A calcium-dependent chloride current in insulin-secreting βTC-3 cells

 Ca²⁺-dependent conductances have been hypothesized to play a role in the bursting pattern of electrical activity of insulin-secreting β cells in response to high plasma glucose. A Maxi K⁺ channel has received the most attention, while a low-conductance Ca²⁺-activated K⁺ current has also been identified. We used an increasingly popular β cell model system, the βTC-3 cell line, and the perforated-patch technique to describe the properties of a novel Ca²⁺-dependent Cl^–current [I _Cl(Ca)] in insulin-secreting pancreatic β cells. The reported I_Cl(Ca) could be activated under physiological Ca²⁺ concentrations and is the first of its kind to be described in pancreatic insulin-secreting cells. We found that long depolarizing steps above –20 mV elicited an outward current which showed slow inward relaxation upon repolarization to negative membrane potentials. Both the outward currents and the inward tails showed dependence on Ca²⁺ influx: their current/voltage (I/V) relations followed that of the ”L-like” Ca²⁺ current (I _Ca) present in these cells; they were blocked completely by the removal of external Ca²⁺ or application of Cd²⁺ at concentrations sufficient for complete block of I _Ca; and their magnitude increased with the depolarizing step duration. Moreover, the inward tail decayed monoexponentially with a time constant which at voltages negative to activation of I _Ca showed a weak linear voltage dependence, while at voltages positive to activation of I _Ca it followed the voltage dependence of I _Ca. This Ca²⁺-dependent current reversed at –21.5 mV and when the external Cl^–concentration was reduced from 159 mM to 62 mM the reversal potential shifted by ≈+20 mV as predicted by the Nernst relation for a Cl^–-selective current. Cl^–channel blockers such as DIDS (100 μM) and niflumic acid (100 μM) blocked this current. We concluded that this current was a Ca²⁺-dependent Cl^–current [I _Cl(Ca)]. From substitution of the external Cl^–with various monovalent anions and from the reversal potentials we obtained the following permeability sequence for I _Cl(Ca): I ^–>NO₃ ^–>Br^–>Cl^–>Acetate^–. Received: 10 October 1996 / Received after revision and accepted: 19 December 1996     

The sustained inward current in sino-atrial node cells of guinea-pig heart

 Single myocytes were dissociated from the sino-atrial (SA) node of guinea-pig hearts. Only a quite small fraction of the cell population showed spontaneous action potentials and these cells were characterized by the presence of the hyperpolarization-activated cation current I _f , the delayed rectifier K⁺ current I _K and the L-type Ca²⁺ current I _Ca,L as well as by the absence of both the transient outward current I _to and the inward rectifier K⁺ current I _K,1. After blocking I _f and I _K, depolarizing pulses from –80 mV revealed a large nicardipine-sensitive late current (NSLC). The NSLC was scarcely affected by decreasing extracellular [Ca²⁺] ([Ca²⁺]_o) from 1.8 to 0.1 mM, while it was decreased significantly by depleting [Na⁺]_o, differently from I _Ca,L. NSLC was blocked by nicardipine and was increased by Bay K 8644. NSLC was increased by isoprenaline and the additional application of acetylcholine reversed the increase of this current. We conclude that NSLC is largely composed of I _st described in the rabbit SA node pacemaker cells, and that I _st is unique for the pacemaker cells in mammalian SA node cells. Most of the quiescent cells showed neither I _f nor I _st. Received: 22 July 1996 / Received after revision: 30 September 1996 / Accepted: 9 October 1996     

Effect of vinpocetine on various types of high-threshold potassium currents in snail neurons

