Control of K+ conductance by cholecystokinin and Ca2+ in single pancreatic acinar cells studied by the patch-clamp technique

Summary The effect of cholecystokinin (CCK) and internal Ca²⁺ on outward K⁺ current in isolated pig pancreatic acinar cells has been investigated using the patch-clamp method for whole-cell current recording under voltage-clamp conditions. CCK (2 × 10^–10 M) applied to the bath evoked a marked increase in the outward K⁺ current associated with depolarizing voltage steps, and this effect was fully reversible and acutely dependent on the presence of external Ca²⁺. When strongly buffered Ca²⁺-EGTA solutions were used inside the cells CCK failed to evoke an effect. Increasing the internal Ca²⁺ concentration ([Ca²⁺] _i ) from 5 × 10^–10 M to 10^–7 and 5 × 10^–7 M mimicked the effect of CCK. It would appear therefore that CCK controls K⁺ conductance in the acinar cells via changes in the internal free ionized Ca²⁺ concentration.     

Inhibition by cAMP of Calcium-Activated Chloride Currents in Cultured Sertoli Cells from Immature Testis

We have characterized a Ca²⁺-dependent Cl⁻ current (Cl_Ca) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 μm free Ca²⁺, exhibited outward currents which were inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca²⁺ strongly reduced the currents indicating that they were Ca²⁺ dependent. With cells dialyzed with Cs⁺ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 μm ionomycin induced outward currents having properties of Ca²⁺-activated Cl⁻ currents. With ATP-free pipette solution, the magnitude of currents was not modified suggesting the direct control by Ca²⁺. By contrast, addition of 0.1 mm cAMP in the pipette solution or the superfusion of cells by a permeant analogue of cAMP strongly reduced the currents. These results may suggest that Cl_Ca is inhibited by cAMP-dependent protein kinase. Finally, our results do not agree with the model of primary fluid secretion by exocrine cells, but are in agreement with a hyperpolarizing effect of cAMP in primary culture of Sertoli cells and the release of a low Cl⁻ and bicarbonate-rich primary fluid by these cells. Received: 30 November 1998/Revised: 2 March 1999     

Calcium-dependent Modulation of the Agonist Affinity of the Mammalian Olfactory Cyclic Nucleotide-gated Channel by Calmodulin and a Novel Endogenous Factor

The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca²⁺ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC₅₀) was reversibly increased from 3 μm to about 30 μm. This Ca²⁺-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure to 3 mm Mg²⁺+ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca²⁺ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca²⁺-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the affinity of the channel for CAM. The affinity shift induced by Ca²⁺-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR, ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated by an as yet unidentified factor distinct from CAM. Received: 26 December 1995/Revised: 14 March 1996     

Large Conductance Ca2+-activated K+ Channels in the Soma of Rat Motoneurones

Properties of large conductance Ca²⁺-activated K⁺ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K⁺ _o //155 mm K⁺ _i ) and 231 ± 4 pS in external high-K _o solution (155 mm K⁺ _o //155 mm K⁺ _i ). The channels were activated by depolarization and by an increase in internal Ca²⁺ concentration. Potentials of half-maximum channel activation (E₅₀) were −13, −34, −64 and −85 mV in the presence of 10⁻⁶, 10⁻⁵, 10⁻⁴ and 10⁻³ m internal Ca²⁺, respectively. Using an internal solution containing 10⁻⁴ m Ca²⁺, averaged K_Ca currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged K_Ca currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with a 50% blocking concentration (IC₅₀) of 0.17 ± 0.02 mm. K_Ca channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that K_Ca channels activated by Ca²⁺ entry during the action potential play an important role in the excitability of motoneurones. Received: 7 November 1996/Revised: 29 October 1997     

