豚鼠耳蜗离体外毛细胞的膜电位和离子电流

利用膜片钳技术对分离的豚鼠耳蜗外毛细胞进行了研究,结果表明：（１）新分离的正常ＯＨＣ呈术状,胞膜光滑,胞核位于底部,静纤毛由顶端表皮板伸出,４小时内形态无明显变化。（２）全细胞电压钳记录结合通道阻断剂实验表明,ＯＨＣ膜电流主要由电压依赖性钾离子流组成。（３）利用全细胞记录方式得到的ＯＨＣ静息电位值为－２６±９ｍＶ．     

豚鼠耳蜗离体外毛细胞的膜电位和离子电流

利用膜片钳技术对分离的豚鼠耳蜗外毛细胞(OHC)进行了研究,结果表明:(1)新分离的正常OHC呈柱状,胞膜先滑,胞核位于底部,静纤毛由顶端表皮板伸出,4小时内形态无明显变化.(2)全细胞电压钳记录结合通道阻断剂实验表明,OHC膜电流主要由电压依赖性钾离子流组成.(3)利用全细胞记录方式得到的OHC静息电位值为-26±9mV((?)±SD,n=10).     

Thiamine triphosphate activates an anion channel of large unit conductance in neuroblastoma cells

In neuroblastoma cells, the intracellular thiamine triphosphate (TTP) concentration was found to be about 0.5 m, which is several times above the amount of cultured neurons or glial cells. In inside-out patches, addition of TTP (1 or 10) m to the bath activated an anion channel of large unit conductance (350–400 pS) in symmetrical 150 mm NaCl solution. The activation occurred after a delay of about 4 min and was not reversed when TTP was washed out. A possible explanation is that the channel has been irreversibly phosphorylated by TTP. The channel open probability (P _o) shows a bell-shaped behavior as a function of pipette potential (V _p). P _o is maximal for –25 mV<V _p<10 mV and steeply decreases outside this potential range. From reversal potentials, permeability ratios of P_Cl/ P_Na = 20 and P_Cl/P_gluconate = 3 were estimated. ATP (5 mm) at the cytoplasmic side of the channel decreased the mean single channel conductance by about 50%, but thiamine derivatives did not affect unit conductance; 4,4 -diisothiocyanostilbene-2,2-disulfonic acid (0.1 mm) increased the flickering of the channel between the open and closed state, finally leading to its closure. Addition of oxythiamine (1 mm), a thiamine antimetabolite, to the pipette filling solution potentiates the time-dependent inactivation of the channel at V _p=–20 mV but had the opposite effect at +30 mV. This finding corresponds to a shift of P _o towards more negative resting membrane potentials. These observations agree with our previous results showing a modulation of chloride permeability by thiamine derivatives in membrane vesicles from rat brain.We would like to thank the National Funds for Scientific Research (Belgium) for financially supporting the stay of L.B. in Konstanz. We wish to thank A. Ngezahayo, F. Mendez and Dr. P. Wins for helpful discussions. This work was in part supported by a research grant from the Fonds special pour la Recherche à l'Université de Liege to L.B., the SFB 156 of the DFG and a grant of the Hermann and Lilly Schilling Stiftung to H.-A.K. Neuroblastoma, PC-12 and glioma cell lines were a gift from Prof. G. Moonen (Department of Human Physiology, University of Liège).     

Structural features of the lateral walls in mammalian cochlear outer hair cells

Summary Freeze-fracture, freeze-etching and thin sections have been used to determine features of the structural organisation of the lateral walls in cochlear outer hair cells. The presence of an organised meshwork of filaments in the lateral cortex of the cell is confirmed in intact unfixed cells. This meshwork showed morphological features similar to the cytoskeletal lattice. The lateral plasma membrane is shown to be protein-rich and to contain cholesterol. The membranes of the subplasmalemmal lateral cisternae contain much less protein, and little cholesterol as judged by their responses to filipin and tomatin. These findings indicate differences in the physical properties of the two membrane systems. On the fracture faces of the plasma membrane there is a high density of intramembrane particles and this particle population is heterogeneous. Some particles show morphological features consistent with those of transmembrane channels. Regularly spaced pillars crossing the space between the plasma and cisternal membranes were identified both in thin sections and in freezeetched preparations, but neither the plasma nor cisternal membrane fracture faces showed any feature corresponding directly to the pillar. This suggests the pillars do not insert directly into either membrane. Freeze-fracture and freeze-etching of unfixed cells indicated that the pillar is indirectly associated with the cytoplasmic surface of the plasma membrane, and, at its inner end, linked to the cortical cytoskeletal lattice on the outer surface of the cisternal membrane.     

