Expression of an inwardly rectifying K+ channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

Hyperpolarization-activated Cl current elicited by pituitary adenylate cyclase activating polypeptide in Xenopus oocytes

We examined the electrophysiological effect of pituitary adenylate cyclase activating polypeptide (PACAP) in isolated Xenopus laevis oocytes in vitro. In conventional two-electrode voltage clamp experiments, PACAP (1–10 μM) activated an inward rectifier current at membrane potentials more negative than −60 mV without causing any significant change in currents at potentials more positive than −60 mV both in the follicle-enclosed oocyte and in the defolliculated oocyte. This current reversed at −22.5 mV, close to the theoretical value of Cl⁻ equilibrium potential and the reversal potential of this current was shifted positively by reducing [Cl⁻]_o. This current was blocked by Cl⁻ channel blocker SITS and Ba²⁺. Furthermore, VIP and adenylate cyclase activator forskolin did not elicit the currents. In conclusion, PACAP elicited the hyperpolarization-activated Cl⁻ current in Xenopus laevis oocytes. This current may modulate the membrane potential of the oocyte, thereby affecting the oocyte physiology.     

Novel ion channels in the protists, Mougeotia and Saprolegnia, using sub-gigaseals.

Protoplasts of the filamentous alga, Mougeotia, and the filamentous fungal oomycete, Saprolegnia ferax, exhibit two K⁺ ion channels (2–6 pA) using the patch-clamp technique when the seals are less than 1 GΩ (about 100 MΩ). The membrane potential of the protoplasts was near 0 mV as measured intracellularly with double-barreled micropipettes; thus, inward K⁺ flux is due solely to concentration differences. Although conductances are in the range expected for K⁺ channels, the activity at 0 mV is not seen in other organisms under gigaseal conditions. This paper draws attention to the usefulness of this subsidiary patch-clamp technique and the novel characteristics of ion channels in Mougeotia and Saprolegnia.     

A negative resistance region underlies the triggering property of membrane potential in human T-lymphocytes

Steady-state current-voltage relationships (SSCVRs) of the plasma membrane of human T-lymphocytes were studied at the physiological temperature of 37°C by using the whole-cell patch-clamp technique. SSCVRs displayed a characteristic N-like shape with a negative resistance region (NRR) in a voltage range of −45 to −35 mV. The majority of cells assayed revealed SSCVR patterns crossing the V-axis at three points (in mV): V₁ = −55 to −45, V₂ = −40 to −35, V₃ = −30 to −10. SSCVRs of T-cells activated by phytohaemagglutinin (48–96 h) also displayed NRR, but crossed the V-axis at one point only (V₁ = −55 to −60 mV). It implies the possibility of two stable levels of membrane potential (V₁ and V₃) for the resting T-cells, but only one (V₁) for activated T-cells. These data thus account for the triggering property of T-cell membrane potential previously reported. The NRR can be explained on the basis of the Hodgkin-Huxley type n⁴j model of K⁺ channel kinetics. According to the model the possibility for a membrane to have on or two stable levels of membrane potential depends on the ratio of selective K⁺ conductance to non-selective leaky conductance (G_k/G_leak). The steady-state level of K⁺ conductance in resting T-lymphocytes proved to be sensitive to Ca²⁺. Buffering Ca²⁺ ions from either external or internal solution resulted in an appreciable increase in K⁺ conductance. The possibility for membrane potential have two stable levels of membrane potential in connection with the Ca²⁺ dependence of K⁺ conductance was supposed to be important for Ca²⁺-signalling during T-cell activation.     

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.     

Electrophysiology of mammalian Schwann cells

Schwann cells are the satellite cell of the peripheral nervous system, and they surround axons and motor nerve terminals. The review summarises evidence for the ion channels expressed by mammalian Schwann cells, their molecular nature and known or speculated functions. In addition, the recent evidence for gap junctions and cytoplasmic diffusion pathways within the myelin and the functional consequences of a lower-resistance myelin sheath are discussed.

The main types of ion channel expressed by Schwann cells are K⁺ channels, Cl⁻ channels, Na⁺ channels and Ca²⁺ channels. Each is represented by a variety of sub-types. The molecular and biophysical characteristics of the cation channels expressed by Schwann cells are closely similar or identical to those of channels expressed in peripheral axons and elsewhere. In addition, Schwann cells express P₂X ligand-gated ion channels. Possible in vivo roles for each ion channel type are discussed. Ion channel expression in culture could have a special function in driving or controlling cell proliferation and recent evidence indicates that some Ca²⁺ channel and Kir channel expression in culture is dependent upon the presence of neurones and local electrical activity.     

