Voltage-dependent, slowly activating K+ current (I Ks) and its augmentation by carbachol in rat pancreatic acini

Acetylcholine-stimulated exocrine secretion of Cl- and water requires the concomitant activation of K+ channels. However, there has not been much investigation of the carbachol- (CCH-) activated K+ channel of rodent pancreatic acini. Here, in a study of rat pancreatic acini, we characterize a voltage-dependent, slowly activating outward current (I(Ks)) that is augmented by CCH. Intact acini were obtained by enzymatic digestion and fast-whole-cell patch-clamp was applied. With symmetrical [Cl-] (32 mmol/l) in the pipette and bath solution, acinar cells had resting membrane voltages of -45+/-0.8 mV (n=97) under current-clamp conditions. CCH (10 micromol/l), which is known to activate Cl- channels via a Ca2+-mediated pathway, sharply depolarized the membrane to -4+/-0.5 mV, which was more negative than E(Cl) (0 mV), and reversed it to -41+/-0.9 mV (n=83) by washout. A clamp voltage of 0 mV activated I(Ks) under control conditions (91+/-8.6 pA, n=83). During CCH application an increase of outward current was observed at 0 mV, and at -50 mV a marked increase of inward Cl current occurred. In the presence of CCH the slow activation of I(Ks) was rarely distinguishable because of interference by the huge Cl- conductance. During CCH washout and decrease of inward current, a persistent augmentation of I(Ks) was revealed (486+/-36.3 pA, n=83). I(Ks) and its augmentation were abolished by substituting K+ in the pipette solution with Cs+. Augmentation of I(Ks) was mimicked by applying ionomycin (0.1 micromol/l), a Ca2+ ionophore. Pharmacological blockers were tested. The chromanol 293B and clotrimazole blocked I(Ks) at micromolar concentrations (IC50=3 micromol/l and 9 micromol/l, respectively) and Ba2+ was a poor blocker (IC50=3 mmol/l). In the presence of CCH (0.2 micromol/l), the membrane was depolarized to around -20 mV and the addition of 293B (10 micromol/l) further depolarized the membrane by 11+/-3 mV (n=5). These data suggest the presence of I(Ks) channels in rat pancreatic acini and their muscarinic activation.     

Effects of ATP and elevated K+ on K+ currents and intracellular Ca2+ in human microglia.

We have used whole-cell patch-clamp recordings and calcium microfluorescence measurements to study the effects of ATP and elevated external K+ on properties of human microglia. The application of ATP (at 0.1 mM) led to the activation of a transient inward non-selective cationic current at a cell holding potential of -60 mV and a delayed, transient expression of an outward K+ current activated with depolarizing steps applied from holding level. The ATP response included an increase in inward K+ conductance and a depolarizing shift in reversal potential as determined using a voltage ramp waveform applied from -120 to -50 mV. Fura-2 microspectrofluorescence measurements showed intracellular calcium to be increased following the application of ATP. This response was characterized by an initial transient phase, which persisted in Ca2+-free media and was due to release of Ca2+ from intracellular storage sites. The response had a later plateau phase, consistent with Ca2+ influx. In addition, ATP-induced changes in intracellular Ca2+ exhibited prominent desensitization. Elevated external K+ (at 40 mM) increased inward K+ conductance and shifted the reversal potential in the depolarizing direction, with no effect on outward K+ current or the level of internal Ca2+. The results of these experiments show the differential responses of human microglia to ATP and elevated K+, two putative factors associated with neuronal damage in the central nervous system.     

Activation of Ca(2+)-dependent K+ channel and Cl- conductance in canine pancreatic acinar cells through a cyclic AMP pathway.