Modulation of small conductance calcium-activated potassium channels in C6 glioma cells

Using the patch clamp technique, we have characterized a small conductance, calcium-activated potassium (SK) channel in the C6 glioma cell line. Elevation of cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by applications of serotonin or ionomycin induced bursts of channel openings recorded in the cell-attached configuration. These channels underlie the serotonin-induced, [Ca²⁺] _i -activated whole-cell K⁺ conductance described previously. [Ca²⁺] _i directly activated SK channels in inside-out patches with a biphasic concentration dependence. Submicromolar [Ca²⁺] _i induced bursts of channel openings with a unitary conductance of about 25 pS, similar to that of the serotonin-induced channels. Supramicromolar [Ca²⁺] _i caused prolonged openings with a unitary conductance of about 35 pS, resulting in a pronounced increase of the average current in patches exposed to [Ca²⁺] _i above 100 m. The two modes of opening reflect the activity of the same SK channel. The channel conductance depended on external K⁺ concentration with K _Dof 5 m. The channel was slightly permeable to cations other than K⁺, with a permeability ratio for K⁺Ca²⁺Na⁺ of 10.0400.030, respectively. ATP was required to maintain channel activity in outside-out patches but was not essential in inside-out patches. The modulation of SK channels in C6 cells by components in their microenvironment may be related to the role of glial cells in controlling the extracellular milieu in the CNS.The authors are grateful to Dr. M. Segal for continuous support, stimulating discussions and criticism throughout the course of this work, to Dr. I. Steinberg for helpful suggestions and to Dr. H. Jarosch, for helping with the Fortran application. N.M.'s research was supported in part by BARD, the U.S.-Israel Binational Agricultural Research and Development Fund, grant no. IS-1670-89RC.     

Sodium ionic and gating currents in mammalian cells

Ionic and gating currents from voltage-gated sodium channels were recorded in mouse neuroblastoma cells using the path-clamp technique. Displacement currents were measured from whole-cell recordings. The gating charge displaced during step depolarizations increased with the applied membrane potential and reached saturating levels above 20 mV Prolonged large depolarizations produced partial immobilization of the gating charge, and only about one third of the displaced charge was quickly reversed upon return to negative holding potentials. The activation and inactivation properties of macroscopic sodium currents were characterized by voltage-clamp analysis of large outside-out patches and the single-channel conductance was estimated from nonstationary noise analysis. The general properties of the sodium channels in mouse neuroblastoma cells are very similar to those previously reported for various preparations of invertebrate and vertebrate nerve cells.Offprint requests to: O. Moran     

Activation of galanin receptor 2 stimulates large conductance Ca-dependent K (BK) channels through the IP3 pathway in human embryonic kidney (HEK293) cells

The large conductance Ca²⁺-activated K⁺ (BK) channels are widely distributed in the brain, and act as intracellular calcium sensors in neurons. They play an important feedback role in controlling Ca²⁺ flux and Ca²⁺-dependent processes, including neurotransmitter release and cellular excitability. In this study, the effects of the neuropeptide galanin on BK channels were examined by determining the whole-cell currents and single-channel activities in human embryonic kidney (HEK293) cells co-expressing GalR2 and the BK alpha subunit. Galanin enhanced the currents of BK channels, in a concentration-dependent and PTX-independent manner, with an ED50 value of 71.8 ± 16.9 nM. This activation was mediated by GalR2, since its agonist AR-M1896 mimicked the effect of galanin, and since galanin did not facilitate BK currents in cells co-expressing cDNAs of BK and GalR1 or GalR3. The galanin-induced BK current persisted after replacement with Ca²⁺-free solution, suggesting that extracellular Ca²⁺ is not essential. Chelating intracellular Ca²⁺ by either the slow Ca²⁺ buffer EGTA or the fast Ca²⁺ buffer BAPTA abolished galanin-mediated activation of BK channels, indicating the important role of intracellular Ca²⁺. The role of Ca²⁺ efflux from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) was confirmed by application of thapsigargin, an irreversible inhibitor that depletes Ca²⁺ from SR/ER. Moreover, the inositol-1,4,5-triphosphate receptor (IP3R) was identified as the mediator responsible for increased intracellular Ca²⁺ activating BK channels. Taken together, activation of GalR2 leads to elevation of intracellular Ca²⁺ is due to Ca²⁺ efflux from ER through IP3R sequentially opening BK channels.     

