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1.
The role of swelling-activated currents in cell volume regulation is unclear. Currents elicited by swelling rabbit ventricular myocytes in solutions with 0.6–0.9× normal osmolarity were studied using amphotericin perforated patch clamp techniques, and cell volume was examined concurrently by digital video microscopy. Graded swelling caused graded activation of an inwardly rectifying, time-independent cation current (I Cir,swell) that was reversibly blocked by Gd 3+, but I Cir,swell was not detected in isotonic or hypertonic media. This current was not related to I K1 because it was insensitive to Ba 2+. The P K/P Na ratio for I Cir,swell was 5.9 ± 0.3, implying that inward current is largely Na + under physiological conditions. Increasing bath K + increased g Cir,swell but decreased rectification. Gd 3+ block was fitted with a K
0.5 of 1.7 ± 0.3 μM and Hill coefficient, n, of 1.7 ± 0.4. Exposure to Gd 3+ also reduced hypotonic swelling by up to ∼30%, and block of current preceded the volume change by ∼1 min. Gd 3+-induced cell shrinkage was proportional to I Cir,swell when I Cir,swell was varied by graded swelling or Gd 3+ concentration and was voltage dependent, reflecting the voltage dependence of I Cir,swell. Integrating the blocked ion flux and calculating the resulting change in osmolarity suggested that I Cir,swell was sufficient to explain the majority of the volume change at –80 mV. In addition, swelling activated an outwardly rectifying Cl − current, I Cl,swell. This current was absent after Cl − replacement, reversed at E Cl, and was blocked by 1 mM 9-anthracene carboxylic acid. Block of I Cl,swell provoked a 28% increase in swelling in hypotonic media. Thus, both cation and anion swelling-activated currents modulated the volume of ventricular myocytes. Besides its effects on cell volume, I Cir,swell is expected to cause diastolic depolarization. Activation of I Cir,swell also is likely to affect contraction and other physiological processes in myocytes. 相似文献
2.
Osmotic cell swelling activates Cl − channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56 lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (I Cl−swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of I Cl−swell is defective in p56 lck-deficient cells. Retransfection of p56 lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of I Cl−swell. Addition of purified p56 lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56 lck washed into the cytoplasm activates I Cl−swell in native and p56 lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56 lck, slow single-channel gating events can be observed revealing a unitary conductance of 25–28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56 lck. We conclude that osmotic swelling activates I Cl−swell in lymphocytes via the tyrosine kinase p56 lck. 相似文献
3.
We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl − current ( I
Cl,swell) in BC 3H1 myoblasts. This Cl − current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl − currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (∼60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10–80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg 2+ concentration or reducing the Cl − concentration of the extracellular solution. Replacing extracellular Cl − by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN − > I − > NO 3
− > Cl − >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg 2+, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of I
Cl,swell are strongly influenced by external pH , external divalent cations, and by the nature of the permeant anion. 相似文献
4.
The influence of NO 3− uptake and reduction on ionic balance in barley seedlings ( Hordeum vulgare, cv. Compana) was studied. KNO 3 and KCl treatment solutions were used for comparison of cation and anion uptake. The rate of Cl − uptake was more rapid than the rate of NO 3− uptake during the first 2 to 4 hours of treatment. There was an acceleration in rate of NO 3− uptake after 4 hours resulting in a sustained rate of NO 3− uptake which exceeded the rate of Cl − uptake. The initial (2 to 4 hours) rate of K + uptake appeared to be independent of the rate of anion uptake. After 4 hours the rate of K + uptake was greater with the KNO 3 treatment than with the KCl treatment, and the solution pH, cell sap pH, and organic acid levels with KNO 3 increased, relative to those with the KCl treatment. When absorption experiments were conducted in darkness, K + uptake from KNO 3 did not exceed K + uptake from KCl. We suggest that the greater uptake and accumulation of K + in NO 3−-treated plants resulted from ( a) a more rapid, sustained uptake and transport of NO 3− providing a mobile counteranion for K + transport, and ( b) the synthesis of organic acids in response to NO 3− reduction increasing the capacity for K + accumulation by providing a source of nondiffusible organic anions. 相似文献
5.
