新鲜分离小鼠胃ICC样细胞的鉴定及其电生理学特性

将免疫荧光及传统全细胞膜片钳技术应用于新鲜分离的小鼠胃Cajal 间质细胞样细胞上,探讨了小鼠胃Cajal 间质细胞样细胞形态和电生理学特性。经胶原酶消化得到的Cajal间质细胞样细胞胞体呈短梭形,且自胞体发出多个较短的毛刺状突起。免疫细胞化学结果表明,Cajal 间质细胞样细胞胞体和突起酪氨酸激酶受体c-kit表达呈阳性。在传统全细胞记录模式、膜电位钳制在-60 mV 的条件下,可以记录到自发、节律性内向电流,即起搏电流。钙调蛋白抑制剂W-7 (50µmol/L）明显增强了起搏电流幅度并引发明显的内向钳制电流。当电极内液中EGTA 的浓度由0.1 mmol/L增加到10 mmol/L时,也明显增强了起搏电流幅度并引发明显的内向钳制电流。实验结果提示,新鲜分离的小鼠胃Cajal 间质细胞样细胞可以产生自发、自律性内向电流,而这种电流对胞内低钙或钙调蛋白抑制剂敏感。这种具有自发性电活动的Cajal 间质细胞样细胞可能就是胃Cajal 间质细胞。     

Pharmacological and biophysical isolation of K+ currents encoded by ether-a-go-go-related genes in murine hepatic portal vein smooth muscle cells

Previous studies have shown that murine portal vein myocytes express ether-à-go-go related genes (ERGs) and exhibit distinctive currents when recorded under symmetrical K⁺ conditions. The aim of the present study was to characterize ERG channel currents evoked from a negative holding potential under conditions more pertinent to a physiological scenario to assess the possible functional impact of this conductance. Currents were recorded with ruptured or perforated patch variants of the whole cell technique from a holding potential of –60 mV. Application of three structurally distinct and selective ERG channel blockers, E-4031, dofetilide, and the peptide toxin BeKM-1, all inhibited a significant proportion of the outward current and abolished inward currents with distinctive "hooked" kinetics recorded on repolarization. Dofetilide-sensitive currents at negative potentials evoked by depolarization to +40 mV had a voltage-dependent time to peak and rate of decay characteristic of ERG channels. Application of the novel ERG channel activator PD-118057 (1–10 µM) markedly enhanced the hooked inward currents evoked by membrane depolarization and hyperpolarized the resting membrane potential recorded by current clamp and the perforated patch configuration by 20 mV. In contrast, ERG channel blockade by dofetilide (1 µM) depolarized the resting membrane potential by 8 mV. These data are the first record of ERG channel currents in smooth muscle cells under quasi-physiological conditions that suggest that ERG channels contribute to the resting membrane potential in these cells. vascular smooth muscle; voltage-dependent K⁺ current; membrane excitability     

Carbachol regulates pacemaker activities in cultured interstitial cells of Cajal from the mouse small intestine

We studied the effect of carbachol on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine by muscarinic stimulation using a whole cell patch clamp technique and Ca²⁺-imaging. ICC generated periodic pacemaker potentials in the current-clamp mode and generated spontaneous inward pacemaker currents at a holding potential of–70 mV. Exposure to carbachol depolarized the membrane and produced tonic inward pacemaker currents with a decrease in the frequency and amplitude of the pacemaker currents. The effects of carbachol were blocked by 1-dimethyl-4-diphenylacetoxypiperidinium, a muscarinic M₃ receptor antagonist, but not by methotramine, a muscarinic M₂ receptor antagonist. Intracellular GDP-β-S suppressed the carbachol-induced effects. Carbachol-induced effects were blocked by external Na⁺-free solution and by flufenamic acid, a non-selective cation channel blocker, and in the presence of thapsigargin, a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum. However, carbachol still produced tonic inward pacemaker currents with the removal of external Ca²⁺. In recording of intracellular Ca²⁺ concentrations using fluo 3-AM dye, carbachol increased intracellular Ca²⁺ concentrations with increasing of Ca²⁺ oscillations. These results suggest that carbachol modulates the pacemaker activity of ICC through the activation of non-selective cation channels via muscarinic M₃ receptors by a G-protein dependent intracellular Ca²⁺ release mechanism.     

