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     

Extracellular acidification elicits a chloride current that shares characteristics with ICl(swell)

A Cl^– current activated by extracellular acidification, I_Cl(pHac), has been characterized in various mammalian cell types. Many of the properties of I_Cl(pHac) are similar to those of the cell swelling-activated Cl^– current I_Cl(swell): ion selectivity (I^– > Br^– > Cl^– > F^–), pharmacology [I_Cl(pHac) is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 1,9-dideoxyforskolin (DDFSK), diphenylamine-2-carboxylic acid (DPC), and niflumic acid], lack of dependence on intra- or extracellular Ca²⁺, and presence in all cell types tested. I_Cl(pHac) differs from I_Cl(swell) in three aspects: 1) its rate of activation and inactivation is very much more rapid, currents reaching a maximum in seconds rather than minutes; 2) it exhibits a slow voltage-dependent activation in contrast to the fast voltage-dependent activation and time- and voltage-dependent inactivation observed for I_Cl(swell); and 3) it shows a more pronounced outward rectification. Despite these differences, study of the transition between the two currents strongly suggests that I_Cl(swell) and I_Cl(pHac) are related and that extracellular acidification reflects a novel stimulus for activating I_Cl(swell) that, additionally, alters the biophysical properties of the channel. cell swelling-activated chloride current; patch clamp; pH     

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     

Activation of chloride currents in murine portal vein smooth muscle cells by membrane depolarization involves intracellular calcium release

The present study describes the first characterization of Ca²⁺-activated Cl^– currents (I_ClCa) in single smooth muscle cells from a murine vascular preparation (portal veins). I_ClCa was recorded using the perforated patch version of the whole cell voltage-clamp technique and was evoked using membrane depolarization. Generation of I_ClCa relied on Ca²⁺ entry through dihydropyridine-sensitive Ca²⁺ channels because I_ClCa was abolished by 1 µM nicardipine and enhanced by raising external Ca²⁺ concentration or by application of BAY K 8644. I_ClCa was characterized by the sensitivity to Cl^– channel blockers and the effect of altering the external anion on reversal potential. Activation of I_ClCa after membrane depolarization was dependent on Ca²⁺ release from intracellular stores. Thus the amplitude of I_ClCa was diminished by the SR-ATPase inhibitor cyclopiazonic acid, the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate (2-APB), and the ryanodine receptor blocker tetracaine. The degree of inhibition produced by the application of 2-APB and tetracaine together was significantly greater than the effect of each agent applied alone. In current-clamp mode, injection of depolarizing current elicited a biphasic action potential, with the later depolarization being sensitive to niflumic acid (NFA; 10 µM). In isometric tension recordings, NFA inhibited spontaneous contractions. These data support a role for this conductance in portal vein excitability.     

Stimulus-induced currents in isolated taste receptor cells of the larval tiger salamander

Gustatory receptor cells, isolated from the lingual epitheliumof larval tiger salamanders (Ambystoma tigrinum), possess avariety of voltage- and ion-dependent conductances, includinga transient Na⁺ -current (I_Na), a voltage-gated Ca²⁺ -current(I_A). a transient K₊ -current (I_A), a delayed rectifier K⁺ -current(I_K), and a Ca₂₊ -activated K₊ -current (I_K(Ca))- By use ofwhole-cell and excised-patch tight-seal recording techniques,we examined the effects of taste stimuli on the conductancesof taste cells from the tiger salamander. Depolarizing receptorpotentials elicited by NaCl were associated with slow, gradedinward currents which were composed of amiloride-sensitive andtetrodoxin-(TTX)-sensitive components. Potassium chloride producedmaintained inward currents, which usually showed both phasicand tonic components and were only partially blocked by tetraethylammoniumchloride (TEA). Citric and acetic acids elicited slow depolarizationsin taste cells. Under voltage-clamp, acids produced graded inwardcurrents which were composed of two components: one attributableto a transient block of voltage-dependent K₊ -channels and asmaller component which may have resulted from an increasedconductance to cations. Quinine hydrochloride elicited slowdepolarization of taste cells which was associated with a slowlydeveloping maintained inward current under voltage-clamp. Quininesuppressed both voltage-dependent inward and outward currents.In some taste cells, L-arginine elicited small outward currentswhich were attributable to an increase in K₊ conductance. Inother cells, L-arginine produced a decrease in voltage-dependentoutward currents and generated depolarizations associated withinward currents. These results indicate that several independentmechanisms, including amiloride-sensitive Na₊ -channels, andstimulus modulation of voltage-dependent K₊ -channels, are involvedin taste cell responses to chemical stimuli. More than one mechanismis typically present in a single cell. ³Present address: Department of Physiology, Tokyo Medical andDental University, 5-45 Yushima 1-chome, Bunkyo-ku, Tokyo 113,Japan     

