The influence of amino-reactive substances on contraction threshold of frog skeletal muscle

Summary The action of the amino-reactive substances pyridoxal phosphate, 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid and 2,4,6-trinitrobenzene sulfonic acid on the contraction threshold, taken as parameter for the initiation of contraction, was investigated in fibers of the sartorius muscle of the frog. The contraction threshold was shifted by 1 to 11 mV tomore negative potentials with 1 to 20mm PDP. Similar shifts from 2 to 17 mV were produced by 0.66 to 20mm SITS. The threshold shift was only partially reversible. The shift of the contraction threshold obtained with 2mm SITS was nearly constant at different [Ca²⁺]_o and [Mg²⁺]_o from 1.5 to 50mm with a tendency to increase at higher divalent cation concentration. TNBS had no effect on the contraction threshold.The action of PDP and SITS on the contraction threshold was successfully described by the surface charge model used earlier to explain the effect of lanthanum, neuraminidase and ruthenium red on the contraction threshold (M. Dörrscheidt-Käfer,Pfluegers Arch. 380:171–179, 181–187, 1979;J. Membrane Biol. 62:95–103, 1981). Here it was assumed that PDP and SITS bind to positive fixed charges on the surface of the T-tubular wall. This results in a shift of the calculated surface potential to more negative values which is thought to account for the measured shift of the contraction threshold.     

Factors controlling the K+ conductance inChara

Summary Previous current/voltage (I/V) investigations of theChara K⁺ state have been extended by increasing the voltage range (up to +200 mV) through blocking the action potential with La³⁺. A region of negative slope was found in theI/V characteristics at positive PD's, similar to that already observed at PD's more negative than the resting level. These decreases in membrane currents at PD's more negative than –150 mV and at PD's close to 0 or positive are thought to arise from the K⁺ channel closure. Both the negative slope regions could be reversibly abolished by 0.1mm K⁺, 20mm Na⁺, more than 10mm Ca²⁺ or 5mm tetraethylammonium (TEA). The K⁺ channels are therefore blocked by TEA, closed by low [K⁺] _o or high [Ca²⁺] _o and are highly selective to K⁺ over Na⁺. With the K⁺ channels closed, the remainingI/V profile was approximately linear over the interval of 400 mV (suggesting a leakage current), but large rectifying currents were observed at PD's more positive than +50 mV. These currents showed a substantial decrease in high [Ca²⁺] _o , sometimes displayed a slight shift to more positive PD's with increasing [K⁺] _o and were unaffected by TEA or changes in [Na⁺] _o . The slope of the linear part of theI/V profile was steeper in low [K⁺] _o than in TEA or high [Na⁺] _o (indicating participation of K⁺, but not Na⁺, in the leak current). Diethylstilbestrol (DES) was employed to inhibit the proton pump, but it was found that the leakage current and later the K⁺ channels were also strongly affected.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

Effects of calcium and bay K-8644 on calcium currents in adrenal medullary chromaffin cells

Summary The kinetic and steady-state characteristics of calcium currents in cultured bovine adrenal chromaffin cells were analyzed by the patch-clamp technique. Whole cell inward Ca²⁺ currents, recorded in the presence of either 5.2 or 2.6mm Ca²⁺ exhibited a single, noninactivating component. To analyze the effects of Ca²⁺ and Bay K-8644 on the kinetics of the Ca²⁺ currents, we used a modified version of the Hodgkin-Huxley empirical model. At physiological [Ca²⁺] (2.5mm) the midpoint of the steady-state Ca²⁺-channel activation curve lay at –6.9 mV. Increasing the [Ca²⁺] to 5.2mm shifted the midpoint by –4.3 mV along the voltage axis. At the midpoint, changes in potential of 7.8 mV (for 5.2mm Ca²⁺) and 9.2 mV (for 2.5mm Ca²⁺) induced ane-fold change in the activation of the current. Increasing [Ca²⁺]₀ from 2.5 to 5.2mm induced a marked increase in the rate constant for turning on the Ca²⁺ permeability. Conductances were estimated from the slope of the linear part of the currentvoltage relationships as 8.7 and 4.2 nS in the presence of 5.2 and 2.5mm Ca²⁺, respectively. Incubation of the cells in the presence of Bay K-8644 at increasing concentrations from 0.001 to 0.1 m increased the slope conductance from 4.2 to 9.6 nS. Further increases in the concentration of Bay K-8644 from 1 to 100 m induced a marked reduction in the conductance to 1.1 nS. In the presence of Bay K-8644 (0.1 m) the midpoint of the activation curve was shifted by 6.1 mV towards more negative potentials, i.e., from –6.9 to –13 mV. At the midpoint potential of –13 mV, a change in potential of 6.9 mV caused ane-fold change in Ca²⁺ permeability. The kinetic analysis showed that Bay K-8644 significantly reduced the size of the rate constant for turning off the Ca²⁺ permeability.     