Stretch-activated chloride,potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L.

Mechanosensitive ion channels in the plasma membrane of Vicia faba guard cell protoplasts were studied by use of the patch clamp technique. Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics and ion selectivity. We found three distinct SA channels, permeable to Cl^–, K⁺ and Ca²⁺ and distinguishable from spontaneous (non-SA) channels for these ions on the basis of conductance, kinetics, and voltage-dependence, as well as sensitivity to membrane stretch. In the attached patch configuration, light suction (2 to 10 kPa) reversibly induced channel opening with multiple amplitudes and complex kinetics. The open probability for SA channels increased nonlinearly with pipette suction. In guard cells in situ, these SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and cytoplasmic calcium. Through such effects, SA channels likely influence volume and turgor regulation of guard cells, and thereby control of leaf gas exchange.Abbreviations EK equilibrium potential for potassium transport - E_Cl equilibrium potential for chloride transport - SA stretchactivated Dedicated to the 80. birthday of Franz HedrichSupported by a grant from the Deutsche Forschungsgemeinschaft to R.H. and a Department of Energy grant to D.J.C. gratefully acknowledges a John S. Guggenheim Fellowship and Fulbright Kommission Senior Professor Award. We thank Ingrid Baumann and Angela Schön for technical assistance, and Klaus Raschke and Heiner Busch for spirited discussions and support.     

The BEACH protein LRBA is required for hair bundle maintenance in cochlear hair cells and for hearing

Lipopolysaccharide‐responsive beige‐like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain‐containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein‐truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model.     

Protein phosphorylation is required for diazoxide to open ATP-sensitive potassium channels in insulin (RINm5F) secreting cells

The patch-clamp open-cell recording configuration has been used to investigate the effects of non-hydrolyzable analogues of ATP on the diazoxide-activation of K_ATP channels in the insulin-secreting cell line RINm5F. K⁺ channels inhibited by 0.1, 0.5 and 1.0 mM ATP were consistently activated by 200 μM diazoxide. During sustained activation of channels, exchange of ATP for either AMP-PNP, AMP-PCP or ATPγS abolished the effects of diazoxide. If diazoxide was added to the membrane in the continued presence of AMP-PNP, AMP-PCP or ATPγS either no effects were observed or alternatively a small transient activation of channels occurred. This study suggests that protein phosphorylation is necessary for diazoxide to activate ATP-sensitive potassium channels in insulin-secreting cells.     

Non-selective cation and dysfunctional chloride cahnnels in the apical membrane of nasal epithelial cells cultured from cystic fibrosis patients

Chloride channels and non-selective cation channels in the apical membranes of cultured nasal epithelial cells from three cystic fibrosis patients were investigated with the patch-clamp techinique. Outwardly rectifying chloride channels were found in 31% of the inside-out patches, but activity of this channel was never observed in cell-attached patches, even after stimulation with adrenaline. In 30% of the patches with chloride channels, activation occurred immediately after excision. Most of the channels, however, activated only after a membrane depolarization of +40 to +120 mV. Once activated, the chloride channels were indistinguishable from thsoe in nasal epithelial cells of control patients. Amiloride-insensitive, calcium- and voltage-dependent, non-selective cation channels were present in 11% of the cell-attached and 43% of the cell-free patches and could not be distinguished from those in controls. The cystic fibrosis chloride channel defect is conserved in cultured nasal epithelial cells, while a non-selective cation channel is apparently not affected.     