Effects of Tetraethylammoniumchloride (TEA), Vanadate, and Alkali Ions on the Lateral Leaflet Movement Rhythm of Desmodium motorium (Houtt.) Merr

The period (∼3-5 min) of the ultradian rhythm of the lateral leaflet movement of Desmodium motorium is strongly lengthened (≤30-40%) by the K⁺ channel blocker tetraethylammoniumchloride (20, 30, and 40 mM) and vanadate (0.5 and 1 mM), which is an effective inhibitor of the plasma membrane-bound H⁺ pump. The alkali ions K⁺, Na⁺, Rb⁺, and Cs⁺ (10-40 mM) shorten the period only slightly (≤ 10-15%). Li⁺ (5-30 mM), however, increases the period of the leaflet rhythm drastically (≤80%). We concluded that the plasmalemma-H⁺-ATP-ase-driven K⁺ transport through K⁺ channels is an essential component of the ultradian oscillator of Desmodium, as has been proposed for the circadian oscillator.     

Dynamic properties of stretch-activated K+ channels in adult rat atrial myocytes

The effect of mechanical stress on the heart's electrical activity has been termed mechanoelectric feedback. The response to stretch depends upon the magnitude and the waveform of the stimulus, and upon the timing relative to the cardiac cycle. Stretch-activated ion channels (SACs) have been regarded as the most likely candidates for serving as the primary transducers of mechanical stress. We explored the steady state and dynamic responses of single channels in adult rat atrial cells using the patch clamp with a pressure clamp. Surprisingly, we only observed K⁺-selective SACs, probably of the 2P domain family. The channels were weakly outward rectifying with flickery bursts. In cell attached mode, the mean conductance was 74±14 and 65±16 pS for +60 and −60 mV, respectively (140 mM [K⁺]_out, 2 mM [Mg²⁺]_out and 0 mM [Ca²⁺]_out). The latency of the response to pressure steps was 50–100 ms and the time to peak 400 ms. About half of the channels in cell-attached patches showed adaptation/inactivation where channel activity declined to a plateau of 20–30% of peak in 1 s. The time dependent behavior of these SACs is generally consistent with whole-cell currents observed in chick and rat ventricular cells, although the net current was outward rather than inward.     

Transient hyperpolarization of yeast by glucose and ethanol

At pH 7, addition of glucose under anaerobic conditions to a suspension of the yeast Saccharomyces cerevisiae causes both a transient hyperpolarization and a transient net efflux of K⁺ from the cells. Hyperpolarization shows a peak at about 3 min and a net K⁺ efflux at 4–5 min. An additional transient hyperpolarization and net K⁺ efflux are found after 60–80 and 100 min, respectively. Addition of 2-deoxyglucose instead of glucose does not lead to hyperpolarization of the cells or K⁺ efflux. At low pH, neither transient hyperpolarization nor a transient K⁺ efflux are found. With ethanol as substrate and applying aerobic conditions, both a transient hyperpolarization and a transient K⁺ efflux are found at pH 7. The fluorescent probe 2-(dimethylaminostyryl)-1-ethylpyridinium appears to be useful for probing changes in the membrane potential of S. cerevisiae. It is hypothesized that the hyperpolarization of the cells is due to opening of K⁺ channels in the plasma membrane. Accordingly, the hyperpolarization of the cells at pH 7 is almost completely abolished by 1.25 mM K⁺, whereas the same amount of Na⁺ does not reduce the hyperpolarization     

植物K+通道AKT1的研究进展

钾(K)是植物生长发育必需的大量营养元素之一, 主要通过根细胞的K⁺通道及转运蛋白介导吸收。AKT1是Shaker型K⁺通道家族的重要成员, 在植物根吸收K⁺和体内跨膜转运中发挥重要作用。该文综述了植物AKT1的分子结构、组织特异性表达、调控机制及生物学功能等方面的研究进展, 并对该通道今后的研究方向进行了展望。     

