Ionic Currents across the Plasmalemma of Chara inflata Cells: II. EFFECTS OF EXTERNAL Na+, Ca2+ AND Cl- ON K+ AND Cl CURRENTS

The electrical conductance of the plasmalemma of cells of Charainflata, due to the diffusion of ions, consists predominantlyof K⁺, Cl^– and leak components. When the membrane electricalpotential difference is stepped in a negative direction witha voltage-clamp, the resulting inward current has componentsI_K, I_Cl and I_L (leak). During such voltage-clamp steps I_K isinactivated, and Ic activated with voltage-dependent half-times.Increases in the external NaCl concentration reduce the magnitudeof I_K and increase the magnitude of I_c, but reduce the half-timeof inactivation or activation. The NaCl-induced changes in I_kand I_Cl and their kinetics were more pronounced at pH₀ =6.5than at pH₀ =9.5. When the concentration of external CaCl₂ wasincreased, I_k, I_Cl and the half-time of inactivation, (T_1/2),of I_k were all reduced. The half-time of activation of I_Cl wasincreased. The NaCI-induced changes could result from increases in bothexternal ion concentration and osmotic pressure. Previous experimentshave shown that an increase in external osmotic pressure alonealters the properties of the conductances. In this paper weattempt to separate the purely ionic effects from the osmoticones. Key words: Chara inflata, ionic effects, K⁺ and Cl^– currents     

Properties of ATP-dependent K(+) channels in adrenocortical cells

Bovine adrenocortical zona fasciculata (AZF)cells express a novel ATP-dependent K⁺-permeable channel(I_AC). Whole cell and single-channel recordings were used to characterize I_AC channels withrespect to ionic selectivity, conductance, and modulation bynucleotides, inorganic phosphates, and angiotensin II (ANG II). Inoutside-out patch recordings, the activity of unitaryI_AC channels is enhanced by ATP in the patchpipette. These channels were K⁺ selective with nomeasurable Na⁺ or Ca²⁺ conductance. Insymmetrical K⁺ solutions with physiological concentrationsof divalent cations (M²⁺), I_ACchannels were outwardly rectifying with outward and inward chordconductances of 94.5 and 27.0 pS, respectively. In the absence ofM²⁺, conductance was nearly ohmic. Hydrolysis-resistantnucleotides including AMP-PNP and NaUTP were more potent than MgATP asactivators of whole cell I_AC currents. Inorganicpolytriphosphate (PPP_i) dramatically enhancedI_AC activity. In current-clamp recordings, nucleotides and PPP_i produced resting potentials in AZFcells that correlated with their effectiveness in activatingI_AC. ANG II (10 nM) inhibited whole cellI_AC currents when patch pipettes contained 5 mMMgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP.Inhibition by ANG II was not reduced by selective kinase antagonists.These results demonstrate that I_AC is adistinctive K⁺-selective channel whose activity isincreased by nucleotide triphosphates and PPP_i.Furthermore, they suggest a model for I_AC gatingthat is controlled through a cycle of ATP binding and hydrolysis.

     

Divalent cation and anion currents are activated during the dark-induced transient hyperpolarization of the plasma membrane of the green alga Eremosphaera viridis

Darkening after illumination induces a transient hyperpolarizationof the plasma membrane of the unicellular green alga Eremosphaeraviridis de Bary. With electro-physiological methods, in particularthe two electrode voltage-clamp technique, we investigated theion fluxes involved in this transient potential change (TP).The question was whether other ion currents besides those carriedby the known Ca²⁺-dependent K⁺ channel take part in this actionpotential-like, but hyperpolarizing, response. At maximum hyperpolarizationvoltage-clamp measurements resulted in ‘N-shaped’I/V curves, known from other botanical systems. The differentinstantaneous current components of the N-shaped I/V curvesoccurred at different times during a single transient potentialchange (TP). Substitution of alkali metal cations in the bathingsolution by NMG/NO₃ showed that the inward currents in the I/Vcurves were not carried by an influx of K⁺ into the cytoplasm.The voltage amplitude of the TP not only depended on the externalK⁺ concentration, but also on the Mg²⁺ concentration in thebathing solution. Increasing Mg²⁺ concentrations shifted themembrane potential in the top of the TP in the direction ofthe Nernst potential of Mg²⁺ and resulted in an increased inwardcurrent component of the N-shaped I/V curves. Another currentcomponent was found to be carried not by cations but by an effluxof anions. It was a voltage-dependent component with a maximumcurrent amplitude at voltages of about –220 to –240mV, and was blocked by the anion channel inhibitors anthracen-9-carboxylicacid (A9C), (5-nitro-2-3-phenylpropylamino) benzoic acid (NPPB)and ZnCI2. Based on these data a model is proposed which explainsthe N-shape of the I/V curves observed during the transientpotential change of the alga E. viridis by the combination ofan inward cation current with an inward anion current and theoutward cation current carried by the Ca²⁺-dependent K⁺ channels. Key words: Anion current, cation current, Eremosphaera viridis, potassium channel, voltage-clamp     