Functional cystic fibrosis transmembrane conductance regulator tagged with an epitope of the vesicular stomatis virus glycoprotein can be addressed to the apical domain of polarized cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel apically localized in epithelial cells. In cystic fibrosis patients, the gene encoding this N-linked glycoprotein is mutated. About 70% of CF patients express a mutated form of CFTR, deleted at the phenylalanine residue at position 508 (deltaF508). CFTR-deltaF508 fails to exit the endoplasmic reticulum; it remains incompletely glycosylated and is rapidly degraded. To optimize CFTR detection for membrane localization studies and biochemical studies, we tagged wild-type and deltaF508 CFTR with the VSV-G epitope at their carboxy-terminal ends. We have generated pig kidney epithelial cell clones (LLCPK1) expressing VSV-G-tagged human wild-type and deltaF508-CFTR. In CFTR-expressing cells, the transfected protein is maturated and transported to the apical membrane where it is concentrated. The cells exhibit a strong anion channel activity after stimulation by cAMP, as demonstrated by a halide sensitive fluorescent dye assay (6-methoxy-N-ethylquinominium, SPQ), and whole-cell patch-clamp approach. This activity of CFTR-VSV-G is indistinguishable from the wild-type CFTR. In contrast, in cells expressing tagged deltaF508-CFTR or in non-transfected cells, no anion channel activity could be detected after stimulation by cAMP. In deltaF508-CFTR-VSV-G-expressing cells, the mutated CFTR remained in the incompletely glycosylated form and was localized in the endoplasmic reticulum. These cell lines reproduce the cellular fate of wild-type and mutated CFTR-deltaF508. To our knowledge, they are the first differentiated epithelial cell lines stably expressing tagged CFTR and CFTR-deltaF508 in which cellular processing and functional activity of these two proteins are reproduced. Thus the addition of the VSV-G epitope does not impair the localization and function of CFTR, and these cell lines can be used to examine CFTR function in vitro.     

An ionotropic GABA receptor in cultured mushroom body Kenyon cells of the honeybee and its modulation by intracellular calcium

GABAergic inhibitory transmission is very abundant within the insect brain. We, therefore, studied the functional properties of the ionotropic GABA receptor of honeybee mushroom body Kenyon cells in vitro. GABA applications elicit rapidly activating and desensitizing currents, which are concentration-dependent between 10 and 500 μM. The mean peak amplitude induced by 500 μM GABA at a holding potential of −110 mV is −1.55 ± 0.23 nA (SEM, n = 29). The GABA-induced current is mediated by Cl⁻ ions because (1) the reversal potential of the GABA-induced current of −40.6 mV is very close to the calculated Nernst potential of chloride (−44.8 mV). (2) With equimolar chloride concentrations the reversal potential shifted to about 0 mV. GABA or muscimol are equally efficient channel agonists, whereas CACA is a partial agonist. Picrotoxin or philanthotoxin (100 μM) completely and reversibly block the GABA-induced current, bicuculline (100 μM) has no effect. Elevating the intracellular Ca²⁺ concentration increases the GABA current amplitude. This modualtory effect is blocked by the kinase blocker K 252a, but not by blockers of CaMkinaseII (KN-93), PKC (bisindolylmaleimide) or PKA (KT 5720). We conclude that Kenyon cells express functional GABA receptors whose properties support an inhibitory role of GABAergic transmission.     

Developmental alterations in the biophysical properties of Cav1.3 Ca2+ channels in mouse inner hair cells

Prior to hearing onset, spontaneous action potentials activate voltage-gated Ca_v1.3 Ca²⁺ channels in mouse inner hair cells (IHCs), which triggers exocytosis of glutamate and excitation of afferent neurons. In mature IHCs, Ca_v1.3 channels open in response to evoked receptor potentials, causing graded changes in exocytosis required for accurate sound transmission. Developmental alterations in Ca_v1.3 properties may support distinct roles of Ca_v1.3 in IHCs in immature and mature IHCs, and have been reported in various species. It is not known whether such changes in Ca_v1.3 properties occur in mouse IHCs, but this knowledge is necessary for understanding the roles of Ca_v1.3 in developing and mature IHCs. Here, we describe age-dependent differences in the biophysical properties of Ca_v1.3 channels in mouse IHCs. In mature IHCs, Ca_v1.3 channels activate more rapidly and exhibit greater Ca²⁺-dependent inactivation (CDI) than in immature IHCs. Consistent with the properties of Ca_v1.3 channels in heterologous expression systems, CDI in mature IHCs is not affected by increasing intracellular Ca²⁺ buffering strength. However, CDI in immature IHCs is significantly reduced by strong intracellular Ca²⁺ buffering, which both slows the onset of, and accelerates recovery from, inactivation. These results signify a developmental decline in the sensitivity of CDI to global elevations in Ca²⁺, which restricts negative feedback regulation of Ca_v1.3 channels to incoming Ca²⁺ ions in mature IHCs. Together with faster Ca_v1.3 activation kinetics, increased reliance of Ca_v1.3 CDI on local Ca²⁺ may sharpen presynaptic Ca²⁺ signals and improve temporal aspects of sound coding in mature IHCs.     