Effects of adenosine 3',5'-cyclic monophosphate (cAMP) on Ca(2+)-dependent K+ channel and Cl- conductance in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp methods. In whole-cell current recordings on isolated cells dialyzed with K(+)-rich solution containing 0.5 mM EGTA, addition of 0.5 mM dibutyryl cAMP (dbcAMP), or 50 microM forskolin to the bath increased outward K+ and inward Cl- currents associated with depolarizing and hyperpolarizing voltage jumps, respectively. In intact cells (cell-attached configurations), addition of 0.5 mM dbcAMP or 50 microM forskolin to the bath increased the opening of single K+ channel. In Ca(2+)-free external solution (bath and pipette) 50 microM forskolin or 0.5 mM dbcAMP application evoked an increase in the opening of single K+ channel in intact cells. Addition of 0.5 mM dbcAMP to the bath solution containing 10 mM EGTA without Ca2+ increased the currents of whole-cell dialyzed with K(+)-rich solution containing 10 mM EGTA. When cell was dialyzed with 20 mM EGTA, dbcAMP, or forskolin application did not increase the whole-cell currents. In excised inside-out patches, addition of the catalytic subunit of cAMP-dependent protein kinase (16 U/ml) in the presence of 0.3 mM ATP to the cytoplasmic face of membrane activated the K+ channel, but 0.1 mM cAMP did not. These results suggest that cAMP-dependent phosphorylation can activate Ca(2+)-dependent K+ channels without increase in intracellular free Ca2+ and cAMP-dependent mechanism can activate Ca(2+)-dependent Cl- conductances without the increase in Ca2+ in canine pancreatic acinar cells.     

Background conductance attributable to spontaneous opening of muscarinic K+ channels in rabbit sino-atrial node cells.

Single myocytes were dissociated from the rabbit sino-atrial node, and the membrane background conductance produced by spontaneous opening of the muscarinic K+ channels was investigated by recording whole-cell and single channel currents in both normal K+ (5.4 mM) and high-K+ (145 mM) external solutions. Increasing external K+ concentration ([K+]o) from 5.4 to 145 mM induced a large inward shift of the whole-cell current accompanied by considerable current fluctuations at -50 mV. The high-K(+)-induced current was both K+ selective and voltage dependent, which was examined by varying [K+]o. This current was almost completely suppressed by 1-5 mM Ba2+ or 2-10 mM Cs+ and it was partly blocked by 10 microM atropine. In high-K+ (145 mM) solution, 20 nM acetylcholine (ACh) further increased the K+ conductance as well as the current noise. The power density spectrum of the noise was fitted with a sum of two Lorentzian functions. The corner frequencies of both the slow (approximately 5 Hz) and fast (approximately 120 Hz) components were comparable between the noise before and during the ACh application. Internal dialysis with a non-hydrolysable derivative of ATP, 5'-adenylylimido-diphosphate (AMP-PNP) or Mg(2+)-free solution markedly decreased both the amplitude and fluctuations of the high-K(+)-induced current. The relation between the variance of the current fluctuations and the mean current amplitude was linear in every experiment using dialysis of AMP-PNP or Mg(2+)-free internal solution, or using superfusion of ACh. The slopes of these relations gave comparable single channel current amplitudes of -0.7 pA at -50 mV. These results indicate that the spontaneous opening of the muscarinic K+ channels is largely responsible for the high-K(+)-induced current. In the high-K+ solution, the variance-mean relation at -50 mV showed that the muscarinic K+ channel provides an inward current of 3.12 +/- 2.13 pA pF-1 (n = 23), which was about 60% of the total inward background current. In the normal K+ solution, the variance-mean relation at -50 mV indicated that an outward current of 6.0 +/- 2.0 pA (0.33 +/- 0.28 pA pF-1, n = 8) was provided by the K+ channel. The single channel current amplitude was estimated to be 0.06 +/- 0.02 pA (n = 9). Cell-attached recordings in the absence of ACh demonstrated sporadic and brief openings of channels identical to the ACh-induced channels. The power density spectra of the single channel currents exhibited kinetic properties comparable with those of the whole-cell currents.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increase in chloride permeability of snail neurons during high potassium-induced hyperpolarization

