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     

Human Kir2.1 channel carries a transient outward potassium current with inward rectification

We have previously reported a depolarization-activated 4-aminopyridine-resistant transient outward K⁺ current with inward rectification (I _to.ir) in canine and guinea pig cardiac myocytes. However, molecular identity of this current is not clear. The present study was designed to investigate whether Kir2.1 channel carries this current in stably transfected human embryonic kidney (HEK) 293 cells using whole-cell patch-clamp technique. It was found that HEK 293 cells stably expressing human Kir2.1 gene had a transient outward current elicited by voltage steps positive to the membrane potential (around −70 mV). The current exhibited a current–voltage relationship with intermediate inward rectification and showed time-dependent inactivation and rapid recovery from inactivation. The half potential (V _0.5) of availability of the current was −49.4 ± 2.1 mV at 5 mM K⁺ in bath solution. Action potential waveform clamp revealed two components of outward currents; one was immediately elicited and then rapidly inactivated during depolarization, and another was slowly activated during repolarization of action potential. These properties were similar to those of I _to.ir observed previously in native cardiac myocytes. Interestingly, inactivation of the I _to.ir was strongly slowed by increasing intracellular free Mg²⁺ (Mg²⁺ _i , from 0.03 to 1.0, 4.0, and 8.0 mM). The component elicited by action potential depolarization increased with the elevation of Mg²⁺ _i . Inclusion of spermine (100 μM) in the pipette solution remarkably inhibited both the I _to.ir and steady-state current. These results demonstrate that the Mg²⁺ _i -dependent current carried by Kir2.1 likely is the molecular identity of I _to.ir observed previously in cardiac myocytes.     

The effects of over-expression of the FK506-binding protein FKBP12.6 on K+ currents in adult rabbit ventricular myocytes

This study examines the effects of the intracellular protein FKBP12.6 on action potential and associated K⁺ currents in isolated adult rabbit ventricular cardiomyocytes. FKBP12.6 was over-expressed by ~6 times using a recombinant adenovirus coding for human FKBP12.6. This over-expression caused prolongation of action potential duration (APD) by ~30%. The amplitude of the transient outward current (I _to) was unchanged, but rate of inactivation at potentials positive to +40 mV was increased. FKBP12.6 over-expression decreased the amplitude of the inward rectifier current (I _K1) by ~25% in the voltage range −70 to −30 mV, an effect prevented by FK506 or lowering intracellular [Ca²⁺] below 1 nM. Over-expression of an FKBP12.6 mutant, which cannot bind calcineurin, prolonged APD and affected I _to and I _K1 in a similar manner to wild-type protein. These data suggest that FKBP12.6 can modulate APD via changes in I _K1 independently of calcineurin binding, suggesting that FKBP12.6 may affect APD by direct interaction with I _K1.     

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     

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     

Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.     

Distal colonic Na+ absorption inhibited by luminal P2Y2 receptors

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na⁺ channel (ENaC)-mediated Na⁺ absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y₂ and a luminal P2Y₄ receptor, stimulate K⁺ secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na⁺ absorption in distal colonic mucosa of mice treated on a low Na⁺ diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V _te): −13.7 ± 1.9 mV (lumen negative), transepithelial resistance (R _te): 24.1 ± 1.8 Ω cm², equivalent short circuit current (I _sc): −563.9 ± 63.8 μA/cm² (n = 21). Amiloride completely inhibited I _sc to −0.5 ± 8.5 μA/cm². Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I _sc by 160.7 ± 29.7 μA/cm² (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC₅₀ values of 10 μM and 3 μM respectively. In P2Y₂ knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na⁺ absorption was absent. In contrast, in P2Y₄ KO mice the inhibitory effect of luminal UTP on Na⁺ absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na⁺ diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y₂ and P2Y₄ receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y₂ receptor mediates inhibition of electrogenic Na⁺ absorption.     