Monovalent Cation Permeation through the Connexin40 Gap Junction Channel : Cs,Rb, K,Na, Li,TEA, TMA,TBA, and Effects of Anions Br,Cl, F,Acetate, Aspartate,Glutamate, and NO3
The unitary conductances and permeability sequences of the rat connexin40 (rCx40) gap junction channel s to seven monovalent cations and anions were studied in rCx40-transfected neuroblastoma 2A (N2A) cell pairs using the dual whole cell recording technique. Chloride salt cation substitutions (115 mM principal salt) resulted in the following junctional maximal single channel current-voltage relationship slope conductances (γ j in pS): CsCl (153), RbCl (148), KCl (142), NaCl (115), LiCl (86), TMACl (71), TEACl (63). Reversible block of the rCx40 channel was observed with TBA. Potassium anion salt γ j are: Kglutamate (160), Kacetate (160), Kaspartate (158), KNO 3 (157), KF (148), KCl (142), and KBr (132). Ion selectivity was verified by measuring reversal potentials for current in rCx40 gap junction channels with asymmetric salt solutions in the two electrodes and using the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The permeabilities relative to Li + are: Cs + (1.38), Rb + (1.32), K + (1.31), Na + (1.16), TMA + (0.53), TEA + (0.45), TBA + (0.03), Cl − (0.19), glutamate − (0.04), and NO 3− (0.14), assuming that the monovalent anions permeate the channel by forming ion pairs with permeant monovalent cations within the pore thereby causing proportionate decreases in the channel conductance. This hypothesis can account for why the predicted increasing conductances with increasing ion mobilities in an essentially aqueous channel were not observed for anions in the rCx40 channel. The rCx40 effective channel radius is estimated to be 6.6 Å from a theoretical fit of the relationship of relative permeability and cation radius. 相似文献
6.
Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl − pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl − activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K
m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P Na/P Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P I (1.8) > P Br (1.3) > P Cl (1.0) > P F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F − flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I −/Cl − permeability ratio (P I/P Cl < 0.4). The switch to low I − permeability was enhanced at potentials that favored Cl − entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl − and I − ions may influence I − permeation and be responsible for the wide range of P I/P Cl ratios that have been reported for the CFTR channel. The low P I/P Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence. 相似文献
7.
Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH 2-terminal–truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179–182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in ∼40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of ∼1.6 e 0. The fully open T442S chimeric channel has the conductance sequence Rb + > NH 4
+ > K +. The opposite conductance sequence, K + > NH 4
+ > Rb +, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb +. 相似文献
8.
The apical membrane of intestinal epithelia expresses intermediate conductance K + channel (KCNN4), which provides the driving force for Cl − secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl − secretion via stimulation of Rap2-phospholipase Cϵ-[Ca 2+] i signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl − secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl − secretion and apical K + conductance (I K(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2′- O- methyladenosine 3′,5′-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K + channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed ∼83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate I K(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated I K(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases. 相似文献
9.
Photosynthesis, stroma-pH, and internal K + and Cl − concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K +, H +, Cl −) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar). Isolated intact chloroplasts are a Donnan system which accumulates cations (K+ or added Tetraphenylphosphonium+) and excludes anions (Cl−) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium+ distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K+ or Na+ and the release of protons all become very small. Light-induced K+ uptake is not abolished by valinomycin suggesting that the K+ uptake is not primarily active. Intact chloroplasts contain higher K+ concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO2 fixation at high K+ concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO2 fixation in intact chloroplasts. 相似文献
10.
The effects of intracellular application of trypsin on the Cl – current induced by hypotonic cell swelling ( I
Cl,swell) in human prostate cancer epithelial cells (LNCaP) was studied using the patch-clamp technique. In cells predialyzed with 1 mg/mL trypsin, I
Cl,swell developed and diminished in response to the application and withdrawal of hypotonic solution about three times faster than that in control cells. In trypsin-infused cells, I
Cl,swell also had about twofold higher current density and displayed considerably slowed voltage-dependent inactivation, which was quite pronounced in control cells at potentials above +60 mV. Trypsin-induced modification of I
Cl,swell could be prevented by coinfusion of 10 mg/mL soybean trypsin inhibitor, suggesting that proteolytic cleavage of essential intracellular structural domains of the I
Cl,swell-carrying volume-regulated anion channel (VRAC) was responsible for this functional modification. The effect of trypsin was not dependent on the presence of intracellular ATP. We conclude that VRACs, similarly to voltage-gated Na +, K +, and Cl – channels, possess intracellular inactivation domain(s) subjected to proteolytic cleavage that may function in conformity with the classical ball-and-chain inactivation model. 相似文献
11.