High K+-induced hyperpolarization was recorded intracellularly from the snail neurons, Euhadra subnimbosa, and the ionic mechanism underlying this hyperpolarization was analyzed in comparison with the ACh-induced hyperpolarization of the same cell, the latter known to be Cl(-)-dependent. The membrane resistance always decreased during both hyperpolarizing responses to high K+ (24mM) and ACh (0.1 mM). Both hyperpolarizing responses to high K+ and ACh were reversed in Cl(-)-free Ringer to the depolarizing responses. Both hyperpolarizing responses to high K+ and ACh were markedly augmented immediately after returning to normal Cl- from Cl(-)-free Ringer perfusion. Increase in intracellular Cl(-)-concentration by a leak from KCl-electrode reversed both hyperpolarizing responses to high K+ and ACh. Reversal potential of high K+-response was always 10-20 mV more positive than that of ACh-response, when measured in normal Ringer perfusion. Intracellular Cl(-)-concentration of the cells which were hyperpolarized by high K+ was estimated to be one half of that of the cells which were depolarized by high K+. Above results indicated that the high K+-induced hyperpolarization is due to the permeability increase of the postsynaptic membrane toward Cl-, masking the depolarizing effect of high K+ on the same membrane.     

Permeability changes produced by L-glutamate at the excitatory post-synaptic membrane of the crayfish muscle.

1. Permeability changes produced by L-glutamate at the neuromuscular junction of the crayfish (Cambarus clarkii) were investigated by application of the drug iontophoretically to the voltage-clamped junction and measuring the resulting 'glutamate current'. 2. Reversal potentials were determined by measuring the glutamate current at different membrane potentials. They were +39-1 +/- 3-6 mV (mean +/- S.E. of mean) in normal solution and +16-5 +/- 2-0 mV in solutions made twice as hypertonic by the addition of sucrose. 3. Decreasing external Na+ concentration shifted the reversal potential in the negative direction; increased Na+ in the positive direction. 4. The relation between the amplitude of the glutamate current and extracellular Na+ concentration was approximately linear. 5. Alteration of the external K+ or Cl- concentration did not affect the amplitude or reversal potential of glutamate current. 6. In Na+-free solution the application of L-glutamate produced a small inward current at the resting potential and its amplitude was augmented by increasing the external Ca2+ concentration. 7. Increasing the Ca2+ concentration in the normal Na+ media produced no appreciable effect on the reversal potential but decreased the amplitude of glutamate current. 8. The results indicate that L-glutamate increases the membrane permeability mainly to Na+ and slightly to Ca2+. 9. The time course of glutamate current was shorter than that of the concentration calculated from the diffusion equation and it was simulated more closely by the square of the concentration.     

A slow calcium-dependent chloride conductance in clonal anterior pituitary cells

1. Whole cell voltage-clamp recordings were made from GH3 cells, a clonal cell line initially derived from a rat anterior pituitary tumor, using patch electrodes filled with CsCl or N-methylglucamine chloride (NMG Cl). The bathing medium contained tetraethylammonium chloride (TEA; 20 mM) and NaCl (120 mM) or NMG Cl (140 mM). These conditions resulted in substantial blockade of outward currents. 2. Depolarizing voltage steps from a holding potential of -50 mV activated transient (T-type) and sustained (L-type) inward Ca2+ currents. In addition, prolonged depolarization (greater than 1 s) invariably elicited a slowly activating inward current that persisted with maintained depolarization, and deactivated slowly on repolarization, resulting in a prominent inward tail current. 3. This tail current could be recorded under conditions where Ca2+ and Cl- were the only membrane-permeant ions (symmetrical NMG Cl). The tail current nulled near 0 mV with symmetrical Cl- and showed a negative reversal potential with nominally Cl--free internal solution. Ba2+ substituted for Ca2+ as a carrier of inward current, but no tail current was expressed. These observations indicate that Cl- is the charge carrier of the slow inward tail current. 4. The voltage dependence for activation of the slow tail current was U-shaped with a peak at approximately -10 mV. This closely paralleled the voltage dependency of the Ca2+ currents. Recordings with 5 mM internal ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) to buffer intracellular Ca2+ to low nM levels exhibited slow inward tail currents that were of lower peak amplitude than with the usual 1.1 mM EGTA-containing pipette solution, but the kinetics of the currents were similar in both cases. In addition, the slow tail current was eliminated on superfusion with the Ca2+ channel blocker Cd2+ or with Ca2+-free medium. These results demonstrate that the current is dependent on Ca2+ influx; it is, therefore, referred to as ICl(Ca). 5. Activation of ICl(Ca) required depolarization of at least 1 s. More prolonged depolarizations activated progressively greater current, to a maximum with 6-s depolarization. In most cases, the decay of the tail current was described by a single exponential function with time constant approximately 0.8-0.9 s within the potential range -80 to -30 mV. At more depolarized potentials the decay was slower (increasing e-fold/20-mV change in membrane potential). 6. In a high proportion of cells, ICl(Ca) rapidly diminished in amplitude on repeated activation. This "rundown" occurred more rapidly than the rundown of the Ca2+ currents.(ABSTRACT TRUNCATED AT 400 WORDS)     