Thermodynamic properties of hyperpolarization-activated current (Ih) in a subgroup of primary sensory neurons

Ih is a poorly selective cation current that activates upon hyperpolarization, present in various types of neurons. Our aim was to perform a detailed thermodynamic analysis of Ih gating kinetics, in order to assess putative structural changes associated with its activation and deactivation. To select dorsal root ganglia neurons that exhibit large Ih, we applied a current signature method by Petruska et al. (J Neurophysiol 84:2365–2379, 2000) and found appropriate neurons in cluster 4. Currents elicited by 3,000-ms hyperpolarizing pulses at 25 and 33°C were fitted with double exponential functions, yielding time constants similar to those of HCN1. The fast activation and deactivation rates showed temperature coefficients (Q ₁₀) of 2.9 and 3.1, respectively, while Q ₁₀ of the absolute conductance was 1.3. Using the Arrhenius–Eyring formalism we computed heights of voltage-independent Gibbs free energy and entropy barriers for each rate. The free energy barriers of the fast rates were just ∼2RT units lower than those of the corresponding slow rates (31.3 vs. 33.2RT for activation, and 24.7 vs. 25.8RT for deactivation, at 25°C). Interestingly, the entropy barriers of the slow rates were negative: −15.2R units for activation and −11.9R units for deactivation, compared to 4.6 and 1.3R units, respectively, for the fast component. The equivalent gating charge (z _g) (3.75 ± 0.32, mean ± SEM, at 25°C) and half-activation potential (V _1/2) (−70.0 ± 1.3 mV at 25°C) did not vary significantly with temperature.     

Effect of stretch on calcium channel currents recorded from the antral circular myocytes of guinea-pig stomach

The effect of membrane stretch on voltage-activated Ba²⁺ current (I _Ba) was studied in antral circular myocytes of guinea-pig using the whole- cell patch-clamp technique. The changes in cell volume were elicited by superfusing the myocytes with anisosmotic solutions. Hyposmotic superfusate (202 mosmol/l) induced cell swelling and increased peak values of I _Ba at 0 mV (from −406.6 ± 45.5 pA to −547.5 ± 65.6 pA, mean ± SEM, n = 8) and hyperosmotic superfusate (350 mosmol/l) induced cell shrinkage and decreased peak values of I _Ba at 0 mV (to −269.5 ± 39.1 pA, n = 8). Such changes were reversible and the extent of change was dependent on the osmolarity of superfusate. The values of normalized I _Ba at 0 mV were 1.43 ± 0.04, 1.30 ± 0.06, 1.23 ± 0.04, 1.19 ± 0.04, 1 and 0.68 ± 0.06 at 202, 220, 245, 267, 290 and 350 mosmol/l, respectively (n = 8). I _Ba was almost completely blocked by nicardipine (5 μM) under hyposmotic conditions. The values of steady-state half-inactivation voltage (−37.7 ± 3.3 and −36.5 ± 2.6 mV, under control and hyposmotic conditions, respectively) or the half-activation voltage (−13.6 ± 2.3 and −13.9 ± 1.9 mV) of I _Ba were not significantly changed (P > 0.05, n = 6). Cell membrane capacitance was slightly increased from 50.00 ± 2.86 pF to 50.22 ± 2.82 pF by a hyposmotic superfusate (P < 0.05, n = 6). It is suggested that cell swelling increases voltage-operated L-type calcium channel current and that such a property is related to the response of gastric smooth muscle to mechanical stimuli. Received: 14 November 1995/Received after revision and accepted: 8 January 1996     

Delayed onset and slow time course of the non-M-type muscarinic current in bullfrog sympathetic neurons