Cytosolic calcium regulates a potassium current in corn (Zea mays) protoplasts

Summary The voltage- and time-dependent K⁺ current,I _K ⁺ _out , elicited by depolarization of corn protoplasts, was inhibited by the addition of calcium channel antagonists (nitrendipine, nifedipine, verapamil, methoxyverapamil, bepridil, but not La³⁺) to the extracellular medium. These results suggested that the influx of external Ca²⁺ was necessary for K⁺ current activation. The IC₅₀, concentration of inhibitor that caused 50% reduction of the current, for nitrendipine was 1 m at a test potential of +60 mV following a 20-min incubation period.In order to test whether intracellular Ca²⁺ actuated the K⁺ current, we altered either the Ca²⁺ buffering capacity or the free Ca²⁺ concentration of the intracellular medium (pipette filling solution). By these means,I _K ⁺ _out could be varied over a 10-fold range. Increasing the free Ca²⁺ concentration from 40 to 400nm also shifted the activation of the K⁺ current toward more negative potentials. Maintaining cytoplasmic Ca²⁺ at 500nm with 40nm EGTA resulted in a more rapid activation of the K⁺ current. Thus the normal rate of activation of this current may reflect changes in cytoplasmic Ca²⁺ on depolarization. Increasing intracellular Ca²⁺ to 500nm or 1 m also led to inactivation of the K⁺ current within a few minutes. It is concluded thatI _K ⁺ _out is regulated by cytosolic Ca²⁺, which is in turn controlled by Ca²⁺ influx through dihydropyridine-, and phenylalkylamine-sensitive channels.     

Activation by Angiotensin II of Ca2+-dependent K+ and Cl− Currents in Zona Fasciculata Cells of Bovine Adrenal Gland

The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These effects were abolished by Losartan (10⁻⁵ m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method, a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau current. In Cl⁻-free solution, the outward current reversed at −78.5 mV, a value close to E _K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K⁺-free solution, the transient outward current, sensitive to Cl⁻ channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E _Cl. Its magnitude changed in the same direction as the driving force for Cl⁻. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca²⁺-free solution (47% of cells) and was reversibly blocked by Cd²⁺ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca²⁺ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin II is due to Ca²⁺-dependent K⁺ and Cl⁻ currents. These two membrane currents are co-activated in response to an internal increase of [Ca²⁺] _i originating from intra- and extracellular stores. Received: 29 May 1997/Revised: 4 November 1997     

Large-conductance Calcium-activated Potassium Channels of Cultured Rat Melanotrophs

A large conductance, Ca²⁺-activated K⁺ channel of the BK type was examined in cultured pituitary melanotrophs obtained from adult male rats. In cell-attached recordings the slope conductance for the BK channel was ≈190 pS and the probability (P _o ) of finding the channel in the open state at the resting membrane potential was low (<<0.1). Channels in inside-out patches and in symmetrical 150 mm K⁺ had a conductance of ≈260 pS. The lower conductance in the cell-attached recordings is provisionally attributed to an intracellular K⁺ concentration of ≈113 mm. The permeability sequence, relative to K⁺, was K⁺ > Rb⁺ (0.87) > NH⁺ ₄ (0.17) > Cs⁺≥ Na⁺ (≤0.02). The slope conductance for Rb⁺ was much less than for K⁺. Neither Na⁺ nor Cs⁺ carried measurable currents and 150 mm internal Cs⁺ caused a flickery block of the channel. Internal tetraethylammonium ions (TEA⁺) produced a fast block for which the dissociation constant at 0 mV (K _D (0 mV)) was 50 mm. The K _D (0 mV) for external TEA⁺ was much lower, 0.25 mm, and the blocking reaction was slower as evidenced by flickery open channel currents. With both internal and external TEA⁺ the blocking reaction was bimolecular and weakly voltage dependent. External charybdotoxin (40 nm) caused a large and reversible decrease of P _o . The P _o was increased by depolarization and/or by increasing the concentration of internal Ca²⁺. In 0.1 μm Ca²⁺ the half-maximal P _o occurred at ≈100 mV; increasing Ca²⁺ to 1 μm shifted the voltage for the half-maximal P _o to −75 mV. The Ca²⁺ dependence of the gating was approximated by a fourth power relationship suggesting the presence of four Ca²⁺ binding sites on the BK channel. Received: 23 October/Revised: 15 December 1995     

Roles of Ca2+ and Protein Tyrosine Kinase in Insulin Action on Cell Volume via Na+ and K+ Channels and Na+/K+/2Cl− Cotransporter in Fetal Rat Alveolar Type II Pneumocyte