Caffeine-induced depolarization in amphibian skeletal muscle fibres: role of Na+/Ca2+ exchange and K+ release.

Caffeine (4 mM) produces a depolarization of about 10 mV in frog muscle fibres (Leptodactylus ocellatus). The aim of this work was to study the mechanisms of this effect. An approximately threefold rise in membrane resistance [Cl--free (SO(4)2-) medium] substantially increased, and both Na+-free medium and Ni2+ (5 mM) reduced, the caffeine-induced depolarization. In voltage-clamped (-60 mV) short fibres from lumbricalis muscle of the toad (Buffo arenarum), caffeine generated an inward current of 4.13 +/- 0.48 microA cm(-2). This caffeine-induced current was reduced by 60% in Na+-free medium, 44% in the presence of 5 mM amiloride and 48% by 5 mM Ni2+, suggesting that the activation of the Na+-Ca2+ exchanger in its forward mode may play a role in the observed electrical effects of the drug. Caffeine also produced a marked release of K+. Net K+ efflux increased from 3.5 +/- 0.2 (control) to 22.1 +/- 2.3 pmol s(-1) cm(-2) (caffeine). It is shown that in the presence of the drug, [K+] in the lumen of the T tubules may well increase to levels which could produce, in part, both the observed depolarization and the caffeine-induced current under voltage clamp conditions. The caffeine-induced K+ efflux was not reduced by 5 mM Ni2+. At a holding potential of 30 mV the caffeine-induced current was reversed (outward) and roughly halved by 5 mM Ni2+. The Ni2+-sensitive fraction of the caffeine-induced current, assumed to represent the Na+-Ca2+ exchanger current, had an estimated reversal potential close to 12 mV ([Na+]o = 115 mM; [Ca2+]o = 1 mM). In conclusion, the depolarizing effect of caffeine described here would be produced by two mechanisms: (a) an inward current generated by the activation of the Na+-Ca2+ exchanger in its forward mode, and (b) the rise of the external [K+] in restricted spaces like the T tubules.     

Dual role for potassium in Balanus photoreceptor: antagonist of calcium and suppression of light-induced current.

1. The mechanism of reduction and final abolition of the depolarizing receptor potential of Balanus eburneus photoreceptors in K+-free saline was examined with electro-physiological techniques including voltage-clamp and ion specific electrodes. 2. An extended exposure to K+-free saline reduces the transient peak and the steady phases of the depolarizing receptor potential by approximately equal amounts. The process can be reversed in normal saline although the wave form of the response is often more rectangular upon recovery. Restoration of K+ induces a transient hyperpolarization of the resting membrane for several minutes. 3. The depolarizing receptor potential can also be restored in K+-free solution by reducing the Ca2+ concentration. This saline depolarizes the resting membrane, and the wave form of the depolarizing receptor potential assumes a rectangular configuration. 4. Voltage-clamp experiments revealed that an extended exposure to K+-free saline produced an extreme reduction of the inward light-induced current (LIC), but no detectable change in the membrane potential at which the current reverses sign. Membrane conductance in darkness showed little change. Reduction of the Ca2+ concentration from 20 to 0-2 mM in K+-free restored the current and produced a negative 8-10 mV shift in the zero current potential. There was also a significant decrease in membrane conductance in darkness. 5. Current-voltage relations of the membrane in K+-free, low Ca2+, or K+-free low Ca2+ salines were somewhat dependent upon the order the salines were presented. 6. Low Ca2+ saline (0-2 mM) by itself produced a -5 mV shift in the zero-current potential. Removing K+ in low Ca2+ produced an additional shift (-5 mV) in the zero-current potential.     