The onset and time course of the muscarinic currents induced by brief applications of acetylcholine (ACh) were examined in voltage-clamped neurons of bullfrog sympathetic ganglia bathed in a solution containing d-tubocurarine. At a potential of –40 mV, the ACh-induced current (I _ACh) appeared within 1.2 s and rapidly increased to its peak with a half-activation time of 2.2 s. This initial current was termed the fastI _ACh and was blocked by 4 mM Ba²⁺. At a potential more negative than –60 mV, the fastI _ACh disappeared and the remainingI _ACh activated with a delay of 3.9 s and slowly increased to its peak with a half-activation time of 8.2 s. This delayed current was termed the slowI _ACh and is thought to be associated with inhibition of a K⁺ current, orI _M, as well as activation of an inward current through non-M-type muscarinic cation channels. The slowI _ACh was not inhibited by Ba²⁺, but its amplitude was reduced with depolarization (the extra-polated reversal potential was +3 mV). In Na⁺-free solution, the amplitude of the slowI _ACh reduced, but its polarity did not reverse in the voltage region examined (-30 to –100 mV). The slow excitatory postsynaptic current was also recorded, and was shown to have a similar delay in onset and slow time course. The results demonstrate that ACh activates the non-M-type muscarinic current three times more slowly than it inhibitsI _M.     

Effects of antidiuretic hormone,parathyroid hormone and glucagon on transepithelial voltage and resistance of the cortical and medullary thick ascending limb of Henle's loop of the mouse nephron

The effect of antidiuretic hormone (arginine vasopressin, AVP, 10⁻¹⁰mol.l⁻¹), parathyroid hormone (PTH, 10⁻⁸ mol.l⁻⁸) and glucagon (10⁻⁸ mol.l⁻¹) on the transepithelial potential difference (PD_te) and the transepithelial resistance (R_te) were tested in in vitro perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. When compared with mTAL segments (PD_te: 8.5±0.4 mV,n=16), cTAL segments displayed a high PD_te of 15.7±0.9 mV (n=11) at the beginning of perfusion experiments which reached a value of 9.4±0.6 mV (n =11) after 38±4 min perfusion. Simultaneously R_te increased significantly from 24±3 to 28±1 Ω cm² (n=11). When PTH, AVP or glucagon were added to the bath solution, PD_te increased with PTH from 10.3±0.8 to 15.2±0.8 mV (n=13), with AVP from 10.2±0.5 to 15.0±0.7 mV (n=24) and with glucagon from 11.3±1.9 to 15.3±2.1 mV (n=8). At the same time R_te decreased from 30±3 to 23±2 Ω cm², from 28±1 to 23±1 Ω cm² and from 23±2 to 18±2 Ω cm², respectively. In mTAL segments, AVP and glucagon increased PD_te from 8.4+0.5 to 13.5±0.9 mV (n=11) and from 8.8±0.6 to 12.8±0.6 mV (n=8) respectively, while R_te decreased significantly from 23±1 to 20±1 Ω cm² and from 27±3 to 21±3 Ω cm². PTH, on the other hand, had no effect on PD_te and R_te. Since the response to PTH appeared to be specific to cTAL segments, paired experiments were performed, in which AVP or glucagon were successively tested with PTH on cTAL and mTAL segments, to ascertain the specificity of the hormonal response. In cTAL segments, PTH and AVP increased the equivalent short-circuit current (I_sc=PD_te/R_te) by 82% and 86% respectively, while PTH and glucagon, in another series, increased I_sc by 95% and 81% respectively. In mTAL segments, I_sc was increased in the presence of AVP and glucagon by 88%, and 93% respectively, whereas PTH had no effect. These results indicate that Nacl reabsorption in cTAL segments is stimulated by AVP, PTH and glucagon and in mTAL segments by AVP and glucagon. The amplitude of the response to the hormones is similar in the two segments. The residual stimulation in cTAL segments, however, persists longer than in mTAL segments.     