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.     

Ca2+-activated K+ channels in the apical membrane ofNecturus choroid plexus

Summary The properties of Ca²⁺-activated K⁺ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K⁺ over Na⁺ and K⁺ over Cs⁺. They were blocked by high cytoplasmic Na⁺ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca²⁺ concentrations. Increasing Ca²⁺ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca²⁺ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba²⁺ reduced both channel-open probability and conductance. The sites for the action of Ba²⁺ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba²⁺ dissociation constants (K _d (0 mV) for the effects on probability and conductance, respectively. TEA⁺ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK _d (0 mV)=5.6mm. A second site, with a higher affinity,K _d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA⁺ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca²⁺-activated K⁺ channels found in other tissues.     

Characterization of the 1,25-dihydroxycholecalciferol-stimulated calcium influx pathway in CaCo-2 cells

The present studies were conducted to investigate the mechanisms underlying the 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃)-induced increase in intracellular Ca²⁺ ([Ca²⁺] _i ) in individual CaCo-2 cells. In the presence of 2mm Ca²⁺, 1,25(OH)₂D₃-induced a rapid transient rise in [Ca²⁺] _i in Fura-2-loaded cells in a concentration-dependent manner, which decreased, but did not return to baseline levels. In Ca²⁺-free buffer, this hormone still induced a transient rise in [Ca²⁺] _i , although of lower magnitude, but [Ca²⁺] _i then subsequently fell to baseline. In addition, 1,25(OH)₂D₃ also rapidly induced⁴⁵Ca uptake by these cells, indicating that the sustained rise in [Ca²⁺] _i was due to Ca²⁺ entry. In Mn²⁺-containing solutions, 1,25(OH)₂D₃ increased the rate of Mn²⁺ influx which was temporally preceded by an increase in [Ca²⁺] _i . The sustained rise in [Ca²⁺] _i was inhibited in the presence of external La³⁺ (0.5mm). 1,25(OH)₂D₃ did not increase Ba²⁺ entry into the cells. Moreover, neither high external K⁺ (75mm), nor the addition of Bay K 8644 (1 μm), an L-type, voltage-dependent Ca²⁺ channel agonist, alone or in combination, were found to increase [Ca²⁺] _i , 1,25(OH)₂D₃ did, however, increase intracellular Na⁺ in the absence, but not in the presence of 2mm Ca²⁺, as assessed by the sodium-sensitive dye, sodium-binding benzofuran isophthalate. These data, therefore, indicate that CaCo-2 cells do not express L-type, voltage-dependent Ca²⁺ channels. 1,25(OH)₂D₃ does appear to activate a La³⁺-inhibitable, cation influx pathway in CaCo-2 cells.     