Sodium-activated potassium current in guinea pig gastric myocytes

This study was designed to identify and characterize Na+-activated K+ current (I(K(Na))) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+([K+]degrees). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68+/-2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steady-state current produced under [K+]degrees (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+- and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+-selective, since reversal potentials (E(rev)) of various concentrations of [K+]degrees-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 microM), a blocker of I(K(Na)), completely and reversibly inhibited this current. The characteristics of the current coincide with those of I(K(Na)) of other cells. Our results indicate the presence of I(K(Na)) in guinea pig gastric myocytes.     

Changes in glutamate-evoked currents of frog motoneurones by extracellular Mg2+

Responses of frog motoneurones to glutamate were studied using a single electrode voltage clamp method. At resting membrane potential glutamate evoked biphasic effects: firstly a transient outward current with reversal potential between -90 and -100 mV. Secondly an inward current with reversal potential near 0 mV and non-linearly related to the holding potential. Mg2+ (1 mM) had no effect on the outward current but reduced the inward current and its slope conductance at holding potentials more negative than -60 mV. These data indicate that the operation of the inward current activated by glutamate was partly but not solely controlled by extracellular Mg2+.     

Single channel analysis of the inward rectifier K current in the rabbit ventricular cells

The inward rectifier K channel in rabbit ventricular cells was studied by the patch-clamp method. Single channel currents were recorded in giga-sealed cell-attached patches with 150 mM K+ in the pipette. The slope conductance in the membrane potential range from -140 to -40 mV was 46.6 +/- 6.7 pS (mean +/- S.D., n = 16), and was reduced by decreasing [K+] in the pipette (20 or 50 mM). The channel was blocked by an application of Cs+ or Ba2+ (0.04-1 mM) in the pipette. Outwardly directed current, recorded with 50 mM K+ in the pipette, revealed the inward rectification of the single channel current. The probability of the channel being open was 0.33 +/- 0.05 (n = 10) at the resting potential (RP=-81.7 +/- 1.7 mV, n = 16) with 150 mM K+ in the pipette, and it decreased with hyperpolarization. The mean open time of the channel was 178 +/- 25 msec (n = 6) at RP. The closed time of the channel seemed to have two exponential components, with time constants of 11.0 +/- 2.0 msec and 1.92 +/- 0.52 sec (n = 6) at RP. The slower time constant was increased with hyperpolarization. The averaged patch current recorded upon hyperpolarizing pulses demonstrated a time-dependent current decay as expected from the single channel kinetics. The results indicated that the inward rectifier K+ current has time- and voltage-dependent kinetics.     

Antagonistic modulation of a hyperpolarization-activated Cl(-) current in Aplysia sensory neurons by SCP(B) and FMRFamide

Whole cell voltage-clamp recordings from Aplysia mechanosensory neurons obtained from the pleural ganglion were used to investigate the actions on membrane currents of the neuropeptides SCP(B) and FMRFamide. At the start of whole cell recording, SCP(B) typically evoked an inward current at a holding potential of -40 mV, due to the cAMP-mediated closure of the S-type K+ channel, whereas FMRFamide evoked an outward current, due to the opening of the S-type K+ channels mediated by 12-lipoxygenase metabolites of arachidonic acid. However, after several minutes of whole cell recording with a high concentration of chloride in the whole cell patch pipette solution, the responses to SCP(B) and FMRF-amide at -40 mV were inverted; SCP(B) evoked an outward current, whereas FMRFamide and YGGFMRFamide evoked inward currents. Ion substitution experiments and reversal potential measurements revealed that these responses were due to the opposing regulation of a Cl(-) current, whose magnitude was greatly enhanced by dialysis with the high Cl(-) - containing pipette solution. SCP(B) inhibited this Cl(-) current through production of cAMP and activation of PKA. YGGFMRFamide activated this Cl(-) current by stimulating a cGMP-activated phosphodiesterase that hydrolyzed cAMP. Thus a cAMP-dependent Cl(-) current undergoes antagonistic modulation by two neuropeptides in Aplysia sensory neurons.     