The Plasmodium falciparum-induced anion channel of human erythrocytes is an ATP-release pathway

Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in the host erythrocyte membrane involving ATP release and autocrine purinergic signaling. P. falciparum-parasitized but not unstimulated uninfected erythrocytes released ATP in a 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 7 μM)-sensitive and serum album (SA; 0.5% w/v)-stimulated manner. Since Plasmodium infection of human erythrocytes induces SA-dependent outwardly (OR) and SA-independent inwardly rectifying (IR) anion conductances, we tested whether the infection-induced OR channels directly generate an ATP release pathway. P. falciparum-parasitized erythrocytes were recorded in whole-cell mode with either Cl⁻ or ATP as the only anion in the bath or pipette. In parasitized cells with predominant OR activity, replacement of bath NaCl by Na–ATP (NMDG–Cl pipette solution) shifted the current reversal potential (V _rev) from −2 ± 1 to +51 ± 3 mV (n = 15). In cells with predominant IR activity, in contrast, the same maneuver induced a shift of V _rev to significantly larger (p ≤ 0.05, two-tailed t test) values (from −3 ± 1 to +66 ± 8 mV; n = 5) and an almost complete inhibition of outward current. The anion channel blocker NPPB reversibly decreased the ATP-generated OR currents from 1.1 ± 0.1 nS to 0.2 ± 0.05 nS and further shifted V _rev to +87 ± 7 mV (n = 12). The NPPB-sensitive fraction of the OR reversed at +48 ± 4 mV suggesting a relative permeability of P _ATP/P _Cl ≈ 0.01. Together, these data raise the possibility that the OR might be the electrophysiological correlate of an erythrocyte ATP release pathway. Canan Akkaya and Ekaterina Shumilina contributed equally to this work and, thus, share first authorship.     

Modulation of the transient outward K+ current by inhibition of endothelin-A receptors in normal and hypertrophied rat hearts

Inhibition of endothelin-A (ET_A) receptors has been shown to reduce ventricular electrical abnormalities associated with cardiac failure. In this study, we investigate the effect of ET_A-receptor inhibition on the development of regional alterations of the transient outward K⁺ current (I _to) in the setting of pressure-induced left ventricular (LV) hypertrophy. Cardiac hypertrophy was induced in female Sprague–Dawley rats by stenosis of the ascending aorta (AS) for 7 days. Treatment with the selective ET_A-receptor antagonist darusentan (LU135252, 35 mg [kg body weight]⁻¹ day⁻¹) was started 1 day before the surgery. AS induced a 46% increase in the relative LV weight (p < 0.001) and caused a significant reduction in I _to (at +40 mV) in epicardial myocytes (19.5 ± 1.2 pA pF⁻¹, n = 32 vs 23.2 ± 1.2 pA pF⁻¹, n = 35, p < 0.05). Darusentan further reduced I _to in AS (15.4 ± 1.3 pA pF⁻¹, n = 37, p < 0.05) and sham-operated animals (19.8 ± 1.6 pA pF⁻¹, n = 48, ns.). The effects of AS and darusentan on I _to were significant and independent as tested by two-way analysis of variance. I _to was not affected in endocardial myocytes. These results indicate that endothelin-1 may exert a tonic effect on the magnitude of I _to in the epicardial region of the left ventricle but that ET_A-receptor activation is not necessary for the development of electrical alterations associated with pressure-induced hypertrophy.     

Evidence for electrogenic sodium-bicarbonate cotransport in cultured rat cerebellar astrocytes

We have studied the regulation of intracellular pH (pH_i), and HCO ₃ ⁻ -dependent membrane currents in cultured astrocytes from neonatal rat cerebellum, using the fluorescent pH-sensitive dye 2,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and the whole-cell patch-clamp technique. The steady-state pH_i was 6.96 in both nominally CO₂/HCO ₃ ⁻ -free, HEPES-buffered saline (6.96 ±0.14;n=48) and in a saline containing 5% CO₂/24 mM HCO ₃ ⁻ (6.96±0.18;n=48) (at pH 7.4). Inhibition of the Na⁺/H⁺ exchange by amiloride (2 mM) caused a significant decrease of pH_i in nominally CO₂/HCO ₃ ⁻ -free saline. Addition of CO₂/HCO ₃ ⁻ in the continuous presence of amiloride induced a large and fast intracellular alkalinization. Removal of external Na⁺ also caused a fall of pH_i, and addition of CO₂/HCO ₃ ⁻ in Na⁺-free saline evoked a further fall of pH_i, while the outward current was reduced or even reversed. The stilbene 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS, 0.3 mM) reduced the pH_i recovery from the CO₂/HCO ₃ ⁻ -evoked acidification, and blocked the prominent intracellular acidification upon removal of CO₂/HCO ₃ ⁻ . Removal of external Cl⁻ had little effect on these pH_i changes. Lowering the external pH from 7.4 to 6.6 in CO₂/HCO ₃ ⁻ -containing saline produced a large and rapid intracellular acidification and inward current, which were both greatly reduced by DIDS and in the absence of CO₂/HCO ₃ ⁻ . The results suggest that the CO₂/HCO ₃ ⁻ -dependent current is partly due to a reversible bidirectional, electrogenic Na⁺-HCO ₃ ⁻ cotransporter, which helps to regulate pH_i in these cells. In addition, a prominent Na⁺/H⁺ exchanger contributes to extrude acid equivalents from these astrocytes to maintain the steadystate pH_i.     