Induction of pacemaker currents by DA-9701, a prokinetic agent, in interstitial cells of Cajal from murine small intestine

The interstitial cells of Cajal (ICC) are pacemaking cells required for gastrointestinal motility. The possibility of whether DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, modulates pacemaker activities in the ICC was tested using the whole cell patch clamp technique. DA-9701 produced membrane depolarization and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid, a non-selective cation channel blocker, but not niflumic acid, abolished the generation of pacemaker currents induced by DA-9701. Pretreatment with a Ca²⁺-free solution and thapsigargin, a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum, abolished the generation of pacemaker currents. In addition, the tonic inward currents were inhibited by U-73122, an active phospholipase C inhibitor, but not by GDP-β-S, which permanently binds G-binding proteins. Furthermore, the protein kinase C inhibitors, chelerythrine and calphostin C, did not block the DA-9701-induced pacemaker currents. These results suggest that DA-9701 might affect gastrointestinal motility by the modulation of pacemaker activity in the ICC, and the activation is associated with the non-selective cationic channels via external Ca²⁺ influx, phospholipase C activation, and Ca²⁺ release from internal storage in a G protein-independent and protein kinase C-independent manner.     

Non-Selective Cation Channels Mediate Chloroquine-Induced Relaxation in Precontracted Mouse Airway Smooth Muscle

Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs) or by inactivating voltage-dependent L-type Ca²⁺ channels (VDLCCs). In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs) precontracted with acetylcholine (ACH). In the presence of nifedipine (10 µM), ACH induced cytosolic Ca²⁺ elevation and cell shortening in single airway smooth muscle cells (ASMCs), and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca²⁺-free conditions, and, following a restoration of Ca²⁺, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs) were blocked by chloroquine. Pyrazole 3 (Pyr3), an inhibitor of transient receptor potential C3 (TRPC3) channels, partially inhibited ACH-induced contraction, intracellular Ca²⁺ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.     

Regulating role of acetylcholine and its antagonists in inward rectified K+ channels from guard cell protoplasts ofVicia faba

The inward rectified potassium current ofVicia faba guard cell protoplasts treated with acetylcholine (ACh) or the antagonists of its receptors were recorded by employing the patch clamp technique. The results show that ACh at lower concentrations increases the inward K⁺ current, in contrast, ACh at higher concentrations inhibits it. Treated with d-Tubocurarine (d-Tub), an antagonist of the nicotine ACh receptor (nAChR) inhibits the inward K⁺ current by 30%. Treated with atropine (Atr), an antagonist of the muscarine (Mus) ACh receptor (mAChR) also inhibits it by 36%. However, if guard cell protoplasts are treated with d-Tub and Atr together, the inward K⁺ current is inhibited by 60% –75%. Tetraethylammonium chloride (TEA), a strong inhibitor of K⁺ channels has no effect on the inward K⁺ current regulated by ACh, suggesting that there are inward K⁺ channels modulated by AChRs on the membrane of the guard cell protoplasts. These data demonstrate an ACh-regulated mechanism for stomatal movement.     

Neurotensin modulates pacemaker activity in interstitial cells of Cajal from the mouse small intestine