Potassium currents across the plasma membrane of protoplasts derived from rye roots: a patch-clamp study

Epidermal-cell protoplasts from rye (Secale cereale L.) rootswere voltage-clamped in both the whole-cell and outside-outmembrane-patch modes. Time-dependent inwardly-rectified (IR)and outwardly-rectified (OR) K⁺-currents were recorded, as wellas a ubiquitous, timeindependent (instantaneous) K⁺-current. The IR current activated at voltages more negative than —100mVwith two exponentially rising components. The time-constantof the shorter component was voltage-independent, whereas thetime-constant of the longer component was voltage-dependent,increasing as the activating voltage became more negative. TheIR current showed no inactivation. The IR current deactivatedwith a single exponential timecourse. The steady-state IR currentcould be fitted to a Boltzmann function with —135 mV asthe voltage at which the current was half-maximal and a minimalgating charge of 1.93. These parameters were insensitive tochanges in E_K. One component of the IR current was K ⁺ , butother ions were also permeable. The IR current was inhibitedby extracellular Ca²⁺ , Ba²⁺ , Cs⁺, and TEA⁺, but was insensitiveto quinine. Single channels with unitary conductances of 56pS and 110 pS (in c.100 mM K⁺) were recorded at negative voltages. Two OR currents were observed. One had sigmoidal activationkinetics and activated at low positive voltages. The other activatedmore rapidly, with apparently exponential kinetics, at voltages50–100 mV more positive than the first. Neither currentshowed inactivation and deactivation of OR currents followeda double exponential time-course. Unitary-conductances of thechannels mediating these OR currents were 24 pS and 57 pS (inc.100 mM K⁺), respectively. Only the first type of OR currentwas studied in detail. This current activated with a sigmoidaltime-course, which could be described using a Hodgkin-Huxleyfunction with the activation variable raised to the second power.Its voltage-dependence was modulated in response to changesin E^K and analysis of single-channel recordings indicated thatthe channel was K⁺-selective. The current was inhibited by Ba²⁺and TEA+, but not Ca²⁺, Cs⁺ or quinine. The instantaneous current was selective for monovalent cationsand K⁺ , Na⁺ and Cs⁺ were all permeant. It was inhibited byextracellular quinine and the instantaneous inward K⁺-currentwas reduced by extracellular Ca²⁺, Ba²⁺ and TEA⁺, as well asby competing permeant monovalent cations. The kinetics and pharmacology of these currents are comparedwith K⁺-currents across the plasma membrane of protoplasts fromother root-derived cells and with K⁺ channels in the plasmamembrane of rye roots studied following incorporation into artificial,planar lipid bilayers. Key words: Ionic currents, patch-clamp, pharmacology, potassium, K⁺, rye, Secale cereale L     