The aim of the present study was to investigate the roles of Ca²⁺ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca²⁺ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca²⁺ (<1 nm). This insulin action in a Ca²⁺-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na⁺/K⁺/2Cl⁻ cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na⁺ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca²⁺-free solution was completely blocked by quinine (1 mm, a blocker of Ca²⁺-activated K⁺ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm extracellular Ca²⁺, that the stimulatory action of insulin on an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter requires Ca²⁺, and that in a Ca²⁺-free solution insulin activates a quinine-sensitive K⁺ channel but not in the presence of 1 mm Ca²⁺. The insulin action on cell volume in a Ca²⁺-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca²⁺-ATPase which depletes the intracellular Ca²⁺ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca²⁺-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K⁺ channel is mediated through PTK activity in a cytosolic Ca²⁺-dependent manner. Lavendustin A, further, completely blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in a Ca²⁺-free solution, but only partially blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm Ca²⁺. This observation suggests that the activity of the Na⁺/K⁺/2Cl⁻ cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca²⁺-independent pathway and the other is a PTK-independent, Ca²⁺-dependent pathway. Further, we observed that removal of extracellular Ca²⁺ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na⁺ channel and the bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter, and that removal of extracellular Ca²⁺ abolished the activity of the quinine-sensitive K⁺ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca²⁺ results from diverse effects on the cotransporter and Na⁺ and K⁺ channels. Received: 2 September 1998/Revised: 30 November 1998     

Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels

The cellular mechanisms that regulate potassium (K⁺) channels in guard cells have been the subject of recent research, as K⁺ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca²⁺ concentrations, Ca²⁺ buffers and GTP-binding protein modulators on inward-rectifying K⁺ channels. Ca²⁺ inhibition of inward-rectifying K⁺ currents depended strongly on the concentration and effectiveness of the Ca²⁺ buffer used, indicating a large Ca²⁺ buffering capacity and pH increases in guard calls. When the cytosolic Ca²⁺ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K⁺ channels were strongly inhibited. However, when EGTA was used as the Ca²⁺ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca²⁺. Under the imposed conditions, GTPγS did not significantly inhibit inward-rectifying K⁺ channel currents when cytosolic Ca²⁺ was buffered to low levels or when using EGTA as the Ca²⁺ buffer. Furthermore, GDPβS reduced inward K⁺ currents at low cytosolic Ca²⁺, indicating a novel mode of inward K⁺ channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca²⁺ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward-rectifying K⁺ channel currents in a population of cells, indicating possible Ca²⁺-dependent action of GTP-binding protein modulators in K⁺ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K⁺ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K⁺ channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of proton-pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G-protein modulators regulate inward K⁺ channels in a more complex and limited, possibly Ca²⁺-dependent, manner than previously proposed.     

Effects of quinine on Ca++-induced K+ efflux from human red blood cells

Summary The Ca⁺⁺-mediated increase in K⁺-permeability of intact red blood cells (Gardos effect) was initiated by exposing cells to known concentrations of Ca⁺⁺ (using EGTA buffers) in the presence of the ionophore A23187. The potency of quinine, an inhibitor of the response, was found to depend on the external K⁺ concentration. In K⁺-free solutions the concentration of quinine to achieve 50% inhibition (K ₅₀) was 5 m, but at 5mm K⁺ the required concentration was increased 20-fold to 100 m. An increase in internal Na⁺ had the opposite effect, allowing a high potency of quinine despite the presence of external K⁺. Alterations in the internal K⁺ level, on the other hand, were without effect on theK ₅₀, suggesting that the membrane potential is not a factor. This conclusion is supported by the lack of effect on quinine inhibition of substitution of Cl^– by NO ₃ ^– , a considerably more permeant anion. The data are consistent with the hypothesis that quinine inhibits by competitively displacing K⁺ from an external binding site, the reported K⁺-activation site for the Ca⁺⁺-mediated K⁺-permeability.     

Sodium-calcium exchange in renal epithelial cells: Dependence on cell sodium and competitive inhibition by magnesium

Summary Kinetic properties of Na⁺–Ca²⁺ exchange in a renal epithelial cell line (LLC-MK₂) were assessed by measuring cytosolic free Ca²⁺ with fura-2 and⁴⁵Ca²⁺ influx. Replacing external Na⁺ with K⁺ produced relatively small increases in free Ca²⁺ and⁴⁵Ca²⁺ uptake unless the cells were incubated with ouabain. Ouabain markedly increased cell Na⁺ and strongly potentiated the effect of replacing external Na⁺ with K⁺ on free Ca²⁺ and⁴⁵Ca²⁺ uptake.⁴⁵Ca²⁺ influx in 140mm K⁺ or N-methyl-d-glucamine minus influx in 140mm Na⁺ was used to quantify Na⁺–Ca²⁺ exchange activity of Na⁺-loaded cells. The dependence of exchange on cell Na⁺ was sigmoidal; theK _0.5 was 26±3 mmol/liter cell water space, and the Hill coefficient was 3.1±0.2. The kinetic features of the dependence of exchange on cell Na⁺ partly account for the small increase in Ca²⁺ influx when all external Na⁺ is replaced by K⁺. Besides raising cell Na⁺ ouabain appears to activate the exchanger. Magnesium competitively inhibited exchange activity. The potency of Mg²⁺ was 8.2-fold lower with potassium instead of N-methyl-d-glucamine or choline as the replacement for external Na⁺. Potassium also increased theV _max of exchange by 86% and had no effect on theK _m for Ca²⁺. The exchanger does not cause detectable²²Na⁺–Mg²⁺ exchange and does not appear to require K⁺ or transport⁸⁶Rb⁺. Although exchange activity was plentiful in the epithelial cells from monkey kidney, others from amphibian, canine, opossum, and porcine kidney had no detectable exchange activity. All of the measured kinetic properties of Na⁺–Ca²⁺ exchange in the renal epithelial cells are very similar to those of the exchanger in rat aortic myocytes.     