Modulation of hepatocellular swelling-activated K+ currents by phosphoinositide pathway-dependent protein kinase C

K⁺ channels participate in the regulatory volume decrease (RVD) accompanying hepatocellular nutrient uptake and bile formation. We recently identified KCNQ1 as a molecular candidate for a significant fraction of the hepatocellular swelling-activated K⁺ current (I_KVol). We have shown that the KCNQ1 inhibitor chromanol 293B significantly inhibited RVD-associated K⁺ flux in isolated perfused rat liver and used patch-clamp techniques to define the signaling pathway linking swelling to I_KVol activation. Patch-electrode dialysis of hepatocytes with solutions that maintain or increase phosphatidylinositol 4,5-bisphosphate (PIP₂) increased I_KVol, whereas conditions that decrease cellular PIP₂ decreased I_KVol. GTP and AlF₄^– stimulated I_KVol development, suggesting a role for G proteins and phospholipase C (PLC). Supporting this, the PLC blocker U-73122 decreased I_KVol and inhibited the stimulatory response to PIP₂ or GTP. Protein kinase C (PKC) is involved, because K⁺ current was enhanced by 1-oleoyl-2-acetyl-sn-glycerol and inhibited after chronic PKC stimulation with phorbol 12-myristate 13-acetate (PMA) or the PKC inhibitor GF 109203X. Both I_KVol and the accompanying membrane capacitance increase were blocked by cytochalasin D or GF 109203X. Acute PMA did not eliminate the cytochalasin D inhibition, suggesting that PKC-mediated I_KVol activation involves the cytoskeleton. Under isotonic conditions, a slowly developing K⁺ current similar to I_KVol was activated by PIP₂, lipid phosphatase inhibitors to counter PIP₂ depletion, a PLC-coupled ₁-adrenoceptor agonist, or PKC activators and was depressed by PKC inhibition, suggesting that hypotonicity is one of a set of stimuli that can activate I_KVol through a PIP₂/PKC-dependent pathway. The results indicate that PIP₂ indirectly activates hepatocellular KCNQ1-like channels via cytoskeletal rearrangement involving PKC activation. KCNQ1; patch clamp; phosphatidylinositol 4,5-bisphosphate; regulatory volume decrease     

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     

Characteristics of hyperpolarization-activated cation currents in portal vein smooth muscle cells

Voltage-clamp studies offreshly isolated smooth muscle cells from rabbit portal veinrevealed the existence of a time-dependent cation current evoked bymembrane hyperpolarization (termed I_h). Both therate of activation and the amplitude of I_h wereenhanced by membrane hyperpolarization. Half-maximal activation ofI_h was about 105 mV with conventional wholecell and 80 mV when the perforated patch technique was used. Incurrent clamp, injection of hyperpolarizing current produced a markeddepolarizing "sag" followed by rebound depolarization. Activationof I_h was augmented by an increase in theextracellular K⁺ concentration and was blocked rapidly byexternally applied Cs⁺ (1-5 mM). The bradycardic agentZD-7288 (10 µM), a selective inhibitor of I_h,produced a characteristically slow inhibition of the portal veinI_h. The depolarizing sag recorded in current clamp was also abolished by application of 5 mM Cs⁺.Cs⁺ significantly decreased the frequency of spontaneouscontractions in both whole rat portal vein and rabbit portal veinsegments. Multiplex RT-PCR of rabbit portal vein myocytes using primers derived from existing genes for hyperpolarization-activated cation channels (HCN1-4) revealed the existence of cDNA clonescorresponding to HCN2, 3, and 4. The present study shows that portalvein myocytes contain genes shown to encode forhyperpolarization-activated channels and exhibit an endogenous currentwith characteristics similar to I_h in other celltypes. This conductance appears to determine, in part, the rhythmicityof this vessel.