Functional ion channels in mouse bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not fully understood. The present study was to investigate the functional ionic channels in undifferentiated mouse bone marrow MSCs using whole cell patch-voltage clamp technique, RT-PCR, and Western immunoblotting analysis. We found that three types of ionic currents were present in mouse MSCs, including a Ca²⁺-activated K⁺ current (I_KCa), an inwardly rectifying K⁺ current (I_Kir), and a chloride current (I_Cl). I_Kir was inhibited by Ba²⁺, and I_KCa was activated by the Ca²⁺ ionophore A-23187 and inhibited by the intermediate-conductance I_KCa channel blocker clotrimazole. I_Cl was activated by hyposmotic (0.8 T) conditions and inhibited by the chloride channel blockers DIDS and NPPB. The corresponding ion channel genes and proteins, KCa3.1 for I_KCa, Kir2.1 for I_Kir, and Clcn3 for I_Cl, were confirmed by RT-PCR and Western immunoblotting analysis in mouse MSCs. These results demonstrate that three types of functional ion channel currents (i.e., I_Kir, I_KCa, and I_Cl) are present in mouse bone marrow MSCs. inward rectifier potassium current; intermediate-conductance calcium-activated potassium current; volume-sensitive chloride current     

Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods

The Na⁺-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity (K_0.5) was in the low micromolar range (12–34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current (I_max) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased I_max. Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I_max. Fludarabine and formycin B were also transported with higher I_max than uridine and moderate affinity (102 ± 10 and 66 ± 6 µM, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about –39 mV and a valence of about –0.8. K_0.5 for Na⁺ was 2.3 mM at –50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two Na⁺ per nucleoside. This discrepancy in the number of Na⁺ molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na⁺ binding sites. two-electrode voltage clamp; concentrative nucleoside transport; presteady-state currents     

Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca²⁺-activated K⁺ channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current (I_VNSCC) and a relatively high-threshold voltage-activated (L-type) Ca²⁺ current (I_L). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect I_VNSCC or I_L in dialyzed cells. ATP (1 mM) increased I_VNSCC and decreased I_L in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on I_VNSCC. ADP decreased I_L but had no effect on I_VNSCC. The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on I_VNSCC. ATP stimulation of I_VNSCC was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y₄ is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates I_VNSCC and depresses I_L via binding of P2Y₄ receptors and stimulation of the phospholipase C/PKC pathway. inhibitory junction potentials; smooth muscle; enteric nervous system     

Mechanism of shortened action potential duration in Na+-Ca2+ exchanger knockout mice

In cardiac-specific Na⁺-Ca²⁺ exchanger (NCX) knockout (KO) mice, the ventricular action potential (AP) is shortened. The shortening of the AP, as well as a decrease of the L-type Ca²⁺ current (I_Ca), provides a critical mechanism for the maintenance of Ca²⁺ homeostasis and contractility in the absence of NCX (Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na⁺-Ca²⁺ exchanger knockout mice: reduced transsarcolemmal Ca²⁺ flux. Circ Res 97: 1288–1295, 2005). To investigate the mechanism that underlies the accelerated AP repolarization, we recorded the transient outward current (I_to) in patch-clamped myocytes isolated from wild-type (WT) and NCX KO mice. Peak I_to was increased by 78% and decay kinetics were slowed in KO vs. WT. Consistent with increased I_to, ECGs from KO mice exhibited shortened QT intervals. Expression of the I_to-generating K⁺ channel subunit Kv4.2 and the K⁺ channel interacting protein was increased in KO. We used a computer model of the murine AP (Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, and Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: 1378–1403, 2004) to determine the relative contributions of increased I_to, reduced I_Ca, and reduced NCX current (I_NCX) on the shape and kinetics of the AP. Reduction of I_Ca and elimination of I_NCX had relatively small effects on the duration of the AP in the computer model. In contrast, AP repolarization was substantially accelerated when I_to was increased in the computer model. Thus, the increase in I_to, and not the reduction of I_Ca or I_NCX, is likely to be the major mechanism of AP shortening in KO myocytes. The upregulation of I_to may comprise an important regulatory mechanism to limit Ca²⁺ influx via a reduction of AP duration, thus preventing Ca²⁺ overload in situations of reduced myocyte Ca²⁺ extrusion capacity. genetically altered mice; cardiac myocytes; short QT interval; transient outward current     

Demonstration of Voltage-Dependent and TTX-Sensitive Na+-Channels in Human Melanocytes