Study of a glucose-activated anion-selective channel in rat pancreatic beta-cells

Cell-attached channel recordings were made of an inward channel current in isolated rat pancreatic beta-cells incubated in the presence of diazoxide to clamp the membrane potential close to the K(+) equilibrium potential. With 42 mM Cl(-) in the pipette solution, a channel of approximately 200 pS was observed in 20-40% of patches which conducted an inward current at a pipette potential of 0 mV. The channel was activated by a rise in glucose concentration over the range 5-20 mM. The channel was also activated by methylglyoxal, possibly due to its metabolism to D-lactate, but not by the non-metabolizable glucose analogue 3- O-methyl glucose. The channel was activated by hypotonic cell swelling and was sensitive to inhibition by the anion channel blockers 4,4'-dithiocyanatostilbene-2,2'-disulphonic acid, 5-nitro-2-(3-phenylpropylamino) benzoic acid and 4-hydroxytamoxifen. Current reversal occurred at a pipette potential of approximately -67 mV. Raising [Cl(-)] in the pipette solution to 142 mM shifted the reversal potential to -52 mV. It is suggested that the channel is the volume-sensitive anion channel previously described in insulin-secreting cells. Activation of the channel by glucose could be important in generating a depolarizing current leading to increased electrical activity and insulin release, particularly at higher concentrations of glucose where K(ATP) channel activity is minimal.     

Patch-clamp study of the calcium-dependent chloride current in AtT-20 pituitary cells

1. Voltage-clamp recordings were made from cultured AtT-20 pituitary cells using the whole-cell patch-clamp technique. Cells were perfused internally with Cs+ to block K+ currents and bathed externally with either 1 microM tetrodotoxin or with tetraethylammonium (TEA) as a Na+ substitute to block voltage-activated Na+ currents. 2. Depolarizing voltage steps from a holding potential of -80 mV to potentials positive to -30 mV evoked two currents: a fast inward current that activated between -30 and +70 mV and a slowly activating current (designated "slow step current") that was inward between -30 and near 0 mV (the Cl- equilibrium potential) and outward positive to about 0 mV. Repolarization to -80 mV revealed a slowly decaying, inward tail current, whose magnitude with respect to step potential closely matched the current-voltage relationship of the voltage-activated Ca2+ current. 3. Activation of the fast inward current, slow step current, and tail current, was prevented by extracellular application of Cd2+ or removal of extracellular Ca2+. Replacement of extracellular Ca2+ with Ba2+ potentiated the fast inward current but blocked the slow step and tail currents. Intracellular perfusion with greater than 1 mM of the Ca2+ chelators ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) or [1,2-bis(2)aminophenoxy]ethane N,N,N',N'-tetraacetic acid (BAPTA) prevented activation of the slow step and tail currents, but not the fast inward current. 4. The reversal potential of the slow inward current was sensitive to changes in the Cl- equilibrium potential but not to substitution of TEA for Na+. The slow step current, but not the fast inward current, was partially blocked by the Cl- channel blocker, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. 5. These data indicate that both the slow inward tail current and the slowly activating, reversible step current were a Ca2+-dependent Cl- current, similar to that described in other neuronal and nonneuronal cell types. The fast inward current was a voltage-activated Ca2+ current, described previously in these and other cells. 6. In the absence of intracellular EGTA, the tail current decayed with complex kinetics, its time course apparently dependent on the magnitude of the voltage-activated Ca2+ current. In the presence of 200 microM intracellular EGTA, the tail current decayed significantly faster and often decayed exponentially.     