Membrane currents and cytoplasmic sodium transients generated by glutamate transport in Bergmann glial cells

Effects of glutamate and kainate (KA) on Bergmann glial cells were investigated in mouse cerebellar slices using the whole-cell configuration of the patch-clamp technique combined with SBFI-based Na⁺ microfluorimetry. l-Glutamate (1 mM) and KA (100 μM) induced inward currents in Bergmann glial cells voltage-clamped at −70 mV. These currents were accompanied by an increase in intracellular Na⁺ concentration ([Na⁺]_i) from the average resting level of 5.2 ± 0.5 mM to 26 ± 5 mM and 33 ± 7 mM, respectively. KA-evoked signals (1) were completely blocked in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM), an antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/KA ionotropic glutamate receptors; (2) reversed at 0 mV, and (3) disappeared in Na⁺-free, N-methyl-D-glucamine (NMDG⁺)-containing solution, but remained almost unchanged in Na⁺-free, Li⁺-containing solution. Conversely, l-glutamate-induced signals (1) were marginally CNQX sensitive (∼10% inhibition), (2) did not reverse at a holding potential of +20 mV, (3) were markedly suppressed by Na⁺ substitution with both NMDG⁺ and Li⁺, and (4) were inhibited by d,l-threo-β-benzyloxyaspartate. Further, d-glutamate, l-, and d-aspartate were also able to induce Na⁺-dependent inward current. Stimulation of parallel fibres triggered inward currents and [Na⁺]_i transients that were insensitive to CNQX and MK-801; hence, we suggested that synaptically released glutamate activates glutamate/Na⁺ transporter in Bergmann glial cells, which produces a substantial increase in intracellular Na+ concentration.     

NH4 + conductance in Xenopus laevis oocytes

 Superfusing Xenopus laevis oocytes with NH₄Cl (10 mmol/l, pH 7.5) resulted in an inward current at a clamp potential of –70 mV. In paired experiments (n=22), the NH₄Cl-induced peak current was –293±94 nA, under control conditions (osmolality: 240 mosmol/kg), and rose to –523±196 nA when osmolality was reduced to 144 mosmol/kg. In parallel with the rise in NH₄Cl-induced inward current, membrane conductance at –70 mV doubled and the zero-current potential changed from +3.3±9.4 mV to –22.0±8.0 mV (n=22) in the presence of NH₄Cl during exposure to a hypoosmolar solution. In the absence of NH₄Cl, oocytes responded to hypoosmolality with a shift in zero-current potential to more negative values and an increased conductance which became partially sensitive to isosorbiddinitrate (ISDN), suggesting the activation of a volume-sensitive K⁺ channel. Membrane conductance in the presence of NH₄Cl was decreased by ISDN to similar extents under isoosmolal and hypoosmolal conditions, indicating that NH₄ ⁺ enters the oocytes through a volume-sensitive conductance separate from the ISDN-sensitive K⁺ channel. Received: 20 July 1998 / Received after revision and accepted: 19 October 1998     

Actions of myristyl-FRCRCFa, a cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchanger, tested in rabbit ventricular myocytes