Neurotensin, a tridecapeptide localized in the gut to discrete enteroendocrine cells of the small bowel mucosa, is a hormone that plays an important role in gastrointestinal secretion, growth, and motility. Neurotensin has inhibitory and excitatory effects on peristaltic activity and produces contractile and relaxant responses in intestinal smooth muscle. Our objective in this study is to investigate the effects of neurotensin in small intestinal interstitial cells of Cajal (ICC) and elucidate the mechanism. To determine the electrophysiological effects of neurotensin on ICC, whole-cell patch clamp recordings were performed in cultured ICC from the small intestine. Exposure to neurotensin depolarized the membrane of pacemaker cells and produced tonic inward pacemaker currents. Only neurotensin receptor1 was identified when RT-PCR and immunocytochemistry were performed with mRNA isolated from small intestinal ICC and c-Kit positive cells. Neurotensin-induced tonic inward pacemaker currents were blocked by external Na⁺- free solution and in the presence of flufenamic acid, an inhibitor of non-selective cation channels. Furthermore, neurotensin-induced action is blocked either by treatment with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, a phospholipase C inhibitor, or thapsigargin, a Ca²⁺-ATPase inhibitor in ICC. We found that neurotensin increased spontaneous intracellular Ca²⁺ oscillations as seen with fluo4/AM recording. These results suggest that neurotensin modulates pacemaker currents via the activation of non-selective cation channels by intracellular Ca²⁺-release through neurotensin receptor1.     

The role of nitric oxide and l-type calcium channel blocker in the contractility of rabbit ileum in vitro

Nitric oxide (NO) and calcium channel blockers are two agents that can affect gastrointestinal motility. The goal of this work was to study the rabbit intestinal smooth muscle contraction response to (1) sodium nitroprusside (SNP), the NO donor, and its potential mechanism of action, and (2) nifedipine, the l-type Ca²⁺ channel blocker; to clarify the degree of participation by extra- and intracellular Ca²⁺ in smooth muscle contraction. We used standard isometric tension and intracellular micro-electrode recordings. To record the activity of the longitudinal smooth muscle of the ileum, segments of 1.5?cm length of the ileum were suspended vertically in organ baths of Krebs solution. The mechanical activity of the isolated ileal longitudinal muscle was recorded. Different substances were added, and the changes produced on spontaneous contraction were recorded. We found that SNP produced significant decrease, while nitric oxide synthase inhibitor produced significant increase in the amplitude of spontaneous contractions. Both apamin, the Ca²⁺-dependent K⁺ channel blocker, and methylene blue, the inhibitor of soluble guanylate cyclase, alone, partially decreased relaxation induced by SNP. Addition of both methylene blue and apamine together abolished the inhibitory effect produced by SNP on spontaneous contractions. Nifedipine produced significant decrease in the amplitude of spontaneous contractions. In conclusion, in longitudinal muscle of rabbit ileum, calcium channels blocker are potent inhibitors of spontaneous activity. However, both extracellular and intracellular Ca²⁺ participates in the spontaneous contractions. NO also has inhibitory effect on spontaneous activity, and this effect is mediated by cGMP generation system and Ca²⁺-dependent K⁺ channels.     

The K+ channel KZM2 is involved in stomatal movement by modulating inward K+ currents in maize guard cells

Stomata are the major gates in plant leaf that allow water and gas exchange, which is essential for plant transpiration and photosynthesis. Stomatal movement is mainly controlled by the ion channels and transporters in guard cells. In Arabidopsis, the inward Shaker K⁺ channels, such as KAT1 and KAT2, are responsible for stomatal opening. However, the characterization of inward K⁺ channels in maize guard cells is limited. In the present study, we identified two KAT1‐like Shaker K⁺ channels, KZM2 and KZM3, which were highly expressed in maize guard cells. Subcellular analysis indicated that KZM2 and KZM3 can localize at the plasma membrane. Electrophysiological characterization in HEK293 cells revealed that both KZM2 and KZM3 were inward K⁺ (K_in) channels, but showing distinct channel kinetics. When expressed in Xenopus oocytes, only KZM3, but not KZM2, can mediate inward K⁺ currents. However, KZM2 can interact with KZM3 forming heteromeric K_in channel. In oocytes, KZM2 inhibited KZM3 channel conductance and negatively shifted the voltage dependence of KZM3. The activation of KZM2–KZM3 heteromeric channel became slower than the KZM3 channel. Patch‐clamping results showed that the inward K⁺ currents of maize guard cells were significantly increased in the KZM2 RNAi lines. In addition, the RNAi lines exhibited faster stomatal opening after light exposure. In conclusion, the presented results demonstrate that KZM2 functions as a negative regulator to modulate the K_in channels in maize guard cells. KZM2 and KZM3 may form heteromeric K_in channel and control stomatal opening in maize.     

Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels

The cellular mechanisms that regulate potassium (K⁺) channels in guard cells have been the subject of recent research, as K⁺ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca²⁺ concentrations, Ca²⁺ buffers and GTP-binding protein modulators on inward-rectifying K⁺ channels. Ca²⁺ inhibition of inward-rectifying K⁺ currents depended strongly on the concentration and effectiveness of the Ca²⁺ buffer used, indicating a large Ca²⁺ buffering capacity and pH increases in guard calls. When the cytosolic Ca²⁺ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K⁺ channels were strongly inhibited. However, when EGTA was used as the Ca²⁺ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca²⁺. Under the imposed conditions, GTPγS did not significantly inhibit inward-rectifying K⁺ channel currents when cytosolic Ca²⁺ was buffered to low levels or when using EGTA as the Ca²⁺ buffer. Furthermore, GDPβS reduced inward K⁺ currents at low cytosolic Ca²⁺, indicating a novel mode of inward K⁺ channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca²⁺ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward-rectifying K⁺ channel currents in a population of cells, indicating possible Ca²⁺-dependent action of GTP-binding protein modulators in K⁺ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K⁺ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K⁺ channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of proton-pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G-protein modulators regulate inward K⁺ channels in a more complex and limited, possibly Ca²⁺-dependent, manner than previously proposed.     

Inward and outward currents of native and cloned K(ATP) channels (Kir6.2/SUR1) share single-channel kinetic properties

BackgroundThe ATP-sensitive K⁺ (K(ATP)) channel is found in a variety of tissues extending from the heart and vascular smooth muscles to the endocrine pancreas and brain. Common to all K(ATP) channels is the pore-forming subunit Kir6.x, a member of the family of small inwardly rectifying K⁺ channels, and the regulatory subunit sulfonylurea receptor (SURx). In insulin secreting β-cells in the endocrine part of the pancreas, where the channel is best studied, the K(ATP) channel consists of Kir6.2 and SUR1. Under physiological conditions, the K(ATP) channel current flow is outward at membrane potentials more positive than the K⁺ equilibrium potential around ?80 mV. However, K(ATP) channel kinetics have been extensively investigated for inward currents and the single-channel kinetic model is based on this type of recording, whereas only a limited amount of work has focused on outward current kinetics.MethodsWe have estimated the kinetic properties of both native and cloned K(ATP) channels under varying ionic gradients and membrane potentials using the patch-clamp technique.ResultsAnalyses of outward currents in K(ATP) and cloned Kir6.2ΔC26 channels, alone or co-expressed with SUR1, show openings that are not grouped in bursts as seen for inward currents. Burst duration for inward current corresponds well to open time for outward current.ConclusionsOutward K(ATP) channel currents are not grouped in bursts regardless of membrane potential, and channel open time for outward currents corresponds to burst duration for inward currents.     

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.     

Voltage-dependent L-type Ca channels and a novel type of non-selective cation channel activated by cAMP-dependent phosphorylation in mesoderm-like (MES-1) cells

Undifferentiated P19 embryonal carcinoma cells (ECC P19), the P19-derived clonal cell lines END-2 (visceral endoderm-like), EPI-7 (epithelioid ectoderm-like), MES-1 (mesoderm-like) and a parietal yolk sac cell line (PYS-2) were used as cellular models to examine the functional expression of voltage-dependent Ca channels and other Ca-permeable cation channels at various stages of early embryonic development. Whole-cell currents were recorded by means of the patch clamp technique. Whereas more than 75% of MES-1 cells possessed Ca channel currents, neither P19, END-2, EPI-7 nor PYS-2 cells had detectable voltage-dependent inward currents. Ca channel currents of MES-1 cells were highly sensitive towards 1,4-dihydropyridines and blocked by cadmium. Adrenaline (10 μM) caused Ca channel stimulation in only 14% of MES-1 cells examined. However, in 62% of the cells adrenaline activated a linear current component which under physiological conditions reversed close to 0 mV. Removal of extracellular Na⁺ suppressed the adrenaline-induced inward current, while reducing extracellular Cl⁻ had no significant effect. These findings suggest that the adrenaline-induced current is carried through non-selective cation channels which were found to be permeable for Na⁺, K⁺, Cs⁺ å Ca²⁺. Remarkably, the intracellular signalling pathway for activation of the non-selective cation current involved the cascade of reactions leading to cAMP-dependent phosphorylation, a regulatory pathway well known for cardiac Ca channels. A possible functional role of adrenaline-induced non-selective cation currents and Ca channels in embryonal development is discussed.     