A high-threshold (−20 mV) K⁺ current was recorded from isolated edible snail neurons by a two-microelectrode voltage clamp technique. This current consisted of three components: fast-inactivating K⁺ currents (I_A), noninactivating K⁺ current (I_KD), and Ca²⁺-dependent K⁺ current (I_K(Ca)). Different cells had one to three components of K⁺ current. Vinpocetine increased I_A, moderately inhibited I_KD (by 30–50%) and strongly suppressed I_K(Ca) (by 60–90%). Inhibition of I_K(Ca) was not related to the effect of vinpocetine on the inward Ca²⁺ current. When K⁺ current consisted of all three components, the effect of vinpocetine on the ionic current amplitude was opposite at different potentials. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 10, pp. 408–411, October, 1998     

Exogenous lysophosphatidylcholine increases non-selective cation current in guinea-pig ventricular myocytes

Whole cell, patch-clamp studies were performed to examine the effect of lysophosphatidylcholine (LPC) on the membrane current in guinea-pig ventricular myocytes. The addition of 10 μM LPC to the external solution induced a membrane current which had a reversal potential of 0 mV. When Na⁺, the main cation in the external solution, was replaced by either K⁺, N-methyl-D-glucamine (NMG) or 90 mM Ca²⁺, LPC induced a current with the reversal potential near 0 mV, indicating that the current passed through a Ca²⁺-permeable non-selective cation channel. The order of the cationic permeability calculated from the reversal potential of the current was Cs⁺ > K⁺ > NMG > Na⁺ > Ca²⁺. Cl⁻ did not pass through the LPC-induced channel. The LPC-induced current was not blocked by Gd³⁺ in the external solution, nor by the absence of Ca²⁺ in the pipette solution. In conclusion, LPC induces a Ca²⁺-permeable non-selective cation channel in guinea-pig ventricular myocytes. Received: 11 September 1995/Received after revision: 3 January 1996/Accepted: 12 February 1996     

Activation of non-selective cation conductance by carbachol in freshly isolated bovine ciliary muscle cells

 In smooth muscle cells freshly isolated from the bovine ciliary body, effects of carbachol (CCh) on the membrane potential and current were examined by the whole-cell clamp method. The resting membrane potential of the muscle cells used was –60 ± 1 mV (n=111). Extracellular application of CCh (2 μM) depolarized the cells to –15 ± 5 mV (n=50) with an apparent increase in membrane conductance. Under voltage-clamp conditions, CCh (2 μM) evoked an inward current which exhibited inward-going rectification and reversed the polarity at about 0 mV. Removal of Na⁺ from the external solution caused a reduction of the amplitude of the current and a shift of the reversal potential to the negative direction. CCh was able to elicit an inward current even under a condition where Ca²⁺ was the only cation producing an inwardly directed electrochemical gradient. The current was not affected by verapamil or by tetrodotoxin. The CCh-induced current was inhibited by antimuscarinic agents with the affinity sequence: atropine ≈4–DAMP >> pirenzepine > AF-DX116, indicating that the response is mediated by a muscarinic cholinoceptor that belongs to the M₃-subtype. Unlike the non-selective cation channel current in intestinal smooth muscles, which is activated by elevation of the intracellular Ca²⁺ concentration ([Ca²⁺]_i), the current of the ciliary muscle was inactivated when the [Ca²⁺]_i was increased. The conductance, which admits Ca²⁺, may serve as a pathway for Ca²⁺ entry required for contraction. Received: 2 December 1996 / Received after revision: 7 January 1997 / Accepted: 8 January 1997     

Activation of Ca2+-sensitive Cl– current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

 We investigated how Ca²⁺-sensitive transient outward current, I _to(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na⁺ (Na⁺ _i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na⁺-free solutions,the application of nicardipine (5 μM) to block L-type Ca²⁺ current (I _Ca) completely inhibited I _to(Ca). In cells dialysed with a [Na⁺]_i≥5 mM, however, I _to(Ca) could be observed after blockade of I _Ca, indicating the activity of an I _Ca-independent component. The amplitude of I _Ca-independent I _to(Ca) increased with voltage in a [Na⁺]_i-dependent manner. The block of Ca²⁺ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked I _Ca-independent I _to(Ca). In Ca²⁺-free bath solution I _to(Ca) was completely abolished. The application of 2 mM Ni²⁺ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na⁺/Ca²⁺ exchange, or perfusion with pipette solution containing XIP (10 μM), a selective blocker of the exchanger, blocked I _Ca-independent I _to(Ca). From these results we conclude that, in the presence of Na⁺ _i, I _to(Ca) can be activated via Ca²⁺-induced Ca²⁺ release triggered by Na⁺/Ca²⁺ exchange operating in the reverse mode after blockade of I _Ca. Received: 20 January 1998 / Received after revision: 6 July 1998 / Accepted: 25 July 1998     