Ca2+-activated K+ conductance of the human red cell membrane: Voltage-dependent Na+ block of outward-going currents

Summary Human red cells were prepared with various cellular Na⁺ and K⁺ concentrations at a constant sum of 156mm. At maximal activation of the K⁺ conductance,g _K(Ca), the net efflux of K⁺ was determined as a function of the cellular Na⁺ and K⁺ concentrations and the membrane potential,V _m , at a fixed [K⁺]_ex of 3.5mm.V _m was only varied from (V _m –E _K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g _K(Ca) as a function of cellular Na⁺ and K⁺ concentrations atV _m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na⁺. Since the present Ca²⁺-activated K⁺ channels have been shown to be of the multi-ion type, the experimental data from each set of Na⁺ and K⁺ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na⁺ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na⁺ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca²⁺ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg _K(Ca) for outward currents at (V _m –E _K)>25 25 mV reflects a voltage-dependent Na⁺ block of the Ca²⁺-activated K⁺ channels.     

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     

Activation of K+ channels by lanthanum contributes to the block of transmitter release in chick and rat sympathetic neurons

Summary We studied the effects of lanthanum (La³⁺) on the release of ³H-norepinephrine(³H-NE), intracellular Ca²⁺ concentration, and voltage clamped Ca²⁺ and K⁺ currents in cultured sympathetic neurons. La³⁺ (0.1 to 10 m) produced concentration-dependent inhibition of depolarization induced Ca²⁺ influx and ³H-NE release. La³⁺ was more potent and more efficacious in blocking ³H-NE release than the Ca²⁺-channel blockers cadmium and verapamil, which never blocked more than 70% of the release. At 3 m, La³⁺ produced a complete block of the electrically stimulated rise in intracellular free Ca²⁺ ([Ca²⁺] _i ) in the cell body and the growth cone. The stimulation-evoked release of ³H-NE was also completely blocked by 3 m La³⁺. However, 3 m La³⁺ produced only a partial block of voltage clamped Ca²⁺ current (I _Ca). Following La³⁺ (10 m) treatment ³H-NE release could be evoked by high K⁺ stimulation of neurons which were refractory to electrical stimulation. La³⁺ (1 m) increased the hyperpolarization activated, 4-aminopyridine (4-AP) sensitive, transient K⁺ current (I _A ) with little effect on the late outward current elicited from depolarized holding potentials. We conclude that the effective block of electrically stimulated ³H-NE release is a result of the unique ability of La³⁺ to activate a stabilizing, outward K⁺ current at the same concentration that it blocks inward Ca²⁺ current.     

Ca2+-activated K+ currents inNecturus choroid plexus

Summary The tight-seal whole-cell recording method has been used to studyNecturus choroid plexus epithelium. A cell potential of –59±2 mV and a whole cell resistance of 56±6 M were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110mm NaCl Ringer solution and the interior of the cell contained a solution of 110mm KCl and 5nm Ca²⁺, stepping the membrane potential from a holding value of –50 to –10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca²⁺-activated K^_ channels. Based on the voltage dependence of the activation of Ca²⁺-activated K⁺ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K⁺ as their reversal potential at different K⁺ concentration gradients followed the Nernst potential for K⁺. These currents were reduced by the addition of TEA⁺ to the bath solution and were eliminated when Cs⁺ or Na⁺ replaced intracellular K⁺. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t _1/2) to the steady value. Increasing the pipette Ca²⁺ also decreased the delay and decreasedt _1/2. For instance, when pipette Ca²⁺ was increased from 5 to 500nm, the delay andt _1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K⁺ currents through Ca²⁺-activated K⁺ channels.At the resting membrane potential of –60 mV, Ca²⁺-activated K⁺ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20–30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca²⁺. Thus the Ca²⁺-activated K⁺ channels may play a specific role in maintaining intracellular K⁺ concentrations during CSF secretion.     