 In cardiac muscle, the electrogenic Na-Ca exchanger plays important roles in determining action potential shape and in the beat-to-beat homeostasis of intracellular calcium. In this study we tested the actions of a putative cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchange, ”Myristyl- (Myr-) FRCRCFa”. Experiments were performed using isolated rabbit right ventricular myocytes and whole-cell patch-clamp at 35–37°C. The Na-Ca exchange current (I _Na-Ca), L-type calcium current (I _Ca,L), inward rectifier potassium current (I _K1) and delayed rectifier potassium current (I _K) were compared in untreated cells and cells incubated in a solution containing N-myristylated FRCRCFa. With other major currents blocked, I _Na-Ca was measured as the Ni-sensitive component of current during a voltage ramp applied from the holding potential of –40 mV, between +80 and –120 mV (ramp velocity 0.1 V s^–1). In untreated cells, I _Na-Ca at +60 mV was 7.1±0.6 pA/pF and at –100 mV was –2.7±0.3 pA/pF (n=9). After a 15-min pre-incubation with 20 μM Myr-FRCRCFa, I _Na-Ca was reduced to 4.2±0.3 pA/pF at +60 mV and –1.5±0.2 pA/pF at –100 mV (P<0.02; n=7). After incubation with 20 μM Myr-FRCRCFa for 1 h, I _Na-Ca at both potentials was further reduced (2.3±0.8 pA/pF at +60 mV; –0.9±0.3 pA/pF at –100 mV; P<0.008 compared with control; n=4). Under selective recording conditions for I _Ca,L, there was little difference in I _Ca,L density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the I _Ca,L/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. I _K1 was also largely unaffected by pre-incubation of cells with Myr-FRCRCFa. I _K, measured as deactivating tail current following 1-s test depolarisations to a range of test potentials, was also not significantly altered by Myr-FRCRCFa. The suppression of I _Na-Ca in cells incubated in Myr-FRCRCFa suggests that addition of the myristyl group to FRCRCFa peptide conveys cell permeancy to the peptide and that Myr-FRCRCFa applied externally to rabbit ventricular myocytes is moderately effective as an I _Na-Ca blocker. I _Ca,L, I _K1 and I _K were largely unaffected by Myr-FRCRCFa. N-Myristylation of such conformationally constrained hexapeptides may, therefore, provide a means of producing cell-permeant inhibitors of the cardiac Na-Ca exchanger. Received: 6 February 1997 / Received after revision: 8 April 1998 / Accepted: 9 April 1998     

Effect of secretin and inhibitors of HCO3 −/H+ transport on the membrane voltage of rat pancreatic duct cells

The aim of the present study was to study the effect of secretin on the electrophysiological response of pancreatic ducts. Furthermore, we investigated the effects of lipid-soluble buffers and inhibitors of HCO₃ ^–/H⁺ transport. Ducts obtained from fresh rat pancreas were perfused in vitro. Secretin depolarized the basolateral membrane voltage, V _bl, by up to 35 mV (n=37); a halfmaximal response was obtained at 3×10^–11 mol/l. In unstimulated ducts a decrease in the luminal Cl^– concentration (120 to 37 mmol/l) had a marginal effect on V _bl, but after maximal secretin stimulation it evoked a 14±2 mV depolarization (n=6), showing that a luminal Cl^– conductance G _Cl- was activated. The depolarizing effect of secretin on V _bl was often preceded by about a 6 mV hyperpolarization, most likely due to an increase in the basolateral G _K+. Perfusion of ducts with DIDS (4,4 — diisothiocyanatostilbene — 2,2 — disulphonic acid, 0.01 mmol/l) or addition of ethoxzolamide (0.1 mmol/l) to the bath medium diminished the effect of secretin. Acetate or pre-treatment of ducts with NH₄ ⁺/NH₃ (10 mmol/l in the bath) depolarized the resting V _bl of –65±2 mV by 16±4 mV (n=7) and 19±3 mV (n=10), respectively. The fractional resistance of the basolateral membrane (FR _bl) doubled, and the depolarizing responses to changes in bath K⁺ concentrations (5 to 20 mmol/l) decreased from 22±1 to 11±2 mV. The Na⁺/H⁺ antiporter blocker EIPA (5-[N-ethyl-N-isopropyl]-amiloride, 0.1 mmol/l) also depolarized V _bl by 10±1 mV, FR_bl increased and the response to K⁺ concentration changes decreased (n=7). Effects of EIPA and ethoxzolamide on V _bl were greater in ducts deprived of exogenous HCO₃ ^–/CO₂. Taken together, the present study shows that secretin increased the basolateral G _K+ and the luminal G _Cl-. The depolarizing effect of secretin was diminished following inhibition of HCO₃ ^– transport (DIDS), or HCO₃ ^–/H⁺ generation (ethoxzolamide). Manoeuvres that presumably led to lowered intracellular pH (NH₄ ⁺/NH₃ removal, acetate, EIPA) decreased the basolateral G _K+. The present data support our previously published model for pancreatic HCO₃ ^– secretion, and indicate that the basolateral membrane possesses a pH-sensitive G _K+.     