Cell swelling activates K+ and Cl− channels as well as nonselective,stretch-activated cation channels in ehrlich ascites tumor cells

Summary Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba²⁺ and hence presumably to Ca²⁺. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca²⁺ permeability, as part of the volume-activated signal system. In isolated insideout patches a Ca²⁺-dependent, inwardly rectifying K⁺ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mm K⁺ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K⁺ channel takes place after an increase in Ca²⁺ from 10^–7 to 10^–6 m which is in the physiological range. Patch-clamp studies in cellattached mode show K⁺ channels with spontaneous activity and with characteristics similar to those of the K⁺ channel seen in excised patches. The single-channel conductance for outward current at 5 mm external K⁺ is estimated at about 7 pS. A K⁺ channel with similar properties can be activated in the cellattached mode by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K⁺ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K⁺ channels following addition of Ca²⁺ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K⁺ channels in the presence of 3 mm Ca²⁺ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca²⁺ and could not be activated by membrane stretch (suction). In cell-attached mode singlechannel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by hypotonic exposure with a single-channel conductance at 7 pS (or less) and with a time delay at about 1 min. The number of open channels during RVD is estimated at 80 per cell. Two other types of Cl^– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl^– channel which show properties similar to the Cl^– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two channels.     

Control of urinary bladder smooth muscle excitability by the TRPM4 channel modulator 9-phenanthrol

The Ca²⁺-activated monovalent cation selective transient receptor potential melastatin 4 (TRPM4) channel has been recently identified in detrusor smooth muscle (DSM) of the urinary bladder. Two recent publications by our research group provide evidence in support of the novel hypothesis that TRPM4 channels enhance DSM excitability and contractility. This is a critical question as prior studies have primarily targeted hyperpolarizing currents facilitated by K⁺ channels, but the depolarizing component in DSM cells is not well understood. For the first time, we utilized the selective TRPM4 channel inhibitor, 9-phenanthrol, to investigate TRPM4 channel functional effects in DSM at both cellular and tissue levels in rodents. Our new data presented here showed that in rat DSM cells, 9-phenanthrol attenuates spontaneous inward currents in the presence of the muscarinic receptor agonist, carbachol, thus reducing DSM cell excitability. In support of our original hypothesis, we found that TRPM4 channel mRNA levels are much higher in DSM vs. vascular smooth muscle and that inhibition of TRPM4 channels can potentially attenuate DSM excitability. Thus, we postulate the novel concept that selective pharmacological inhibition of TRPM4 channels can limit both excitability and contractility of DSM.     

Macroscopic Ca2+ -Na+ and K+ currents in single heart and aortic cells

Summary The whole-cell voltage clamp technique was used to study the slow inward currents and K⁺ outward currents in single heart cells of embryonic chick and in rabbit aortic cells. In single heart cells of 3-day-old chick embryo three types of slow inward Na⁺ currents were found. The kinetics and the pharmacology of the slow I_Na, were different from those of the slow Ica in older embryos. Two types of slow inward currents were found in aortic single cells of rabbit; angiotensin 11 increased the sustained type and d-cAMP and d-cGMP decreased the slow transient component. Two types of outward K⁺ currents were found in both aortic and heart cells. Single channel analysis demonstrated the presence of a high single K⁺ channel conductance in aortic cells. In cardiac and vascular smooth muscles, slow inward currents do share some pharmacological properties, although the regulation of these channels by cyclic nucleotides and several drugs seems to be different.     