The electrophysiological properties of cultured human melanocytes were investigated using the whole-cell configuration of the patch-clamp technique. Depolarizations to membrane potentials more positive than -30 mV resulted in the rapid development (<1 ms to peak) of an inward current. The maximum peak current was observed at +10 mV and reached an average amplitude of about 270 pA. During the depolarizations, the current inactivated with a time constant of about 2 ms. The current was abolished by the addition of 0.3 μM tetrodotoxin, a blocker of voltage-gated Na⁺-channels, and disappeared when Na⁺ was omitted from the extracellular medium. In addition, the melanocytes contain at least two types of outward K⁺-current. The first type, observed in every cell, was highly sensitive (K_i 1 mM) to the K⁺-channel blocker TEA, required depolarizations beyond zero to be activated and did not inactivate. The second type was less regularly observed (10% of the cells). This current activated at more negative voltages (–20 mV), was resistant to TEA (20 mM) but was blocked by 2 mM 4-aminopyridine and inactivated rapidly during depolarizations. We conclude that human melanocytes are equipped with voltage-dependent Na⁺-channels, a delayed rectifying K⁺-current and a K⁺-current similar to the A-current in neurones.     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents     

Involvement of Cyclic AMP in ABA- and Ca2--Mediated Signal Transduction of Stomatal Regulation in Vicia faba

The focus of this study is to investigate possible involvementof cyclic AMP in regulation of Vicia stomatal movements. Thepresence of 0.1 mM 8-Br-cAMP, a membrane-permeable analogueof cAMP, alone in the incubation medium did not affect stomatalopening in the light in leaf epidermal peel experiments. However,addition of 0.1 mM 8-Br-cAMP completely reversed exogenous ABA-and C^a2+-induced inhibition of stomatal opening. Consistentwith these results, patch-clamping experiments showed that intracellularaddition of 0.5 mM or 1 mM cAMP significantly reversed the inhibitionof whole-cell inward K⁺ currents by internally supplied 13 µMCa²⁺ or 10 µM ABA in stomatal guard cell protoplasts,respectively. Furthermore, intracellular addition of either10 µM prostaglandin E₁ (PGE1, an adenylate cyclase activator)or 1 mM 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesteraseinhibitor) mimicked the effect of exogenous cAMP on the removalof ABA- or Ca²⁺ inhibition of inward K⁺-current. These resultssuggest that a cAMP signaling pathway is involved in signaltransduction in stomatal regulation by interacting with ABAand Ca²⁺ signaling cascades. A hypothetical mechanism by whichcAMP may regulate K⁺ in stomatal guard cells is also discussed. (Received May 6, 1999; Accepted August 27, 1999)     

Cation selectivity and inhibition of malignant glioma Na+ channels by Psalmotoxin 1

Psalmotoxin 1 (a component of the venom of a West Indies tarantula) is a 40-amino acid peptide that inhibits cation currents mediated by acid-sensing ion channels (ASIC). In this study we performed electrophysiological experiments to test the hypothesis that Psalmotoxin 1 (PcTX1) inhibits Na⁺ currents in high-grade human astrocytoma cells (glioblastoma multiforme, or GBM). In whole cell patch-clamped cultured GBM cells, the peptide toxin quickly and reversibly inhibited both inward and outward current with an IC₅₀ of 36 ± 2 pM. The same inhibition was observed in freshly resected GBM cells. However, when the same experiment was performed on normal human astrocytes, the toxin failed to inhibit the whole cell current. We also determined a cationic selectivity sequence for inward currents in three cultured GBM cell lines (SK-MG-1, U87-MG, and U251-MG). The selectivity sequence yielded a unique biophysical fingerprint with inward K⁺ conductance approximately fourfold greater than that of Na⁺, Li⁺, and Ca²⁺. These observations suggest that PcTX1 may prove useful in determining whether GBM cells express a specific ASIC-containing ion channel type that can serve as a target for both diagnostic and therapeutic treatments of aggressive malignant gliomas. patch clamp; amiloride; ion channels; acid-sensing ion channels     

Modeling transmural heterogeneity of K(ATP) current in rabbit ventricular myocytes