Calcium and voltage-dependent alterations of cell volume in neuroblastoma×glioma hybrid NG108-15 cells

Intracellular calcium ([Ca2+](i)), cell volume, membrane potential and currents were measured in neuroblastomaxglioma hybrid cells to gain insight into how [Ca2+](i) controls cell volume. [Ca2+](i) was increased by fluid shear stress, mechanical stimulation of the cells, the Ca2+ ionophore A23187, caffeine and thapsigargin. The increase in [Ca2+](i) induced by mechanical stimulation was decreased by about 50% by caffeine and abolished after incubation of the cells in a Ca2+-free solution. Mechanical stimulation by stirring the cell suspension induced cell shrinkage that was abolished by caffeine, but induced cell swelling in Ca2+-free solution. In the presence of caffeine, A23187 induced cell shrinkage whereas thapsigargin induced cell swelling. Both cell volume changes were inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid. The cells were hyperpolarized by fluid shear stress and A23187 and depolarized by caffeine, thapsigargin and intracellular EGTA. Under all these conditions, the membrane input resistance was decreased. Voltage-clamp experiments suggested that, in addition to an increased anionic current, fluid shear stress and A23187 increased a K+ current, whereas caffeine and intracellular Ca2+ chelation increased a non-selective cation current and thapsigargin increased both a K+ and a non-selective cation current. Taken together, these results suggest that, if cell volume is closely dependent on [Ca2+](i) and the activity of Cl- channels, its relative value is dependent on the ionic selectivity of co-activated channels and the membrane potential.     

Block by the neuropeptide TRH of an apparently novel K+ conductance of rat motoneurones

Using a single electrode voltage clamp technique the actions of rapidly superfused thyrotropin-releasing hormone (TRH, 1 microM) on lumbar motoneurones of the isolated neonatal rat spinal cord were investigated. TRH induced a slowly developing inward current (associated with an input conductance fall) with slow recovery on washout. In the presence of TRH the normally linear current-voltage relations displayed strong inward rectification up to about -40 mV. The TRH-induced current peaked at -50 mV, reversed at -120 mV and was not blocked by Cs+, tetraethylammonium, 4-aminopyridine, Cd2+, or low Na+. Its reversal potential was sensitive to changes in extracellular K+. Ba2+ (0.2-1.5 mM) depressed the effects of TRH. It is suggested that in rat motoneurones TRH blocked an apparently novel K+ conductance (IK(T)) active at resting membrane potential.     

1-EBIO stimulates Cl– secretion by activating a basolateral K+ channel in the mouse jejunum

We investigated the effects of 1-ethyl-2-benzimidazolinone (1-EBIO) on ion transport in the mouse jejunum through the use of the short-circuit (Isc) current technique and the application of the patch-clamp technique to isolated jejunal crypts. In HCO3- Ringer's, 1-EBIO stimulated a dose-dependent (EC50 964 micromol/l), bumetanide-sensitive increase in Isc consistent with stimulation of Cl- secretion. In contrast, in Cl(-)-free HCO3-Ringer's containing glucose, 1-EBIO (500 micromol/l) did not increase the phloridzin (100 micromol/l) sensitive Isc, suggesting that electrogenic Na+ absorption was unaltered. Measurement of the membrane potential (Vm) with the perforated-patch technique indicated that in isolated crypts, 1-EBIO caused a reversible hyperpolarization of Vm and an increase in the change in Vm associated with step changes in bath K+, consistent with an increase in K+ conductance. In on-cell patch experiments with KCI Ringer's in the patch pipette and crypts bathed with NaCl Ringer's, 1-EBIO (500 micromol/l) increased the open probability (NPo; 0.01+/-0.01 to 0.45+/-0.11, n=7) of an inwardly rectified intermediate conductance (g) channel. In inside-out patches with KCl Ringer's in the patch pipette and KCI Ringer's containing 100 nmol/l Ca2+ in the bath, the current-voltage relationship of the channel was inwardly rectified (g of 10 and 52 pS at -Vp of 100 and -100 mV, respectively) and reversed at 0 mV (n=5). Replacement of bath K+ with Na+ shifted the reversal potential toward the equilibrium potential for K+. In the presence of 1-EBIO, reducing the bath Ca2+ from 200 nmol/l to nominally Ca(2+)-free conditions decreased NPo from 0.90+/-0.27 to 0.07+/-0.03 (n=3). We conclude that in the mouse jejunum, I-EBIO does not stimulate electrogenic Na+ absorption. It does, however, stimulate secretion primarily through the activation of a basolateral, intermediate conductance Ca(2+)-sensitive K+ channel.     