Chloroquine (CQ), a bitter tasting drug widely used in treatment of malaria, is associated gastrointestinal side effects including nausea or diarrhea. In the present study, we investigated the effect of CQ on electrolyte transport in rat ileum using the Ussing chamber technique. The results showed that CQ evoked an increase in short circuit current ( ISC) in rat ileum at lower concentration (≤5×10 −4 M ) but induced a decrease at higher concentrations (≥10 −3 M). These responses were not affected by tetrodotoxin (TTX). Other bitter compounds, such as denatoniumbenzoate and quinine, exhibited similar effects. CQ-evoked increase in ISC was partly reduced by amiloride(10 −4 M), a blocker of epithelial Na + channels. Furosemide (10 −4 M), an inhibitor of Na +-K + -2Cl − co-transporter, also inhibited the increased ISC response to CQ, whereas another Cl − channel inhibitor, CFTR(inh)-172(10 −5M), had no effect. Intriguingly, CQ-evoked increases were almost completely abolished by niflumic acid (10 −4M), a relatively specific Ca 2+-activated Cl − channel (CaCC) inhibitor. Furthermore, other CaCC inhibitors, such as DIDS and NPPB, also exhibited similar effects. CQ-induced increases in ISC were also abolished by thapsigargin(10 −6M), a Ca 2+ pump inhibitor and in the absence of either Cl − or Ca 2+ from bathing solutions. Further studies demonstrated that T2R and CaCC-TMEM16A were colocalized in small intestinal epithelial cells and the T2R agonist CQ evoked an increase of intracelluar Ca 2+ in small intestinal epithelial cells. Taken together, these results demonstrate that CQ induces Cl − secretion in rat ileum through CaCC at low concentrations, suggesting a novel explanation for CQ-associated gastrointestinal side-effects during the treatment of malaria. 相似文献
12.
When cells swell in hypo-osmotic solutions, chloride-selective ion channels (Cl swell) activate to reduce intracellular osmolality and prevent catastrophic cell rupture. Despite intensive efforts to assign a molecular identity to the mammalian Cl swell channel, it remains unknown. In an unbiased genome-wide RNA interference (RNAi) screen of Drosophila cells stably expressing an anion-sensitive fluorescent indicator, we identify Bestrophin 1 (dBest1) as the Drosophila Cl swell channel. Of the 23 screen hits with mammalian homologs and predicted transmembrane domains, only RNAi specifically targeting dBest1 eliminated the Cl swell current (I Clswell). We further demonstrate the essential contribution of dBest1 to Drosophila I Clswell with the introduction of a human Bestrophin disease-associated mutation (W94C). Overexpression of the W94C construct in Drosophila cells significantly reduced the endogenous I Clswell. We confirm that exogenous expression of dBest1 alone in human embryonic kidney (HEK293) cells creates a clearly identifiable Drosophila–like I Clswell. In contrast, activation of mouse Bestrophin 2 (mBest2), the closest mammalian ortholog of dBest1, is swell-insensitive. The first 64 residues of dBest1 conferred swell activation to mBest2. The chimera, however, maintains mBest2-like pore properties, strongly indicating that the Bestrophin protein forms the Cl swell channel itself rather than functioning as an essential auxiliary subunit. dBest1 is an anion channel clearly responsive to swell; this activation depends upon its N-terminus. 相似文献
13.
Transcellular Cl − movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na +-K +-2Cl − cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl − above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl − uptake pathway concentrates Cl − ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl −/HCO 3− exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4−/− mice. The decrease in salivation was not related to loss of Na +-K +-2Cl − cotransporter or Na +/H + exchanger activity in Ae4−/− mice but correlated with reduced Cl − uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl −/HCO 3− exchanger activity revealed that HCO 3−-dependent Cl − uptake was reduced in the acinar cells of Ae2−/− and Ae4−/− mice. Moreover, Cl −/HCO 3− exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl −/HCO 3− exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion. 相似文献
14.
A detailed examination was conducted on the linear, or first-order kinetic component for K +( 86Rb +) influx into root segments of both low- and high-salt grown corn seedlings ( Zea mays [A632 × Oh 43]). In tissue from both low- and high-salt grown roots, replacement of Cl − in the uptake solution by either SO 42−, H 2PO 4−, or NO 3− caused a significant (50-60%) and specific inhibition of the linear component of K + influx. The anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid, was found to abolish saturable Cl − influx in corn roots while causing a significant (50-60%) and specific inhibition of the linear K + uptake system; this inhibition was identical to that observed when Cl − was replaced by other anions in the K + uptake solution. Additionally, the quaternary ammonium cation, tetraethylammonium, which has been shown to block K + channels in nerve axons, also caused a dramatic (70%) and specific inhibition of the linear component of K + influx, but this was obtained only in high-salt roots. The reasons for this difference are discussed with respect to the differing abilities of low- and high-salt roots to absorb tetraethylammonium. Our present results indicate that the linear component of K+ influx may occur by a passive process involving transmembrane K+ channels. Fluxes through these K+ channels may be partly coupled to a saturating Cl− influx mechanism. 相似文献
15.