Comparative study of the effects of cromakalim (BRL 34915) and diazoxide on membrane potential, [Ca2+] i and ATP-sensitive potassium currents in insulin-secreting cells

Summary Patch-clamp and single cell [Ca²⁺] _i measurements have been used to investigate the effects of the potassium channel modulators cromakalim, diazoxide and tolbutamide on the insulin-secreting cell line RINm5F. In intact cells, with an average cellular transmembrane potential of –62±2 mV (n=42) and an average basal [Ca²⁺] _i of 102±6nm (n=37), glucose (2.5–10mm): (i) depolarized the membrane, through a decrease in the outward K_ATP current, (ii) evoked Ca²⁺ spike potentials, and (iii) caused a sharp rise in [Ca²⁺] _i . In the continued presence of glucose both cromakalim (100–200 m) and diazoxide (100 m) repolarized the membrane, terminated Ca²⁺ spike potentials and attenuated the secretagogue-induced rise in [Ca²⁺] _i . In whole cells (voltage-clamp records) and excised outside-out membrane patches, both cromakalim and diazoxide enhanced the current by opening ATP-sensitive K⁺ channels. Diazoxide was consistently found to be more potent than cromakalim. Tolbutamide, a specific inhibitor of ATP-sensitive K⁺ channels, reversed the effects of cromakalim on membrane potential and K_ATP currents.     

Characterization of the Increase in [Ca2+] i During Hypotonic Shock and the Involvement of Ca2+-activated K+ Channels in the Regulatory Volume Decrease in Human Osteoblast-like Cells

The calcium indicator fura-2 was used to study the effect of hypotonic solutions on the intracellular calcium concentration, [Ca²⁺] _i , in a human osteoblast-like cell line. Decreasing the tonicity of the extracellular solution to 50% leads to an increase in [Ca²⁺] _i from ∼150 nm up to 1.3 μm. This increase in [Ca²⁺] _i was mainly due to an influx of extracellular Ca²⁺ since removing of extracellular Ca²⁺ reduced this increase to ∼250 nm. After cell swelling most of the cells were able to regulate their volume to the initial level within 800 sec. The whole-cell recording mode of the patch-clamp technique was also used to study the effect of an increase in [Ca²⁺] _i on membrane currents in these cells. An increase in [Ca²⁺] _i revealed two types of Ca²⁺-activated K⁺ channels, K(Ca) channels. Current through both channel types could not be observed below voltage of +80 mV with [Ca²⁺] _i buffered to 100 nm or less. With patch-electrodes filled with solutions buffering [Ca²⁺] _i to 10 μm both channels types could be readily observed. The activation of the first type was apparently voltage-independent since current could be observed over the entire voltage range used from −160 to +100 mV. In addition, the current was also blocked by charybdotoxin (CTX). The second type of K(Ca) channels in these cells could be activated with depolarizations more positive than −40 mV from a holding potential of −80 mV. This type was blocked by CTX and paxilline. Adding paxilline to the extracellular solution inhibited regulatory volume decrease (RVD), but could not abolish RVD. We conclude that two K(Ca) channel types exist in human osteoblasts, an intermediate conductance K(Ca) channel and a MaxiK-like K(Ca) channel. MaxiK channels might get activated either directly or by an increase in [Ca²⁺] _i elicited through hypotonic solutions. In combination with the volume-regulated Cl⁻ conductance in the same cells this K⁺ channel seems to play a vital role in volume regulation in human osteoblasts. Received: 8 February 2000/Revised: 13 July 2000     

Effect of Calcium and Calcium Channel Blockers on Transient Outward Current of F76 and D1 Neuronal Soma Membranes in the Subesophageal Ganglia of Helix aspersa