Effects of ruthenium red on membrane ionic currents in urinary bladder smooth muscle cells of the guinea-pig

 Three major ionic currents, Ca²⁺-dependent K⁺ current (I _K-Ca), delayed rectifier type K⁺ current (I _kd) and Ca²⁺ current (I _Ca), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary bladder. Externally applied ruthenium red (RuR) reduced the amplitude of I _K-Ca and I _Ca at 0 mV (IC₅₀ values were 4.2 and 5.6 μM, respectively), but did not affect I _Kd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (I _caf) at –30 mV were reduced by external 10 μM RuR. When 10 μM RuR was added to the pipette solution, I _K-Ca during depolarization, STOCs and I _caf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca²⁺-dependent K⁺ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic Ca²⁺ concentration was increased. These results indicate that RuR blocks BK and Ca²⁺ channels in urinary bladder smooth muscle cells. The decrease in I _K-Ca, STOCs and I _caf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca²⁺ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR with the Ca²⁺-binding sites of the channel protein. Received: 15 October 1997 / Received after revision and accepted: 25 November 1997     

Membrane currents in immature oocytes of the Rana perezi frog

 Immature oocytes of the Rana perezi frog were studied electrophysiologically to see if some of the unusual ionic channels found in Xenopus oocytes were also expressed in these cells. Growing oocytes showed a fairly linear current/voltage relationship (from –200 to +60 mV), whereas fully grown cells had several voltage-dependent conductances. Depolarizing pulses elicited a potassium current blocked by tetraethylammonium (TEA) and two kinetically different Ca²⁺-dependent Cl^–currents (I _Cl(Ca)), both sensitive to niflumic acid. I _Cl(Ca), which have not been previously observed in Rana immature oocytes, were also found in response to acetylcholine or rabbit serum superfusion or intracellular injection of Ca²⁺. In addition, three different Cl^–currents were activated in these cells by hyperpolarization: (1) a transient inward current dependent on a critical intracellular Ca²⁺ concentration; (2) an inward rectifier Cl^–current, which was Ca²⁺ independent; and (3) a high threshold (over –140 mV), slow Cl^–current, blocked by several divalent cations, 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanatostilbene-2,2′-disulphonic acid (SITS). The presence of most of these infrequent currents in immature oocytes of several frogs and toads suggests that they are not merely the result of random genomic expression but a programmed decision, probably related to a definite functional role. Received: 27 November 1996 / Received after revision and accepted: 9 April 1997     

Store depletion triggers the calcium release-activated calcium current (I CRAC) in macrovascular endothelial cells: a comparison with Jurkat and embryonic kidney cell lines

 In endothelial cells, different types of Ca²⁺ conductances have been described, but none of them has been clearly identified as I _CRAC, the Ca²⁺ release-activated Ca²⁺ current originally described in mast and lymphoma cells. Here we show that in bovine pulmonary artery endothelial cells (CPAE) depletion of intracellular Ca²⁺ stores by inositol 1,4,5-trisphosphate (InsP ₃), Ca²⁺ ionophores and Ca²⁺ pump inhibitors activates a Ca²⁺-selective conductance in the presence of the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The current shows inward rectification, a highly positive reversal potential and is blocked by micromolar concentrations of La³⁺. The conditions used in studies of endothelial cells were also employed in those of HEK-293, an embryonic kidney cell line commonly used to express putative store-operated channels, and Jurkat cells, the reference cell model. Similar to CPAE, HEK cells also have an I _CRAC-like current. At 0 mV holding potential the estimated current density is –0.1 and –0.2 pA/pF in CPAE and HEK cells respectively, i.e. 15 and 30% of that measured in Jurkat cells. As shown in studies of Jurkat cells, larger Na⁺ currents are detectable in CPAE and HEK cells following store depletion in Ca²⁺- and Mg²⁺-free medium. The current carried by Na⁺ ions is similarly blocked by micromolar La³⁺, is inwardly rectifying and has a positive reversal potential. Received: 12 November 1997 / Received after revision: 11 February 1998 / Accepted: 12 February 1998     

