pig pancreas the voltage and Ca²⁺-activated K⁺ channel was studied. The outward currents observed after depolarization in the presence of quasi-physiological Na⁺/K⁺ gradients were immediately abolished when all the K⁺ in the bath fluid was replaced by Rb⁺ (bath fluid in contact with inside of plasma membrane). This effect was immediately and fully reversible upon return to the high K⁺ solution.The voltage and Ca²⁺-activated K⁺ channel was also studied in asymmetrical K⁺/Rb⁺ and Rb⁺/K⁺ solutions. In the first case inward (K⁺) currents could be observed but not outward (Rb⁺) currents, while in the other case inward (Rb⁺) currents could not be seen whereas outward (K⁺) currents were measured. The current-voltage relationships were approximately linear and the null potential was close to 0 mV in both situations. In contrast the null potential for current through the K⁺ channel in the presence of asymmetrical Na⁺/K⁺ or Li⁺/K⁺ solutions was about –70 mV and with reversed gradients about +60 mV.Outward K⁺ currents of reduced size (through the voltage and Ca²⁺-activated K⁺ channel) could be observed when the bath fluid contained 75 mM K⁺ and 75 mM Rb⁺, but not (in the same membrane patches) when 150 mM Rb⁺ and no K⁺ was present.It is concluded that the large voltage- and Ca²⁺-activated K⁺ channel has an extremely low Rb⁺ conductance. It is possible, however, that the permeability for Rb⁺ may be about the same as for K⁺. The voltage-insensitive Ca²⁺-activated cation channel does not discriminate between K⁺ and Rb⁺.     

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.     

Electrophysiological properties of membrane currents in single myometrial cells isolated from pregnant rats

The whole-cell voltage-clamp method was applied to single smooth muscle cells prepared from the longitudinal layer of the pregnant rat myometrium (17–20 days of gestation). It was found that the transient inward current mainly consists of Ca²⁺ current, because the removal of Ca²⁺ ions from the external medium and 10 M nifedipine eliminated this inward current. Its steady-state inactivation curve was obtained by the standard method, in which the membrane potential of half inactivation and the slope factor were estimated to be –58.0±4.9 mV (n=11) and 8.9±1.4 mV (n=11), respectively. In a small number of preparations (in 2 out of 30 preparations), there remained a very fast inward current in Ca²⁺-free medium containing Mg²⁺. Tetrodotoxin (TTX, 10 M) can abolish this current, suggesting that the channel for this current is equivalent to the Na⁺ channel in nerve cells. Two major phases of outward currents were identified by voltage jumps from negative holding levels to more positive levels. The first phase was a fast transient outward current. This current remained intact after external tetraethylammonium (TEA, 20 mM) was added. Following the transient current, a large delayed rectified outward current reached its peak over a period of 50 ms and then decayed. The reversal potential for this outward current was determined by observing the change of polarity of the tail currents with the change in extracellular K⁺ concentration ([K⁺]₀). The slope for the change of reversal potential per ten-fold change in [K⁺]₀ is 57.7 mV at more than 23.2 mM [K⁺]_o, indicating that this current is mostly carried by K⁺ ions. Voltage-dependent inactivation of the delayed rectified outward current was determined by the standard method. The membrane potential for half inactivation and the slope factor were estimated to be –42.8±3.9 mV (n=3) and 10.1±1.5 mV (n=3), respectively. External TEA (20 mM) effectively eliminated the delayed rectified outward currents. Nifedipine (10 M) suppressed not only Ca²⁺ current but also outward K⁺ currents.     

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.     

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.     

Electrogenic Na+/K+-transport in human endothelial cells

Na⁺/K⁺ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca²⁺]_i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na⁺] did not induce significant changes of [Ca²⁺]_i. Superfusing the cells with K⁺-free solutions also did not significantly affect [Ca²⁺]_i. Reapplication of K⁺ after superfusion of the cells with K⁺-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mol/l dihydroouabain (DHO) and was therefore identified as a Na⁺/K⁺ pump current. During block and reactivation of the Na⁺/K⁺ pump no changes in [Ca²⁺]_i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10–1000 U/l) did not affect the pump currents. An increase of the intracellular Na⁺ concentration ([Na⁺]_i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na⁺]_i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K⁺ was 2.4±1.2 mmol/l, and 0.4±0.1 and 8.7±0.7 mmol/l for Tl⁺ and NH₄ ⁺ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than –150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method. If however 100 mol/l guanosine 5-O-(3-thiotriphosphate) (GTPS) were added to the pipette solution, the currents measured in the ruptured-whole-cell-mode were not significantly different from the currents measured with the perforated-patch technique. We suppose that the use of the perforated-patch technique prevents wash out of a guanine nucleotide-binding protein (G-protein)-connected intracellular regulator that is necessary for pump activation.