Ca entry through the store-mediated pathway directly activates only the K current but the subsequent Ca release from the store activates both K and Cl currents in submandibular gland acinar cells of the rat

The store-mediated Ca²⁺ entry was detected in single and cluster of rat submandibular acinar cells by measuring the Ca²⁺ activated ionic membrane currents. In the cells where intracellular Ca²⁺ was partly depleted by stimulation with submaximal concentration of acetylcholine (ACh) under a Ca²⁺-free extracellular condition, an employment of external Ca²⁺ in the absence of ACh caused a sustained increase of the K⁺ current without affecting the Cl⁻ current. A renewed ACh challenge without external Ca²⁺ caused repetitive spikes of both K⁺ and Cl⁻ currents due to the Ca²⁺ release. SK & F 96365 inhibited the generation of the sustained K⁺ current and refilling of the Ca²⁺ store following the Ca²⁺ readmission. It is suggested that the Ca²⁺ enters the cell through the store-mediated pathway near the K⁺ channels and is taken up by the store. Thus, only Ca²⁺ released from the store can activate both the K⁺ and Cl⁻ currents.     

Trafficking of the plant potassium inward rectifier KAT1 in guard cell protoplasts of Vicia faba

Trafficking of K+ inward (Kin+) rectifying channels was analyzed in guard cells of Vicia faba transfected with the Kin+ rectifier from Arabidopsis thaliana KAT1 fused to the green fluorescent protein (GFP). Confocal images and whole-cell patch-clamp measurements confirmed the incorporation of active KAT1 channels into the plasma membrane of transfected guard cell protoplasts. The Kin+ rectifier current density of the plasma membrane was much larger in transfected protoplasts than in wild-type (wt) protoplasts. This shows a coupling between K+ channel synthesis and incorporation of the channel into the plasma membrane. Pressure-driven increase and decrease in surface area led to the incorporation and removal of vesicular membrane carrying active Kin+ rectifier in wt and transfected protoplasts. These vesicular membranes revealed a higher channel density than the plasma membrane, suggesting that Kin+ rectifier remains in clusters during trafficking to and from the plasma membrane. The observed results can be explained by a model illustrating that vesicles of a pre-plasma membrane pool carry K+ channels preferentially in clusters during constitutive and pressure-driven exo- and endocytosis.     

The blocking effect of BmP02, one novel short-chain scorpion peptide on transient outward K(+) channel of adult rat ventricular myocyte

Two components F-2-7-4 and F-2-7-5, each composed of 28 amino acid residues, were purified from the venom of Buthus martensi Karsch by an opportune procedure with cation-exchange column chromatography and repeated HPLC. Both components were totally accounted to about 0.88% dry weight of the crude venom.The molecular weights of both components were determined to be 2950 and 2935 by mass spectrometry, which were fully coincidence with that of the known novel short-chain peptides BmP02 and BmP03, respectively [Romi-Lebrun R, Martin-Eauclaire M-F, Escoubas P, Wu FQ, Lebrun B, Hisada M, Nakajima T. Characterization of four toxins from Buthus martensi scorpion venom, which act on apamin-sensitive Ca²⁺-activated K⁺ channels. Eur J Biochem 1997;145:457–464]. In addition, the sequence of component F-2-7-4 was analyzed to be the same as that of BmP02. The components F-2-7-4 and F-2-7-5 purified in this study were, thus, finally distinguished to be BmP02 and BmP03 from the same venom. Using whole cell patch-clamp recording, it was found that BmP02 diminished the current of transient outward K⁺ channel in adult rat ventricular myocyte in a concentration-dependent manner. The inhibitory effect was reversible. Dynamic studies showed that the activation, inactivation and recovery processes of the transient outward K⁺ channel were not changed significantly after applying of BmP02. In addition, when BmP02 was applied to guinea pig ventricular myocyte, both delayed and inward rectified K⁺ currents showed no change compared with the control. The results suggest strongly that BmP02 or -like peptides from scorpion venom may provide a useful probe for the studying of transient outward K⁺ channel in rat ventricular myocyte.     

Molecular coupling between voltage sensor and pore opening in the Arabidopsis inward rectifier K+ channel KAT1

Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (tau = 318 +/- 34 micros at -180 mV) and a slower component (4.5 +/- 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible ( approximately 10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage-sensing charges of S4 move inward when the KAT1 channels open.     