Endothelin depolarizes myocytes from porcine coronary and human mesenteric arteries through a Ca-activated chloride current

The effect of endothelin (ET) on membrane potential and current was studied in myocytes isolated from porcine coronary or from human mesenteric arteries at 3.6 mM extracellular Ca²⁺ concentration and 37° C. ET (1–100 nM) induced cell shortening and membrane depolarization from a resting potential of –50 mV to about –15 mV. Ca currents (I _Ca, L-type) were transiently reduced by ET. At –50 mV, ET induced an inward current that peaked within 2 s and fell within 10 s to a sustained level. The current could be enlarged by reducing bath extracellular Cl^– ion concentration, but removal of extracellular Na⁺ ions had no effect. The voltage dependence suggests that the ET-induced current is a Cl current (I _Cl) at potentials negative to –30 mV; at more positive potentials K currents (I _{K, Ca}) are superimposed. The effects of ET on I _Ca, I _Cl, I _{K, Ca} contraction were prevented by intracellular Ca chelators, suggesting a Ca-dependent activation mechanism. The ET effects were abolished by pretreatment with 20 mM caffeine or prior cell-dialysis with heparin [thought to block inositol triphosphate-induced sarcoplasmic reticular Ca release]. The results suggest that ET releases Ca from the SR through a phosphoinositol response and that the released Ca acts as second messenger in modulating the membrane currents.     

Comparison of Na+-Ca2+ exchange current elicited from isolated rabbit ventricular myocytes by voltage ramp and step protocols

 In this study, the effects of three different voltage protocols on the Na⁺-Ca²⁺ exchange current (I _Na-Ca) of rabbit right ventricular myocytes were studied. Whole-cell patch-clamp recordings were made using a Cs⁺-based internal dialysis solution and external solutions designed to block major interfering currents. I _Na-Ca was measured at 35–37°C as (5 mM) Ni-sensitive current elicited by: a 2 s descending ramp (DR: +80 to –120 mV); a 2 s ascending ramp (AR: –120 to +80 mV) and 500 ms voltage steps (VS) between –120 and +80 mV. DR and AR were applied from –40 mV and elicited I _Na-Ca with reversal potentials (E _rev) of –17.6±2.5 mV (mean±SEM; n=16) and –46.2±4.1 mV (n=10; P=0.0001) respectively. This difference was maintained when the holding potential was –80 mV (–44.0±2.1 mV, n=24 and –86.3±4.8 mV, n=10; P=0.0001), when the internal Ca chelator (EGTA) was replaced with BAPTA (–19.5±1.8 mV and –46.3±1.6 mV, n=6; P=0.0003) and when DR and AR were applied alternately to the same cell. Experiments using modified ramp waveforms suggested a possible mechanism for these differences. Increases in subsarcolemmal Ca caused by Ca entry (coupled to Na extrusion) during the initial positive potential phase of the DR might have induced I _Na-Ca reversal at less negative potentials than observed with AR, during the initial phase of which subsarcolemmal Ca would not have accumulated. These data suggest that I _Na-Ca during voltage-clamp experiments can be significantly influenced by the type of voltage protocol chosen, as the protocol appears to induce subsarcolemmal changes in Ca and Na concentration that are independent of Ca buffering in the bulk cytosol and can occur on a pulse-to-pulse basis. Received: 23 October 1998 / Received after revision: 8 January 1999 / Accepted: 11 January 1999     