Intestinal Cl − secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca 2+] i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl − secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (I sc) measurement in response to agonist-stimulated Cl − secretion. FSK-stimulated Cl − secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca 2+] i chelator, 1,2-bis-(o-aminophenoxy)-ethane- N, N, N’, N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca 2+] i, activated Ras-related protein 2, and induced Cl − secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced I sc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl − secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl − conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl −>Br −>I − permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca 2+] i signaling pathway is involved in cAMP-stimulated Cl − secretion, which is carried by a novel, previously undescribed Cl − channel. 相似文献
16.
Current-voltage curves for DIDS-insensitive Cl − conductance have been determined in human red
blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl − conductance among the cells. After pretreatment for
30 min with DIDS, net effluxes of K + and Cl − were induced by valinomycin and were measured in the continued
presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl − effluxes, induced by valinomycin or gramicidin at varied [K +] o,
were compared with predictions based on ( 1) the theory of electrodiffusion, ( 2) a single barrier model, ( 3) single
occupancy, multiple barrier models, and ( 4) a voltage-gated mechanism. Electrodiffusion precisely describes the
relationship between the measured transmembrane voltage and [K +] o. Under our experimental conditions (pH
7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio
P K/P Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P Cl. Fitting the constant field current-voltage equation to the measured Cl − currents yields P
Cl = 0.13 h −1 with DIDS, compared to 0.49 h −1 without DIDS,
in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl − current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a
voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the
channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3. 相似文献
17.
A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl − as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO 3
−, BrO 3
−); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl − at the regulatory binding site but impair Cl − passage through the channel pore (Br −, NO 3
−, ClO 3
−, I −, ClO 4
−, SCN −). The permeability sequence for rClC-1, SCN − ∼ ClO 4
− > Cl − > Br − > NO 3
− ∼ ClO 3
− > I − >> BrO 3
− > HCO 3
− >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the P
open curve, SCN − ∼ ClO 4
− > I − > NO 3
− ∼ ClO 3
− ∼ methanesulfonate > Br − > cyclamate > BrO 3
− > HCO 3
− > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl − and SCN − or ClO 4
− suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size. 相似文献
18.
Members of the CLC protein family of Cl − channels and transporters display the remarkable ability to function as either chloride channels or Cl −/H + antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl −/H + exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H + transport was abolished by mutations in either the “gating” or “proton” glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl − > I − conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the “anion-coordinating” tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO 3− conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl −/H + antiporter. 相似文献
19.
The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na + with Li +, K +, Rb +, or Cs + altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P K/P Na = 0.1 and G K/G Na = 0.7 and P Rb/P Na = 0.03 and G Rb/G Na = 0.3. Stimulation of Cl − currents when Na + was replaced with Ca 2+, Sr 2+, or Ba 2+ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na + in a hyperbolic manner, consistent with an apparent affinity (K m) for Na + conduction of 25 mM. Nitrogen-containing cations, including NH 4+, NH 3OH +, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na + currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na + (K m = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule. 相似文献
20.
Protoplasts were enzymically isolated from suspension cultured cells of Nicotiana glutinosa L. and aspects of transport selectivity and kinetics were studied. In the presence of Ca 2+, transport was selective for K + ( 86Rb) over Na +. 36Cl − transport was inhibited by Br − or I − but not by H 2PO 4−. The kinetic data for short term (30 minutes) K + influx over the range of 0.05 to 100 millimolar KCl were complex but similar to those observed in other plant tissues. In contrast, the kinetic data for Cl − and H 232PO 4− over the same concentration range were different from those observed for K +, and could be accounted for by a single isotherm in the range of 0.05 to 4 millimolar and by an almost linear increase in influx rate above 4 millimolar. The kinetic data for Cl − transport into intact cultured cells were identical in character to those observed for isolated protoplasts. The results support the view that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast. 相似文献
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