Twin-electrode voltage-clamp techniques were used to study the effect of calcium and calcium channel blockers on the transient outward current in isolated F76 and D1 neurones of Helix aspersa subesophageal ganglia in vitro (soma only preparation with no cell processes). On lowering extracellular Ca²⁺ concentration from 10 to 2 mm or removing extracellular calcium from the bathing medium, the threshold for this current shifted in a negative direction by 11.5 and 20 mV, respectively. On the other hand, increasing the extracellular Ca²⁺ concentration from 10 to 20 and to 40 mm shifted the steady-state inactivation curves in positive directions on the voltage axis by 7 and 15 mV, respectively. Upon application of calcium channel blockers, Co²⁺, La³⁺, Ni²⁺ and Cd²⁺, transient potassium current amplitude was reduced in a voltage-dependent manner, being more effective at voltages close to the threshold. The current was elicited even at a holding potential of −34 mV. The specific calcium channel blockers, amiloride and nifedipine did not shift the activation and steady-state inactivation curves and did not reduce the transient outward current amplitude. It was concluded that the transient outward current is not dependent on intracellular Ca²⁺ but that it is modulated by Ca²⁺ and di- and trivalent ions extracellularly. The effects of these ions are very unlikely to be due to a surface charge effect because the addition of La³⁺ (200 μm) completely reverses the shift in a hyperpolarizing direction when the extracellular Ca²⁺ concentration was reduced from 10 to 1 mm and additionally shifts the kinetics further still in a depolarizing direction. The responses seen here are consistent with a specific effect of di- and trivalent ions on the transient outward current channels leading to a modification of gating. Received: 30 March 1999/Revised: 5 October 1999     

Regulation of maxi-K+ channels on pancreatic duct cells by cyclic AMP-dependent phosphorylation

Summary Using the patch-clamp technique we have identified a Ca²⁺-sensitive, voltage-dependent, maxi-K⁺ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of 200 pS in excised patches bathed in symmetrical 150mm K⁺, and was blocked by 1mm Ba²⁺. Channel openstate probability (P _o ) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted theP _o /voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration,P _o remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca²⁺ concentration from 2mm to 0.1 m. However, activated channels were still blocked by 1mm Ba²⁺. ChannelP _o was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase., We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K⁺ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca²⁺. Physiologically, these K⁺ channels may contribute to the basolateral K⁺ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.     

Reversible inhibition by lanthanum of the hydrosmotic response to serosal hypertonicity in toad urinary bladder

Summary In the urinary bladder of amphibia, hypertonicity of the serosal bath (SH) evokes an increase in transepithelial water permeability, the characteristics of which resemble the response to antidiuretic hormone (ADH). The ionic dependency, in particular for Ca²⁺, appears very similar forSH- and ADH-induced water fluxes. In the present experiments La³⁺ was used as a probe to study the Ca²⁺-dependency of the hydrosmotic response toSH in isolated urinary bladder of the toadBufo marinus.Addition of La³⁺ (5mm) on the serosal side of the membrane produced a significant and reversible increase in basal transepithelial water flux. The hydrosmotic response elicited by adding 250mm mannitol to the serosal Ringer's solution was inhibited by 30% in the absence of serosal Ca²⁺. Similarly, the hydrosmotic response toSH was inhibited by 37%, 30% and 40% when 5mm La³⁺ was added to the serosal medium 30 min before, concommitantly with, or 60 min after induction ofSH. The inhibition of transepithelial water flux observed in the absence of serosal Ca²⁺ or in the presence of serosal La³⁺ was reversible.The results support a critical role for Ca²⁺ in the modulation of transepithelial water permeability in the urinary bladder of amphibia. Ca²⁺ presumably exerts its effects at a post-cyclic AMP step.     

Studies on Electrogenesis under High K+ Concentrations in Internodal Cells of Chara corallina

+ concentration ([K⁺]_o) on the membrane potential (E_m) of Chara corallina was studied. E_m more negative than -100 mV was maintained even at 100 mM [K⁺]_o. Addition of Ca²⁺ to the external medium further increased this tendency. However, E_m responded sensitively to the increase in [K⁺]_o, when the electrogenic proton pump of the plasma membrane was inhibited by treating cells with dicyclohexylcarbodiimide, an inhibitor of proton pump. Analysis using equivalent circuit model of the plasma membrane suggested that the electrogenic proton pump was activated by the increase in [K⁺]_o. In the presence of 100 mM K⁺, action potentials were generated by electric stimuli. The ionic mechanism of generation of action potentials in the presence of K⁺ at high concentration was discussed. Received 3 October 2000/ Accepted in revised form 6 January 2001     