Voltage-sensitive chloride ion channels in Anopheles gambiae Sua-1B cells

In this study, we performed electrophysiological analysis of Anopheles gambiae Sua-1B cells having “neuron-like” morphologies using the patch clamp method. The recorded cells (n = 79) had processes resembling axons/dendrites, with 63 % unipolar, 22 % bipolar, and 15 % multipolar. While no inward currents were observed following step depolarizations (holding potential = ?80 mV), a slowly activating outward current was observed in 96 % of the cells, especially at depolarized potentials. The amplitude of the current was attenuated nearly 70 % by reducing extracellular Cl^? ion concentration, or by incubating with 100 μM DIDS, a known voltage-sensitive chloride channel blocker, suggesting that the current was mediated by chloride ions. No qualitative difference was found between recordings made with Cs⁺ ions in the intracellular pipette solution (inhibits K⁺ currents) and those made with normal physiological solution, indicating a deficiency of potassium channels. Additionally, recordings made with Ca²⁺-free extracellular bath solution eliminated the slowly activating outward current. A subset of cells (n = 3) lacked this current, but had outward currents with voltage-dependent properties similar to those of volume-regulated chloride channels. Taken together, our results suggest that the voltage-sensitive currents observed in the majority of Sua-1B cells are mediated primarily by chloride channels of the calcium-dependent subtype.     

Voltage-dependent and Src-mediated regulation of NMDA receptor single channel outward currents in cortical neurons

A voltage-dependent but Ca²⁺-independent regulation of N-methyl-D-aspartate (NMDA) receptor outward activity was studied at the single channel level using outside-out patches of cultured mouse cortical neurons. Unlike the inward activity associated with Ca²⁺ and Na⁺ influx, the NMDA receptor outward K⁺ conductance was unaffected by changes in Ca²⁺ concentration. Following a depolarizing pre-pulse, the single channel open probability (NP _o), amplitude, and open duration of the NMDA inward current decreased, whereas the same pre-depolarization increased those parameters of the NMDA outward current (pre-pulse facilitation). The outward NP _o was increased by the pre-pulse facilitation, disregarding Ca²⁺ changes. The voltage–current relationships of the inward and outward currents were shifted by the pre-depolarization toward opposite directions. The Src family kinase inhibitor, PP1, and the Src kinase antibody, but not the anti-Fyn antibody, blocked the pre-pulse facilitation of the NMDA outward activity. On the other hand, a hyperpolarizing pre-pulse showed no effect on NMDA inward currents but inhibited outward currents (pre-pulse depression). Application of Src kinase, but not Fyn kinase, prevented the pre-pulse depression. We additionally showed that a depolarization pre-pulse potentiated miniature excitatory synaptic currents (mEPSCs). The effect was blocked by application of the NMDA receptor antagonist AP-5 during depolarization. These data suggest a voltage-sensitive regulation of NMDA receptor channels mediated by Src kinase. The selective changes in the NMDA receptor-mediated K⁺ efflux may represent a physiological and pathophysiological plasticity at the receptor level in response to dynamic changes in the membrane potential of central neurons.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Inward Rectifying K Channels in the Plasma Membrane of Arabidopsis thaliana

Ion channels in the plasma membrane of protoplasts isolated from cultured cells of Arabidopsis thaliana were studied by means of the patch-clamp technique applied in the whole-cell configuration. In some protoplasts, depolarizing pulses and, in other protoplasts, hyperpolarizing pulses elicited time-dependent currents; both kinds of current were only rarely observed in the same protoplast. The hyperpolarization-activated inward rectifying currents, the focus of this paper, appeared to be due to the relatively slow opening of channels (activation time constant = 150 to 300 milliseconds), which closed at positive potentials. The reversal potential of this current, measured in the presence of different ion concentrations (symmetrical or asymmetrical K⁺ and Cl⁻ or gluconate), was always close to the electrochemical equilibrium potential of K⁺. The currents were inhibited by 10 millimolar tetraethylammonium, a K⁺ channel blocker. These data show that the hyperpolarization-activated currents flow through K⁺ channels, which can provide a pathway for the passive diffusion of K⁺ down its electrochemical gradient.