To investigate the mechanisms regulating excitation-metabolic coupling in rabbit epicardial, midmyocardial, and endocardial ventricular myocytes we extended the LabHEART model (Puglisi JL and Bers DM. Am J Physiol Cell Physiol 281: C2049–C2060, 2001). We incorporated equations for Ca²⁺ and Mg²⁺ buffering by ATP and ADP, equations for nucleotide regulation of ATP-sensitive K⁺ channel and L-type Ca²⁺ channel, Na⁺-K⁺-ATPase, and sarcolemmal and sarcoplasmic Ca²⁺-ATPases, and equations describing the basic pathways (creatine and adenylate kinase reactions) known to communicate the flux changes generated by intracellular ATPases. Under normal conditions and during 20 min of ischemia, the three regions were characterized by different I_Na, I_to, I_Kr, I_Ks, and I_Kp channel properties. The results indicate that the ATP-sensitive K⁺ channel is activated by the smallest reduction in ATP in epicardial cells and largest in endocardial cells when cytosolic ADP, AMP, PCr, Cr, P_i, total Mg²⁺, Na⁺, K⁺, Ca²⁺, and pH diastolic levels are normal. The model predicts that only K_ATP ionophore (Kir6.2 subunit) and not the regulatory subunit (SUR2A) might differ from endocardium to epicardium. The analysis suggests that during ischemia, the inhomogeneous accumulation of the metabolites in the tissue sublayers may alter in a very irregular manner the K_ATP channel opening through metabolic interactions with the endogenous PI cascade (PIP₂, PIP) that in turn may cause differential action potential shortening among the ventricular myocyte subtypes. The model predictions are in qualitative agreement with experimental data measured under normal and ischemic conditions in rabbit ventricular myocytes. ATP-sensitive K⁺ channel; creatine and adenylate kinase reactions; phosphatidylinositol phosphates; heart; mathematical model     

Sequential and opposite regulation of two outward K(+) currents by ET-1 in cultured striatal astrocytes

In the brain,astrocytes represent a major target for endothelins (ETs), a family ofpeptides that can be released by several cell types and that havepotent and multiple effects on astrocytic functions. Four types ofK⁺ currents (I_K) were detected invarious proportions by patch-clamp recordings of cultured striatalastrocytes, including the A-type I_K, theinwardly rectifying I_K IR, theCa²⁺-dependent I_K(I_K Ca), and the delayed-rectifiedI_K (I_K DR). Variationsin the shape of current-voltage relationships were related mainly todifferences in the proportion of these currents. ET-1 was found toregulate with opposite effects the two more frequently recorded outwardK⁺ currents in striatal astrocytes. Indeed, this peptideinduced an initial activation of I_K Ca(composed of SK and BK channels) and a delayed long-lasting inhibitionof I_K DR. In current-clamp recordings, theactivation of I_K Ca correlated with a transient hyperpolarization, whereas the inhibition ofI_K DR correlated with a sustaineddepolarization. These ET-1-induced sequential changes inmembrane potential in astrocytes may be important for the regulation ofvoltage gradients in astrocytic networks and the maintenance ofK⁺ homeostasis in the brain microenvironment.

     

Functional characterization of voltage-gated K+ channels in mouse pulmonary artery smooth muscle cells