The effects of baclofen on calcium channel currents in dorsal sensory cells of the spinal cord in the lamprey

Dorsal sensory cells isolated from the spinal cord of the lamprey speciesIchthyomyzon unicuspis andLampetra fluviatilis were used for whole-cell patch-clamp studies of the effects of baclofen on calcium channel currents, evoked in conditions in which Na⁺, K⁺ currents were blocked, by depolarizing membranes from constant holding potentials of −100 or −80 mV to +30 mV. Ba ions were used as carriers of currents through calcium channels. These studies demonstrated that baclofen (0.5 mM) decreased the peak amplitude of the Ba²⁺ current by an average of 22.5±4.2% (n=12) in dorsal sensory cells of the lampreyIchthyomyzon unicuspis and by 28.4±3.3% in the dorsal sensory cells ofLampetra fluviatilis (n=25). The conductivity of dorsal sensory cell membranes in the presence of baclofen (and GABA) did not change. The blocking action of baclofen persisted in the presence of bicuculline (100 μM) and was lifted by addition of δ-aminovaleric acid and 2-hydroxysaclofen to the perfusing solution. These results are interpreted as evidence for the presence of GABA_B receptors in dorsal sensory cell membranes. The data were compared with published results, and the question of the functional significance of GABA_B receptors in the dorsal sensory cells (primary afferent cells) of cyclostomata is discussed. Translated from Rossiiskii Fiziologischeskii Zhurnal imeni I. M. Sechenova, Vol. 83, No. 11-12, pp. 92–104, November–December, 1997.     

Identification of a voltage- and calcium-dependent non-selective cation channel in cultured adult and fetal human nasal epithelial cells

The apical membranes of cultured human nasal epithelial cells from adults and fetuses were investigated with the patch-clamp technique. Amiloride-insensitive, calcium- and voltage-dependent, non-selective cation channels were found in 4% of the cell-attached, and 18% of the inside-out and outside-out patches (n=412). Multiple functional channels were present in more than 90% of these patches, with a mean of 3.9 channels per patch (n=55). The current-voltage relationship can be described by the Goldman equations and the single channel conductance was 20.1±0.3 pS (n=29) in adult and 20.7±0.4 pS (n=44) in fetal cells in symmetrical 150mM NaCl solutions. The channels were highly selective for cations: P_Na/P_Cl was 30 in adult and 45 in fetal experiments. They were equally permeable for K⁺ and Na⁺, somewhat less for Cs⁺, and impermeable for choline⁺ and tetraethylammonium⁺. The open probability was voltage dependent: it increased approximately 2-fold with 30mV depolarization in the potential range from −60mV to +60mV. The channels were activated by Ca²⁺ concentrations of about 10⁻⁴M at the cytoplasmic side, but were insensitive to extracellular Ca²⁺ and amiloride (10⁻⁴M). The non-selective cation channels found in apical membranes of cultured fetal nasal epithelial cells were not different from the adult ones.