A hyperpolarization-activated cation current (I h) contributes to resting membrane potential in rat superior cervical sympathetic neurones

 Using perforated-patch voltage-clamp recording, a prominent hyperpolarization-activated inward cation current (I _h) has been identified in dissociated, cultured and replated, superior cervical sympathetic (SCG) neurones from 17-day-old rats. I _h was identified as a slowly activated inward current on hyperpolarizing from –60 mV, with an extrapolated null potential (in 3 mM [K⁺]_out) of –42 mV. The activation range for I _h was –40 to –100 mV, with a half-activation voltage (V _0.5) of –63 mV. The current was suppressed by 1 mM Cs⁺ but not by 1 mM Ba²⁺. The reversal potential for the current change induced by Cs⁺ agreed with the null potential for I _h. I _h conferred strong inward rectification to the current-voltage curve negative to –55 mV in both voltage-clamp and current-clamp recording. This inward rectification was reduced by 1 mM Cs⁺. In a sample of eight cells with initial resting membrane potentials between –51 and –64 mV, Cs⁺ increased the resting potential of all cells by between 2.5 and 21 mV. These results indicate that I _h contributes a tonic inward (depolarizing) component to the maintenance of the resting membrane potential in SCG neurones. Received: 16 January 1998 / Received after revision and accepted: 1 April 1998     

NH4 + conductance in Xenopus laevis oocytes

 Current-clamp and voltage-clamp techniques were used to study the effects of NH₄ ⁺ on the cell membrane conductance in Xenopus laevis oocytes. Superfusing the oocytes with NH₄Cl resulted in a depolarization of the oocyte’s cell membrane potential and, at a clamp potential of –70 mV, in an inward current. The magnitude of the inward current was proportional to the NH₄Cl concentration in the extracellular solution and on membrane potential. The reversal potential, E _rev , was –35.5 ± 11.6 mV under control conditions and –3.1 ± 11.0 mV (n = 19) in the presence of NH₄Cl (10 mmol/l). Superfusion of the oocytes with nominally Ca²⁺-free solution affected the NH₄Cl-evoked response only marginally. Replacement of extracellular Na⁺ by N-methyl-D-glucamine⁺ markedly reduced, but did not eliminate, the NH₄Cl-sensitive current and shifted the reversal potential to more negative potentials. The NH₄Cl-induced current was substantially inhibited by 0.1 mmol/l flufenamate, and was less affected by blockers of the endogenous K⁺ conductance, Ba²⁺ and isosorbiddinitrate (ISDN). The results are compatible with the activation of a conductance by NH₄Cl for Na⁺ and NH₄ ⁺. The mechanism by which NH₄Cl activates the conductance remains unknown. Received: 9 December 1996 / Received after revision: 10 March 1997 / Accepted: 13 March 1997     

Properties of a calcium- and voltage-activated potassium current in Helix pomatia neurons

A calcium- and voltage-dependent current was found to be the principal outward current in identified Helix neurons. The current depends on the presence of [Ca²⁺]₀, with half maximal activation at 1 mM [Ca²⁺]₀, and it saturates beyond about 5 mM. The current is termed I_K(Ca) since the charge carried by it corresponds to the amount of potassium ions transferred from the cell interior, as determined from the increase in K⁺ concentration in the external volume with K⁺ liquid ion-exchanger microelectrodes. I_K(Ca) is characterized by bell shaped isochronal I/V curves. The peaks of these curves move from +30 mV to about +70 mV with an increase of the time of measurement from 30–200 ms. I_K(Ca) rise times have a minimum of 10–15 ms at low depolarization around 0 mV, but increase about exponentially with more positive potentials. A tenfold decrease in [Ca²⁺]₀ over the range of 30 to 0.3 mM also produces an increase in rise time, equivalent to a positive shift of potential by 20 mV. On repolarization of the membrane I_K(Ca) disappears much faster than the intracellularly accumulated Ca²⁺, with a time constant which is similar to the minimum activation time constant.     