Purification and partial characterization of K+ channel blockers from the venom of Dendroaspis angusticeps.

Two polypeptides (designated DTX-A and DTX-B) were purified from crude snake venom of Dendroaspis angusticeps using gel filtration, cation exchange colum chromatography and cation exchange high performance liquid chromatography, and their blocking actions of K⁺ channels were investigated in rat brain synaptosomes. Both DTX-A and DTX-B inhibited the voltage-dependent ⁴²K efflux from the synaptosomes. DTX-A blocked ⁴²K efflux of both the rapidly inactivating phase (component T) and the slowly inactivating phase (component S). The inhibitory effect of DTX-A on component T was pronounced compared with that on component S. However, DTX-B selectively blocked ⁴²K efflux of component S. The molecular weights of DTX-A and DTX-B were estimated to be ca 10,000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition of these toxins is different from that of polypeptide purified from the venom of D. angusticeps (-, β, γ- and δ-DTX). These results suggest that DTX-A and DTX-B are new polypeptides which block voltage-dependent K⁺ channels selectively, and that they are useful tools for investigating the K⁺ channel.     

The sodium cycle. II. Na-coupled oxidative phosphorylation in Vibrio alginolyticus cells

The role of Na⁺ in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na⁺-motive respiratory chain of V. alginolyticus. Na⁺ loaded cells incubated in a K⁺ or Li⁺ medium fail to synthesize ATP in response to lactate addition. The addition of Na⁺ at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na⁺ level. Neither lactate oxidation nor Δω generation coupled with this oxidation is increased by external Na⁺ in the Na⁺-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse ΔPNa, i.e., [Na⁺]_in > [Na⁺]_out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K⁺-loaded cells incubated in a high Na⁺ medium, i.e., when ΔpNa of the proper direction ([Na⁺]_in < [Na⁺]_out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K⁺ medium (ΔpNa is low) is decreased by CCCP even without monensin. Artificial formation of ΔpNa by adding 0.25 M NaCl to the K⁺-loaded cells (Na⁺ pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na⁺ pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na⁺ instead of H⁺ as the coupling ion.     

Simulation analysis of intracellular Na+ and Cl- homeostasis during beta 1-adrenergic stimulation of cardiac myocyte

To quantitatively understand intracellular Na⁺ and Cl⁻ homeostasis as well as roles of Na⁺/K⁺ pump and cystic fibrosis transmembrane conductance regulator Cl⁻ channel (I_CFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca²⁺ transient and cell shortening, and the decreases in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. Increasing the density of I_CFTR shortened APD and augmented the peak amplitudes of the L-type Ca²⁺ current (I_CaL) and the Ca²⁺ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I_CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na⁺ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na⁺ affinity of Na⁺/K⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at higher beating rate because of larger Na⁺ influx through forward Na⁺/Ca²⁺ exchange. It was demonstrated that dynamic changes in Na⁺ and Cl⁻ fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.     

Purification and reconstitution of potassium channel proteins from squid axon membranes

Voltage-dependent K⁺ channels are responsible for repolarization of the cell membrane during the late phase of the action potential. Here we report the purification of proteins from squid axon membranes which bind the K⁺-channel blocker noxiustoxin (NTX), and their subsequent functional reconstitution in planar bilayers. The NXT-affinity purified proteins had M_r values of 60000 ± 6000, 160000 ± 15000 and 220000 ± 20000. Their incorporation into bilayers resulted in single-channel currents with three conductances, the most frequent one of 11 pS in 300/100 mM KCl (cis/trans). The voltage dependence, reversal potential and bursting behavior suggest that these are the K⁺ channels involved in the squid axon action potential.     

The BK channel: a vital link between cellular calcium and electrical signaling

Large-conductance Ca²⁺-activated K⁺ channels (BK channels) constitute an key physiological link between cellular Ca²⁺ signaling and electrical signaling at the plasma membrane. Thus these channels are critical to the control of action potential firing and neurotransmitter release in several types of neurons, as well as the dynamic control of smooth muscle tone in resistance arteries, airway, and bladder. Recent advances in our understanding of K⁺ channel structure and function have led to new insight toward the molecular mechanisms of opening and closing (gating) of these channels. Here we will focus on mechanisms of BK channel gating by Ca²⁺, transmembrane voltage, and auxiliary subunit proteins.