Mice are useful animal models to study pathogenic mechanisms involved in pulmonary vascular disease. Altered expression and function of voltage-gated K⁺ (K_V) channels in pulmonary artery smooth muscle cells (PASMCs) have been implicated in the development of pulmonary arterial hypertension. K_V currents (I_K(V)) in mouse PASMCs have not been comprehensively characterized. The main focus of this study was to determine the biophysical and pharmacological properties of I_K(V) in freshly dissociated mouse PASMCs with the patch-clamp technique. Three distinct whole cell I_K(V) were identified based on the kinetics of activation and inactivation: rapidly activating and noninactivating currents (in 58% of the cells tested), rapidly activating and slowly inactivating currents (23%), and slowly activating and noninactivating currents (17%). Of the cells that demonstrated the rapidly activating noninactivating current, 69% showed I_K(V) inhibition with 4-aminopyridine (4-AP), while 31% were unaffected. Whole cell I_K(V) were very sensitive to tetraethylammonium (TEA), as 1 mM TEA decreased the current amplitude by 32% while it took 10 mM 4-AP to decrease I_K(V) by a similar amount (37%). Contribution of Ca²⁺-activated K⁺ (K_Ca) channels to whole cell I_K(V) was minimal, as neither pharmacological inhibition with charybdotoxin or iberiotoxin nor perfusion with Ca²⁺-free solution had an effect on the whole cell I_K(V). Steady-state activation and inactivation curves revealed a window K⁺ current between –40 and –10 mV with a peak at –31.5 mV. Single-channel recordings revealed large-, intermediate-, and small-amplitude currents, with an averaged slope conductance of 119.4 ± 2.7, 79.8 ± 2.8, 46.0 ± 2.2, and 23.6 ± 0.6 pS, respectively. These studies provide detailed electrophysiological and pharmacological profiles of the native K_V currents in mouse PASMCs. K_V channels     

Appearance and maturation of voltage-dependent conductances in solitary spiking cells during retinal regeneration in the adult newt

Summary Electrical membrane properties of solitary spiking cells during newt (Cynops pyrrhogaster) retinal regeneration were studied with whole-cell patch-clamp methods in comparison with those in the normal retina.The membrane currents of normal spiking cells consisted of 5 components: inward Na⁺ and Ca⁺⁺ currents and 3 outward K⁺ currents of tetraethylammonium (TEA)-sensitive, 4-aminopyridine (4-AP)-sensitive, and Ca⁺⁺-activated varieties. The resting potential was about -40mV. The activation voltage for Na⁺ and Ca⁺⁺ currents was about -30 and -17 mV, respectively. The maximum Na⁺ and Ca⁺⁺ currents were about 1057 and 179 pA, respectively.In regenerating retinae after 19–20 days of surgery, solitary cells with depigmented cytoplasm showed slowrising action potentials of long duration. The ionic dependence of this activity displayed two voltage-dependent components: slow inward Na⁺ and TEA-sensitive outward K⁺ currents. The maximum inward current (about 156 pA) was much smaller than that of the control. There was no indication of an inward Ca⁺⁺ current.During subsequent regeneration, the inward Ca⁺⁺ current appeared in most spiking cells, and the magnitude of the inward Na⁺, Ca⁺⁺, and outward K⁺ currents all increased. By 30 days of regeneration, the electrical activities of spiking cells became identical to those in the normal retina. No significant difference in the resting potential and the activation voltage for Na⁺ and Ca⁺⁺ currents was found during the regenerating period examined.     

Changes in Outward K+ Currents in Response to Two Types of Sweeteners in Sweet Taste Transduction of Gerbil Taste Cells

Using the whole cell patch clamp technique, we measured changesin outward K⁺ currents of gerbil taste cells in response todifferent kinds of sweeteners. Outward K⁺ currents of the tastecell induced by depolarizing pulses were suppressed by sweetstimuli such as 10 mM Na-saccharin. The membrane-permeable analogof cAMP, cpt-cAMP, also decreased outward K⁺ currents. On theother hand, the K+ currents were enhanced by amino acid sweetenerssuch as 10 mM D-tryptophan. The outward K⁺ current was enhancedby external application of Ca²⁺-transporting ionophore, 5 µMionomycin, and intracellular application of 5 µM inositol-1,4,5-trisphosphate(IP₃). The outward K⁺ currents were no longer suppressed by10 mM Na-saccharin containing 20 µM gurmarin, but werestill enhanced by 10 mM D-tryptophan containing 20 µMgurmarin. These results suggest that sweet taste transductionfor one group of sweeteners such as Na-saccharin in gerbilsis concerned with an increase of the intracellular cAMP level,and that the transduction for the other group of sweetenerssuch as D-tryptophan is concerned with an increase of the intracellularIP₃ level which releases Ca²⁺ from the internal stores. Chem.Senses 22: 163–169, 1997.     