     

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk^– cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC₅₀ of 4.2 µM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC₅₀ of 1.4 µM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 µM/s and 7.5 s^–1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 µM) also inhibited an ultrarapid delayed rectifier K⁺ current (I_K,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous I_K,ur in a cytoskeleton-independent manner as an open-channel blocker. voltage-gated K⁺ channel; heart; open channel block     

Oscillatory Mechanisms in Pairs of Neurons Connected with Fast Inhibitory Synapses

We study dynamical mechanisms underlying oscillatory behavior in reciprocal inhibitory pairs of neurons, using a two-dimensionalcell model. We introduce one-and-two dimensional phase portraits to illustratethe behaviors, thus reducing the study of dynamical mechanisms to planar geometrical properties. We examined whether other mechanisms besides the escape and release mechanisms (Wang and Rinzel, 1992) might be needed for some cases of reciprocal inhibition, and show that, within the confines of a simple two-dimensional cell model, escape and releaseare sufficient for all cases. We divided the behaviors of a singlecell into six different types and examined the joint behaviors arising from every combination of pairs of cells with behaviors drawn from thesesix types. For the case of two quiescent cells or two cells eachhaving plateau potentials, bifurcation diagrams demonstrate therelations between synaptic threshold and synaptic strength necessaryfor oscillations by escape, oscillations by release, ornetwork-generated plateau potentials. Thus we clarify therelationship between plateau potentials and oscillations in a cell.Using the two dimensional cell model we examine 1:N beating betweencells and find that our simple model displays many of the essentialdynamical properties displayed by more sophisticated models, some ofwhich relate to thalamocortical spindling.     

Cell cycle-related changes in transient K(+) current density in the GH3 pituitary cell line

Our aim was to determinewhether the expression of K⁺ currents is related to thecell cycle in the excitable GH3 pituitary cell line. K⁺currents were studied by electrophysiology, and bromodeoxyuridine (BrdU) labeling was used to compare their expression in cells thereafter identified as being in the S or non-S phase of the cellcycle. We show that the peak density of the transient outward K⁺ current (I_to) was 33% lower incells in S phase (BrdU+) than in cells in other phases of the cellcycle (BrdU). The voltage-dependence of I_towas not modified. However, of the two kinetic components ofI_to inactivation, the characteristics of thefast component differed significantly between BrdU+ and BrdU cells.Recovery from inactivation of I_to showedbiexponential and monoexponential function in BrdU and BrdU+ cells,respectively. This suggests that the molecular basis of this currentvaries during the cell cycle. We further demonstrated that4-aminopyridine, which blocks I_to, inhibited GH3cell proliferation without altering the membrane potential. These datasuggest that I_to may play a role in GH3 cellproliferation processes.

     

Interaction of Extracellular Potassium and Cesium with the Kinetics of Inward Rectifying K+ Channels in the Plasma Membrane of Mesophyll Protoplasts of Avena sativa

Using the patch-clamp technique the kinetics of whole-cell andsingle channel inwardly rectifying K⁺ currents were measuredin enzymatically-isolated protoplasts from Avena sativa mesophyllleaf cells. The hyperpolarization-activated whole-cell currenthad an initial K⁺ component (I_KI) and a time-dependent K⁺ componentwhich reaches steady state (I_KSS) within 500 ms. After an initialdelay, the activation of IKss and the deactivation of the tailK⁺ current (I_KT) followed an exponential time course. The time-constantsof activation (     

Modeling the leech heartbeat elemental oscillator I. Interactions of intrinsic and synaptic currents

We have developed a biophysical model of a pair of reciprocally inhibitory interneurons comprising an elemental heartbeat oscillator of the leech. We incorporate various intrinsic and synaptic ionic currents based on voltage-clamp data. Synaptic transmission between the interneurons consists of both a graded and a spike-mediated component. By using maximal conductances as parameters, we have constructed a canonical model whose activity appears close to the real neurons. Oscillations in the model arise from interactions between synaptic and intrinsic currents. The inhibitory synaptic currents hyperpolarize the cell, resulting in activation of a hyperpolarization-activated inward currentI _h and the removal of inactivation from regenerative inward currents. These inward currents depolarize the cell to produce spiking and inhibit the opposite cell. Spike-mediated IPSPs in the inhibited neuron cause inactivation of low-threshold Ca⁺⁺ currents that are responsible for generating the graded synaptic inhibition in the opposite cell. Thus, although the model cells can potentially generate large graded IPSPs, synaptic inhibition during canonical oscillations is dominated by the spike-mediated component.     