K+-Sensitive Gating of the K+ Outward Rectifier in Vicia Guard Cells

The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K⁺ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K⁺ current was monitored under voltage clamp in 0.1–30 mm K⁺ and in equivalent concentrations of Rb⁺, Cs⁺ and Na⁺. From a conditioning voltage of −200 mV, clamp steps to voltages between −150 and +50 mV in 0.1 mm K⁺ activated current through outward-rectifying K⁺ channels (I _{K, out}) at the plasma membrane in a voltage-dependent fashion. Increasing [K⁺] _o shifted the voltage-sensitivity of I _{K, out} in parallel with the equilibrium potential for K⁺ across the membrane. A similar effect of [K⁺] _o was evident in the kinetics of I _{K, out} activation and deactivation, as well as the steady-state conductance- (g _K ⁻) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near −130 mV in 0.1 mm K⁺, −80 mV in 1.0 mm K⁺, and −20 mV in 10 mm K⁺. Similar data were obtained with Rb⁺ and Cs⁺, but not with Na⁺, consistent with the relative efficacy of cation binding under equilibrium conditions (K⁺≥ Rb⁺ > Cs⁺ > > Na⁺). Changing Ca²⁺ or Mg²⁺ concentrations outside between 0.1 and 10 mm was without effect on the voltage-dependence of g _K or on I _{K, out} activation kinetics, although 10 mm [Ca²⁺] _o accelerated current deactivation at voltages negative of −75 mV. At any one voltage, increasing [K⁺] _o suppressed g _K completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K⁺ was sensitive to voltage, varying from 0.5 to 20 mm with clamp voltages near −100 to 0 mV, respectively. These, and additional data indicate that extracellular K⁺ acts as a ligand and alters the voltage-dependence of I _{K, out} gating; the results implicate K⁺-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of two K⁺ ions outside affects the distribution between closed states of the channel. Received: 27 November 1996/Revised: 4 March 1997     

ADH Action on Whole-Cell Currents by Cytosolic Ca2+-dependent Pathways in Aldosterone-treated A6 Cells

We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca²⁺ concentrations ([Ca²⁺] _c , 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca²⁺] _c , basal conductances mainly consisted of Cl⁻ conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca²⁺] _c to 2 nm abolished the basal Cl⁻ conductance. AVT evoked Cl⁻ conductances at 2 as well as 30 nm [Ca²⁺] _c . In addition to Cl⁻ conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca²⁺] _c but not at 2 nm [Ca²⁺] _c . Thus, cytosolic Ca²⁺ regulates NSC and Cl⁻ conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca²⁺] _c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells. Received: 6 May 1996/Revised: 28 June 1996     

Na+ - and Ca2+ -dependent components in action potentials of the ovulation hormone producing caudo-dorsal cells in Lymnaea stagnalis (Gastropoda)

Action potentials in the afterdischarge of the ovulation hormone producing caudo–dorsal cells (CDC) of Lymnaea stagnalis are strikingly different from electrically evoked spikes in the silent resting and inhibited states of these cells. Spikes evoked in the silent states consist of one fast peak (80–100 mV; 10–15 ms). The overshoot is Na⁺ - and Ca²⁺ -dependent. Spikes are blocked in Na⁺ -free saline and by TTX. Repolarization is retarded by TEA. Co²⁺ increases the overshoot. Active state action potentials (60–80 mV) last up to 125 ms, due to activation of a slow component following the TTX-sensitive spike. The slow component is Na⁺ - and Ca²⁺ -dependent. In normal saline it is blocked by Co²⁺ and La³⁺. In Ca²⁺ -free saline the remaining part of the slow component is blocked by La³⁺ only. The slow component is voltage-dependent in a graded fashion. Activation is bound to the active state in which the CDC are depolarized by 20 mV. TEA and Ca²⁺ -free saline greatly increase spike duration in the active state. This suggests that, in addition to the classical TEA-sensitive channel, a Ca²⁺ -dependent K⁺ channel is involved in repolarization of active state action potentials. The underlying membrane properties and the functional significance are discussed in relation to the pacemaking mechanism of the CDC.