Blockade of HERG channels expressed in Xenopus oocytes by external H+

 We have investigated the effect of external H⁺ concentration ([H⁺]_o)on the human-ether-a-go-go-related gene (HERG) current (I _HERG), the molecular equivalent of the cardiac delayed rectifier potassium current (I _Kr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H⁺]_o was increased, the amplitude of the I _HERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pH_o) values (the potential required for half-maximum activation, V _1/2, was: –41.8 mV, –38.0 mV, –33.7 mV, –26.7 mV in pH_o 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (g _max) was also affected by [H⁺]_o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pH_o from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca²⁺ concentration, which is also known to block HERG channels. When the extracellular Ca²⁺ concentration was increased from 0.5 mM to 5 mM, the shift in V _1/2 caused by increasing [H⁺]_o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in g _max caused by increasing [H⁺]_o was not significantly affected by changing external Ca²⁺ levels. The results indicate that HERG channels are inhibited by [H⁺]_o by two different mechanisms: voltage-dependent blockade (shift of V _1/2) and the decrease in g _max. With respect to the voltage-dependent blockade, the interaction between H⁺ and Ca²⁺ is competitive, whereas for the decreasing g _max, their interaction is non-competitive. Received: 12 January 1999 / Received after revision: 15 February 1999 / Accepted: 16 February 1999     

Electrophysiological study on the high K+ sensitivity of rat glomerulosa cells

 Elevation of extracellular potassium concentration by as little as some tenth of mM activates rat adrenal glomerulosa cells. In the present study some factors responsible for this high K⁺ sensitivity were examined. Using whole-cell voltage-clamp technique we found that both T-type and L-type voltage-dependent Ca²⁺ channels have very low threshold potential (–71 and –58 mV, resp.). By means of patch-clamp technique combined with single-cell fluorimetry we also provided evidence that the activation of I_gl, a K⁺-activated inward rectifying current is associated with Ca²⁺ influx. Both the low activation threshold of voltage-dependent Ca²⁺ channels and the function of I_gl contribute to the exceptional K⁺ sensitivity of the glomerulosa cells. Received: 30 September 1997 / Accepted: 4 November 1997     

Slow inactivation of muscle μ1 Na+ channels in permanently transfected mammalian cells

The slow inactivation of cloned muscle α-subunit Na⁺ channels was investigated using a Chinese hamster ovary cell line permanently transfected with rat muscle μ1 cDNA. Expression of μ1 Na⁺ channels was found in cells maintained for more than 6 months after transfection; > 70% of cells expressed ≥ 3 nA of Na⁺ current at +30 mV under whole-cell patch-clamp conditions. As expected, Na⁺ currents in these cells were blocked by tetrodotoxin as well as by μ-conotoxin. After prolonged depolarization (10 s at +30 mV) to inactivate voltage-gated Na⁺ channels, Na⁺ currents slowly reappeared over a time course of several minutes, during which time the cell was repolarized to the holding potential of −100 mV. This recovery from slow inactivation was best fitted by a double exponential function with τ₁ = 2.5 s (amplitude = 53%) and τ₂ = 83.4 s (amplitude = 38%). In contrast, the development of slow inactivation at +30 mV was best fitted by a single exponential function, with τ = 3.0 s. Steady-state slow inactivation (s _∞) had a midpoint potential (s ₀.₅) of −52 mV and a slope factor (k) of 7.8 mV. Elimination of fast inactivation by treatment with chloramine-T accelerated the development of slow inactivation significantly (by ≈four fold) but had little effect on recovery or on steady-state slow inactivation. Finally, as in cloned brain NaIIA Na⁺ channels, batrachotoxin abolished both fast and slow inactivation of μ1 Na⁺ channels. These results together suggest that slow inactivation takes place in the α-subunit of μ1 muscle Na⁺ channels and is governed by a μl protein region different from that governing fast inactivation. Received: 15 November 1995/Received after revision and accepted: 2 April 1996     