Variety of Ca2+-permeable channels in human carcinoma A431 cells

Summary Patch-clamp methods were used to search for and characterize channels that mediate calcium influx through the plasma membrane of human carcinoma A431 cells. Here we present four Ca²⁺-permeable channel types referred to as SG, G, I and BI. With 105mm Ca²⁺ as the charge carrier, at 30–33°C their mean unitary conductances (in pS) are: 1.3 (SG), 2.4 (G), 3.7 (I) and 12.8 (BI). SG and G channels are activated by nonhydrolyzable analogues of guanosine 5-triphosphate (GTP) applied to the inside of the membrane, suggesting an involvement of G-proteins in the control of their activity. I and BI channels are activated by inositol 1,4,5-trisphosphate (InsP₃). G, I, BI and possibly SG channels are activated from the extracellular side of the membrane by epidermal growth factor (EGF) and histamine. It is assumed that all identified Ca²⁺ channels take part in the generation of the agonist-induced intracellular Ca²⁺ signal. The variety of Ca-channel types seems to be necessary to tune cell responses according to the respective type and level of an external signal, on the one hand, and to the functional state of the cell, on the other.     

Characterization of ion channels in the plasma membrane of epidermal cells of expanding pea (Pisum sativum arg) leaves

Ion channels in isolated patches of the plasma membrane of pea (Pisum sativum arg) epidermal cells were studied with the patch-clamp technique. One anion and one cation channel were dominantly present in most trials. The anion channel conducts nitrate, halides and malate, with a conductance in symmetrical 100 mm Cl^– of 300 pS and can be blocked by SITS when applied to the cytoplasmic side of the membrane. The cation channel poorly discriminates between potassium, sodium and lithium, is not blocked by either TEA or Ba²⁺, and has a conductance of 35 pS in symmetrical 100 mm K⁺. The open probability of the cation channel increases with increase of the Ca²⁺ concentration on the cytoplasmic side of the membrane from 0.1 to 1 m. The possible role of these two channels in the physiology of epidermal cells is discussed.This work was supported by NSF grant DCB-890 3744 to E.V.     

Melanocyte stem cells: a melanocyte reservoir in hair follicles for hair and skin pigmentation

Most mammals are coated with pigmented hair. Melanocytes in each hair follicle produce melanin pigments for the hair during each hair cycle. The key to understanding the mechanism of cyclic melanin production is the melanocyte stem cell (MelSC) population, previously known as 'amelanotic melanocytes'. The MelSCs directly adhere to hair follicle stem cells, the niche cells for MelSCs and reside in the hair follicle bulge-subbulge area, the lower permanent portion of the hair follicle, to serve as a melanocyte reservoir for skin and hair pigmentation. MelSCs form a stem cell system within individual hair follicles and provide a 'hair pigmentary unit' for each cycle of hair pigmentation. This review focuses on the identification of MelSCs and their characteristics and explains the importance of the MelSC population in the mechanisms of hair pigmentation, hair greying, and skin repigmentation.     

Activation of a nonselective cation channel by swelling in atrial cells

Cell swelling has been shown to increase the permeability of the plasma membrane to ions such as K⁺, Na⁺, Ca²⁺ or Cl^– in many types of cells. In cardiac cells, swelling has been reported to increase Cl^– conductance, but whether cation-selective currents are activated by swelling is not known. Low Cl^– or Cl^–-free solutions were used to study the presence of such currents. Lowering the osmolarity of the extracellular medium from 299 to 219 mOsm resulted in cell swelling and concurrent activation of a cation-selective whole-cell current. When cell-attached patches were formed on swollen cells, opening of bursting single channel currents were observed in 18% of the patches studied. Ion substitution experiments indicated that the channel discriminated poorly among monovalent cations, and was impermeable to Cl^–. The channel was permeable to Ca²⁺. In symmetrical 140 mM K⁺, the current-voltage relation was linear with a single channel conductance of 36 ± 3 pS. Depolarization increased channel open probability. Interestingly, depending on the membrane patch studied, application of negative pressure to the pipette caused either an increase or a decrease in the open probability of the channel already activated by swelling. Thus, the sensitivity to tension of the swelling-activated channel was different from those of previously reported stretch-activated channels. These findings suggest that nonselective cation channels exist in rat atrial cells and may be involved in swelling-induced changes in cell function.Dr. Kim is an Established Investigator of the American Heart Association.