Tachykinins modulate multiple ionic conductances in voltage-clamped rat spinal dorsal horn neurons

1. The membrane actions of substance P (SP) and a related tachykinin, neurokinin A (NKA), have been investigated by means of a single-electrode, voltage-clamp technique in the immature rat dorsal horn neurons using an in vitro spinal cord slice preparation. 2. When the membrane potential was held at the resting level of between -75 and -55 mV, bath application of SP or NKA (10(-7) to 10(-5) M, for 1-3 min) induced an inward shift in the holding current lasting several minutes. The magnitude of this effect varied between 10 and 400 pA depending on the concentration of the peptides and the holding potential. 3. When a dorsal horn neuron was held at the resting level and subjected to 1-s depolarizing commands to membrane potentials between -60 and -35 mV, slow inward relaxations and inward tail currents, the latter on repolarization to the holding potential, were recorded. During the tachykinin-induced inward shift in the holding current, the inward relaxation and the tail current were augmented in a dose-related manner. 4. The SP-induced augmentation of the slow inward relaxation and the inward tail current is likely to be due to the enhancement of the activation of the Ca2+ current, because the effect was present, and even augmented in a zero-Ca2+, Ba2+-containing solution, it was reduced or completely abolished by zero-Ca2+, Co2+-, or Mg2+-containing solutions and is largely independent of the changes in external Na+, K+, or Cl- ions. Moreover, in the presence of the K+-channel blocker, tetraethylammonium (TEA), the effect is increased. 5. Depolarizing voltage commands to potentials positive to -35 mV evoked a large, outward K+ current response in the dorsal horn neurons, which was in part Ca2+-sensitive. The outward current response was augmented by SP. The SP effect persists, although being reduced in a zero-Ca2+, Ba2+- or Co2+-containing solutions. 6. In a zero-Ca2+ solution containing Co2+ and TEA, the augmentation of the Ca2+ current and the outward K+ current by SP was abolished. However, the SP-induced increase in a Ca2+-sensitive, voltage-insensitive conductance remained, although being reduced, and the response showed a reversal at about -28 mV. This current may be a result of a tachykinin-activated nonspecific increase in cationic permeability of the membrane of dorsal horn neurons, because the current is reduced by more than one-half when Na+ or Ca2+ is removed from the bathing medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Parotid acinar cells: ionic dependence of acetylcholine-evoked membrane potential changes

Segments of mouse parotid were placed in a superfusion chamber. Surface acini were impaled by one or two micro-electrodes for measurement of membrane potential and resistance. The acinus under investigation was stimulated by micro-iontophoretic application of acetylcholine (ACh) or adrenaline.Neighbouring acinar cells were electrically coupled. Electrical coupling between acinar cells only occurred within restricted domains probably corresponding to an acinus or a group of acini.Passing direct current through one intracellular electrode, the resting potential of an acinus could be set at desired levels and the dependency of the ACh-evoked potential change on the resting potential investigated. The ACh null potential (initial effect) was about –60mV. A delayed hyperpolarizing effect of ACh could not be reversed.The initial ACh-evoked potential change was sensitive to alterations in extracellular Na, K and Cl concentration. The delayed ACh-evoked hyperpolarization was blocked by ouabain, exposure to Na-free or K-free solutions.It is concluded that ACh increases mainly K and Na membrane conductance causing K efflux and Na influx with a subsequent Na activation of an electrogenic Na pump.