Analysis and use of the perforated patch technique for recording ionic currents in pancreaticβ-cells

Summary To investigate the voltage dependence of the Na^–/K^– pump, current-voltage relations were determined in prophasearrested oocytes ofXenopus laevis. All solutions contained 5mm Ba^2– and 20mm tetraethylammonium (TEA) to block K^– channels. If. in addition, the Na⁺/K⁺ pump is blocked by ouabain, K⁺-sensitive currents no larger than 50 nA/cm² remain. Reductions in steady-state current (on the order of 700 nA/cm²) produced by 50 m ouabain or dihydro-ouabain or by K⁺ removal, therefore, primarily represent current generated by the Na^–/K^– pump. In Na^–-free solution containing 5mm K⁺, Na⁺/K⁺ pump current is relatively voltage independent over the potential range from –160 to +40 mV. If external [K⁺] is reduced below 0.5mm, negative slopes are observed over this entire voltage range. Similar results are seen in Na⁺- and Ca²⁺-free solutions in the presence of 2mm Ni²⁺, an experimental condition designed to prevent Na⁺/Ca²⁺ exchange. The occurrence of a negative slope can be explained by the voltage dependence of the apparent affinity for activation of the Na⁺/K⁺ pump by external K⁺, consistent with the existence of an external ion well for K^– binding. In 90mm Na⁺, 5mm K⁺ solution, Na⁺/K⁺ pump current-voltage curves at negative membrane potentials have a positive slope and can be described by a monotonically increasing sigmoidal function. At an extracellular [K⁺] of 1.3mm, a negative slope was observed at positive potentials. These findings suggest that in addition to a voltage-dependent step associated with Na⁺ translocation, a second voltage-dependent step that is dependent on external [K⁺], possibly external K⁺ binding, participates in the overall reaction mechanism of the Na⁺/K⁺ pump.     

Single nonselective cation channels and Ca2+-activated K+ channels in aortic endothelial cells

Summary In cultured bovine aortic endothelial cells, elementary K⁺ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150±10 pS and a small channel with a mean unitary conductance of 12.5±1.1 pS. The 150-pS channel proved to be voltag- and Ca²⁺-activatable and seems to be a K⁺ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca²⁺ concentration at the cytoplasmic side of the membrane from 10^–7 to 10^–4 m. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced rundown nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K⁺ and Na⁺ whose open probability was minimal near –60 mV but increased on membrane hyperpolarization. An increase in internal Ca²⁺ from 10^–7 to 10^–4 m left the open probability unchanged. Although the K⁺ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca²⁺-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na⁺.     

Ionic permeabilities of the plasma membrane of isolated intact bovine rod outer segments as studied with a novel optical probe

Summary The permeability properties of the plasma membrane of intact rod outer segments purified from bovine retinas (ROS) were studied with the aid of the optical probe neutral red as described in the companion paper. The following observations were made: (1) Electrical shunting of ROS membranes greatly stimulated Na⁺ and K⁺ transport, suggesting that this transport reflects Na⁺ and K⁺ currents, respectively. The dissipation of a Na⁺ gradient across the plasma membrane occurred with a half-time of 30 sec at 25°C. (2) The Na⁺ permeability was progressively inhibited when the external Ca²⁺ concentration was raised from 1 m to 20mm. A similar Ca²⁺ dependence was observed for H⁺ and Li⁺ transport. The Na⁺ permeability was not affected when the total internal Ca²⁺ content of ROS was varied between 0.1 mol Ca²⁺/mol rhodopsin and 7 mol Ca²⁺/mol rhodopsin, or when the free internal Ca²⁺ concentration was varied between 0.1 and 50 m. (3) The K⁺ permeability was progressively stimulated when the external Ca²⁺ concentration was raised from 0.001 to 1 m, whereas a further increase to 20mm was without effect. A similar Ca²⁺ dependence was observed for Rb⁺ and Cs⁺ transport. (4) At an external Ca²⁺ concentration in the micromolar range the rate of transport decreased in the order: Na⁺>K⁺=H⁺>Cs⁺>Li⁺. (5) Na⁺ fluxes depended in a sigmoidal way on the external Na⁺ concentration, suggesting that sodium ions move in pairs. The concentration dependence of uniport Na⁺ transport and that of Na⁺-stimulated Ca²⁺ efflux (exchange or antiport transport) were very similar.     