An increase in [Ca2+]i activates basolateral chloride channels and inhibits apical sodium channels in frog skin epithelium

 The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca²⁺]_i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca²⁺]_i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 μM) increased [Ca²⁺]_i from 66 ± 9 to 137 ± 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (I _sc) to drop from 26.4 to 10.6 μA cm^–2 (n = 19, P<0.001) concomitant with a depolarization of the cells from –79 ± 1 to –31 ± 2 mV (n = 18, P<0.001). Apical sodium permeability (P ^a _Na) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. P ^a _Na decreased from 8.01·10^–7 to 3.74·10^–7 cm s^–1 (n = 7, P = 0.04) following an increase in [Ca²⁺]_i. A decrease in apical sodium permeability per se would tend to decrease I _sc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca²⁺]_i. This was confirmed in wash-out experiments with ³⁶Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 ± 5.01 to 62.63 ± 13.3 nmol·min^–1 cm^–2 (n = 5, P = 0.04). We conclude that a small increase in [Ca²⁺]_i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption. Received: 25 June 1996 / Received after revision: 28 August 1996 / Accepted: 2 September 1996     

Sodium-sensitive, calcium-independent potassium conductance in the leech giant glial cell

 We have measured membrane current, membrane potential and intracellular Na⁺ and Ca²⁺ concentrations, [Na⁺]_i and [Ca²⁺]_i, of the giant glial cell in the nervous system of the leech Hirudo medicinalis using conventional microelectrodes and the fluorescent dyes sodium-binding benzofuran isophthalate (SBFI) and fura-2. When the Na⁺ was removed from the saline, the membrane conductance increased twofold from 1.29±0.1 μS to 2.57±0.18 μS (mean ± SEM; n=27). The rise in membrane conductance was accompanied by a current, which reversed around –74 mV, and the amplitude of K⁺-induced depolarizations or currents increased during Na⁺ removal, suggesting an increase in the K⁺ conductance of the glial membrane. We also monitored [Ca²⁺]_i when removing external Na⁺ in the presence and absence of external Ca²⁺, and during injection of the Ca²⁺-chelator BAPTA into the cells. Our results indicate that Na⁺ modulates a K⁺ conductance of these glial cells, independent of intra- and extracellular Ca²⁺. Received: 1 April 1998 / Received after revision and accepted: 22 May 1998     

Reversible inhibition of I K,I AHP,I h and I Ca currents by internally applied gluconate in rat hippocampal pyramidal neurones

 Previously, we reported that the spike frequency adaptation and slow afterhyperpolarizations (sAHP) in hippocampal pyramidal neurones are best preserved during whole-cell recording with a methylsulfate (MeSO₄ ^–)- based internal solution, but undergo a fast rundown when gluconate- (Gluc^–)- based internal solution is used. Here we show, with internal perfusion of patch pipettes, the reversibility of the inhibitory effects of Gluc^–on spike frequency adaptation and sAHP, and extend these observations to fast and medium-duration AHPs. Contrary to what might be expected based on Gluc^–binding of Ca²⁺, the sAHP and its underlying current could be temporarily enhanced by adding 1–3 mM of the calcium chelator BAPTA to the internal solution in the presence of Gluc^–. Replacement of internal MeSO₄ ^–with Gluc^–did not affect the membrane resting potential or the amplitude and duration of action potentials, but reversibly increased the cell input resistance and decreased the threshold current for spike generation. Gluc^–reversibly inhibited the hyperpolarization-activated non-selective cationic current (I _h), the depolarization-activated delayed rectifier K⁺ current (I _K), the high-voltage-activated Ca²⁺ current and the Ca²⁺-activated K⁺ current that underlies the sAHP. The combination of these effects of Gluc^–significantly alters the electrophysiological ”fingerprint” of the neurone. Received: 19 April 1996 / Received after revision: 12 July 1996 / Accepted: 3 September 1996