Malignant human gliomas express an amiloride-sensitive Na+ conductance

Human astrocytoma cells were studied using whole cellpatch-clamp recording. An inward, amiloride-sensitiveNa⁺ current was identified in fourcontinuous cell lines originally derived from human glioblastoma cells(CH235, CRT, SKMG-1, and U251-MG) and in three primary cultures ofcells obtained from glioblastoma multiforme tumors (up to 4 passages).In addition, cells freshly isolated from a resected medulloblastomatumor displayed this same characteristic inward current. In contrast,amiloride-sensitive currents were not observed in normal humanastrocytes, low-grade astrocytomas, or juvenile pilocytic astrocytomas.The only amiloride-sensitive Na⁺channels thus far molecularly identified in brain are the brain Na⁺ channels (BNaCs). RT-PCRanalyses demonstrated the presence of mRNA for either BNaC1 or BNaC2 inthese tumors and in normal astrocytes. These results indicate that thefunctional expression of amiloride-sensitive Na⁺ currents is a characteristicfeature of malignant brain tumor cells and that this pathway may be apotentially useful target for therapeutic intervention.

     

An extracellular sulfhydryl group modulates background Na(+) conductance and cytosolic Ca(2+) in pituitary cells

Treatment of GH₃ pituitarycells with p-chloromercurybenzenesulfonate (PCMBS) increasedthe cytosolic Ca²⁺ concentration([Ca²⁺]_i). This effect was reversed bydithiothreitol and blocked by L-type Ca²⁺ channelantagonists or Na⁺ removal. PCMBS increased membraneconductance and depolarized the plasma membrane. Apart from minoreffects on K⁺ and Ca²⁺ channels, PCMBSincreased (6 times at 80 mV) an inward Na⁺ current whoseproperties were similar to those of a background Na⁺conductance (BNC) described previously, necessary for generation ofspontaneous electrical activity. In rat lactotropes and somatotropes inprimary culture, PCMBS also produced a Na⁺-dependent[Ca²⁺]_i increase, whereas little or no effectwas observed in thyrotropes, corticotropes, and gonadotropes. TheNa⁺ conductance elicited by PCMBS in somatotropes seemed tobe the same as that stimulated by the hypothalamic growth hormone(GH)-releasing hormone, which regulates membrane excitability and GHsecretion. The BNC studied here could play a physiological role,regulating excitability and spontaneous activity, and explainssatisfactorily the [Ca²⁺]_i-increasing actionsof the mercurials reported previously in several excitable tissues.

     

Mechanisms of solute efflux from seed coats: whole-cell K+ currents in transfer cell protoplasts derived from coats of developing seeds of Vicia faba L.

In developing seed ofVicia faba L., solutes imported throughthe phloem of the coats move symplastically from the sieve elementsto a specialized set of cells (the thin-walled parenchyma transfercells) for release to the seed apoplast. Potassium (K⁺) is thepredominant cation released from the seed coats. To elucidatethe mechanisms of K⁺ efflux from seed coat to seed apoplast,whole-cell currents across the plasma membranes of protoplastsof thin-walled parenchyma transfer cells were measured usingthe whole-cell patch-clamp technique. Membrane depolarizationelicited a time-dependent and an instantaneous outward current.The reversal potential (E_R of the time-dependent outward currentwas close to the potassium equilibrium potential (E_K and itshifted in the same direction as E_K upon changing the externalK⁺ concentration, indicating that this current was largely carriedby an efflux of K⁺. The activation of the time-dependent outwardK⁺ current could be well fitted by two exponential componentsplus a constant. The instantaneous outward current could alsobe carried by K⁺ efflux as suggested by ion substitution experiments.These K⁺ outward rectifier currents elicited by membrane depolarizationare probably too small to represent the mechanism for the normalK⁺ efflux from seed coat cells. Membrane hyperpolarization morenegative than –80 mV activated a time-dependent inwardcurrent. K⁺ influx was responsible for the inward current asthe current reversed at membrane voltage close to E_K and shiftedin the same direction as E_K when external [K⁺] was varied. Activationof this K⁺inward rectifier current was well fitted with twoexponential components plus a constant. A regulating functionfor this current is suggested. Key words: Potassium outward rectifier, potassium inward rectifier, transfer cell protoplast, seed coat, Vicia faba L