Large Conductance Ca2+-Activated K+ Channels in Human Meningioma Cells

Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K⁺ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC₅₀= 5.3 mm). Raising the internal Ca²⁺ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V _1/2) of the K⁺ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca²⁺-activated (BK _Ca ) K⁺ channel with a conductance of 296 pS (130 mm K⁺ at both sides of the patch). V _1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca²⁺, respectively, at the internal face of the patch. In cell-attached patches the open probability (P _o ) of BK _Ca channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K⁺ currents with 10 nm Ca²⁺ in the pipette. Application of 20 μm cytochalasin D increased P _o of BK _Ca channels in cell-attached patches within minutes. These data suggest that the activation of BK _Ca channels in meningioma cells does not only depend on voltage and internal Ca²⁺ but is also controlled by the cytoskeleton. Received 18 June 1999/Revised: 18 January 2000     

Membrane Potassium Channels and Human Bladder Tumor Cells. I. Electrical Properties

These experiments were conducted to determine the membrane K⁺ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K⁺ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca²⁺-dependent outward K⁺ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca²⁺-activated, voltage-dependent K⁺ channel (K_Ca) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K_Ca activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K⁺ channel, 21 ± 1 pS at positive transmembrane potential (V _m ) and 145 ± 13 pS at negative V _m . Glibenclamide (50 μm), Ba²⁺ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K⁺ channels in inside/out patches in a dose-dependent manner, and the IC₅₀= 46 μm. The identity of this K⁺ channel with an ATP-sensitive K⁺ channel (K_ATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the K_Ca and a lower conductance K⁺ channel with properties consistent with a sulfonylurea receptor-linked K_ATP. Received: 12 June 1997/Revised: 21 October 1997     

On the Role of Calcium in the Regulatory Volume Decrease (RVD) Response in Ehrlich Mouse Ascites Tumor Cells

The putative role for Ca²⁺ entry and Ca²⁺ mobilization in the activation of the regulatory volume decrease (RVD) response has been assessed in Ehrlich cells. Following hypotonic exposure (50% osmolarity) there is: (i) no increase in cellular Ins(1,4,5)P₃ content, as measured in extracts from [2-³H]myoinositol-labeled cells, a finding at variance with earlier reports from our group; (ii) no evidence of Ca²⁺-signaling recorded in a suspension of fura-2-loaded cells; (iii) Ca²⁺-signaling in only about 6% of the single, fura-2-loaded cells at 1-mm Ca²⁺ (1% only at 0.1-mm Ca²⁺ and in Ca²⁺-free medium), as monitored by fluorescence-ratio imaging; (iv) no effect of removing external Ca²⁺ upon the volume-induced K⁺ loss; (v) no significant inhibition of the RVD response in cells loaded with the Ca²⁺ chelator BAPTA when the BAPTA-loading is performed in K⁺ equilibrium medium; (vi) an inhibition of the swelling-induced K⁺ loss (about 50%) at 1-mm Ba²⁺, but almost no effect of charybdotoxin (100 nm) or of clotrimazole (10 μm), reported inhibitors of the K⁺ loss induced by Ca²⁺-mobilizing agonists. Thus, Ca²⁺signaling by Ca²⁺ release or Ca²⁺ entry appears to play no role in the activation mechanism for the RVD response in Ehrlich cells. Received: 8 December 1996/Revised: 14 January 1997     

Characteristics of a Low Affinity Passive Ca2+ Influx Component in Rat Parotid Gland Basolateral Plasma Membrane Vesicles

We have previously reported the presence of two Ca²⁺ influx components with relatively high (K_Ca= 152 ± 79 μm) and low (K_Ca= 2.4 ± 0.9 mm) affinities for Ca²⁺ in internal Ca²⁺ pool-depleted rat parotid acinar cells [Chauthaiwale et al. (1996) Pfluegers Arch. 432:105–111]. We have also reported the presence of a high affinity Ca²⁺ influx component with K_Ca= 279 ± 43 μm in rat parotid gland basolateral plasma membrane vesicles (BLMV). [Lockwich, Kim & Ambudkar (1994) J. Membrane Biol. 141:289–296]. The present studies show that a low affinity Ca²⁺ influx component is also present in BLMV with K_Ca= 2.3 ± 0.41 mm (V_max= 16.36 ± 4.11 nmoles of Ca²⁺/mg protein/min). Our data demonstrate that this low affinity component is similar to the low affinity Ca²⁺ influx component that is activated by internal Ca²⁺ store depletion in dispersed parotid gland acini by the following criteria: (i) similar K_Ca for calcium flux, (ii) similar IC₅₀ for inhibition by Ni²⁺ and Zn²⁺; (iii) increase in K_Ca at high external K⁺, (iv) similar effects of external pH. The high affinity Ca²⁺ influx in cells is different from the low affinity Ca²⁺ influx component cells in its sensitivity to pH, KCl, Zn²⁺ and Ni²⁺. The low and high affinity Ca²⁺ influx components in BLMV can also be distinguished from each other based on the effects of Zn²⁺, Ni²⁺, KCl, and dicyclohexylcarbodiimide. In aggregate, these data demonstrate the presence of a low affinity passive Ca²⁺ influx pathway in BLMV which displays characteristics similar to the low affinity Ca²⁺ influx component detected in parotid acinar cells following internal Ca²⁺ store depletion. Received: 19 March 1997/Revised: 25 November 1997     

Sodium-selective channels in membranes of rat macrophages

With the use of the patch-clamp technique, highly selective nonvoltage-gated sodium channels were found in the membrane of rat peritoneal macrophages. The inward single channel currents were measured in cell-attached and outside-out mode experiments at different holding membrane potentials within the range of-60 to +40 mV. The channels had a unitary conductance of 10.2 ± 0.2 pS with 145 mm Na⁺ in the external solution at 23–24°C. The results of ion-substitution experiments confirmed that this novel type of cation channel in macrophages is characterized by high selectivity for Na⁺ over K⁺ (as for Cs⁺, NH₄ ⁺, Ca²⁺, Ba²⁺) ions, whose conduction through these sodium-permeable channels was not measurable. Lithium is the only other ion that is transported by this pathway; the unitary conductance was equal to 3.9 ± 0.2 pS in the Li⁺-containing external solution. Single channel currents and conductance were found to be linearly dependent on the external sodium concentration. Sodium channels in macrophage membrane patches were not blocked by tetrodotoxin (0.01–1 m). Single sodium currents were reversibly inhibited by the external application of amiloride (0.1–2 mm) and its derivative ethylisopropilamiloride (0.01–0.1 Mm). The mechanism of channel block by amiloride and its analogue seems to be different.We thank Dr. G.N. Mozhayeva and Dr. A.P. Naumov for useful discussions. This work has been supported by a grant from the Russian Basic Research Foundation, 93-04-21722.     

High-conductance K+ channel in pancreatic islet cells can be activated and inactivated by internal calcium

Summary The Ca²⁺-activated K⁺ channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage (I/V) relationship showed outward rectification and the null potential was more negative than –40 mV. In symmetrical K⁺-rich solutions the single-channelI/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca²⁺-free solution containing 2mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities (P) above 0.1. Raising the free Ca²⁺ concentration in contact with the membrane inside ([Ca²⁺]_i) to 1.5×10^–7 m had little effect on the relationship between membrane potential andP. When [Ca²⁺]_i was increased to 3×10^–7 m and 6×10^–7 m smaller potential changes were required to open the channels. Increasing [Ca²⁺]_i further to 8×10^–7 m again activated the channels, but the relationship between membrane potential andP was complex. Changing the membrane potential from –50 mV to +20 mV increasedP from near 0 to 0.6 but further polarization to +50 mV decreasedP to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca²⁺]_i=1 and 2 m. In this situation a membrane potential change from –70 to +20 mV increasedP from near 0 to about 0.7 but further polarization to +80 mV reducedP to less than 0.1. The high-conductance K⁺ channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca²⁺]_i within the range 0.1 to 1 m which suggests a physiological role for this channel in regulating the membrane potential and Ca²⁺ influx through voltage-activated Ca²⁺ channels.