Ca(2+)-dependent activation of Cl(-) currents in Xenopus oocytes is modulated by voltage

Ca²⁺-activatedCl currents (I_Cl,Ca) wereexamined using fluorescence confocal microscopy to monitorintracellular Ca²⁺ liberation evoked by flash photolysis ofcaged inositol 1,4,5-trisphosphate (InsP₃) involtage-clamped Xenopus oocytes. Currents at +40 mV exhibited asteep dependence on InsP₃ concentration([InsP₃]), whereas currents at140 mV exhibited a higher threshold and more graded relationshipwith [InsP₃]. Ca²⁺ levelsrequired to half-maximally activate I_Cl,Ca wereabout 50% larger at 140 mV than at +40 mV, and currents evokedby small Ca²⁺ elevations were reduced >25-fold. Thehalf-decay time of Ca²⁺ signals shortened at increasinglypositive potentials, whereas the decay of I_Cl,Calengthened. The steady-state current-voltage (I-V) relationshipfor I_Cl,Ca exhibited outward rectification withweak photolysis flashes but became more linear with stronger stimuli.Instantaneous I-V relationships were linear with both strongand weak stimuli. Current relaxations following voltage steps duringactivation of I_Cl,Ca decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at 160 mV. We conclude that InsP₃-mediated Ca²⁺liberation activates a single population of Clchannels, which exhibit voltage-dependent Ca²⁺ activationand voltage-independent instantaneous conductance.

     

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.

     

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.

     

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     

Ca2+-activated Cl- current in cultured myenteric neurons from murine proximal colon

Whole cell patch-clamprecordings were made from cultured myenteric neurons taken from murineproximal colon. The micropipette contained Cs⁺ to removeK⁺ currents. Depolarization elicited a slowly activatingtime-dependent outward current (I_tdo), whereasrepolarization was followed by a slowly deactivating tail current(I_tail). I_tdo andI_tail were present in ~70% of neurons. Weidentified these currents as Cl currents(I_Cl), because changing the transmembraneCl gradient altered the measured reversal potential(E_rev) of both I_tdo andI_tail with that for I_tailshifted close to the calculated Cl equilibrium potential(E_Cl). I_Cl areCa²⁺-activated Cl current[I_Cl(Ca)] because they were Ca²⁺dependent. E_Cl, which was measured from theE_rev of I_Cl(Ca) using agramicidin perforated patch, was 33 mV. This value is more positivethan the resting membrane potential (56.3 ± 2.7 mV), suggestingmyenteric neurons accumulate intracellular Cl.-Conotoxin GIVA [0.3 µM; N-type Ca²⁺ channelblocker] and niflumic acid [10 µM; knownI_Cl(Ca) blocker], decreased theI_Cl(Ca). In conclusion, these neurons haveI_Cl(Ca) that are activated by Ca²⁺entry through N-type Ca²⁺ channels. These currents likelyregulate postspike frequency adaptation.

     

Calcium influx through If channels in rat ventricular myocytes

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I_f)/neuronal (I_h) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I_h channels in neurons. This raises the possibility of Ca²⁺ permeation in I_f, the I_h counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca²⁺ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I_f in rat ventricular myocytes. We observed Ca²⁺ influx when HCN/I_f channels were activated. Ca²⁺ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I_f channel blocker, inhibited I_f and Ca²⁺ influx at the same time. Quantitative analysis revealed that Ca²⁺ flux contributed to 0.5% of current produced by the HCN2 channel or I_f. The associated increase in Ca²⁺ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I_f current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca²⁺ chelator), preactivation of I_f channels significantly reduced the action potential duration, and the effect was blocked by another selective I_f channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I_f channels had no effects on action potential duration. Our data extend our previous discovery of Ca²⁺ influx in I_h channels in neurons to I_f channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels