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1.
Summary Exposure of porcine renal brush-border membrane vesicles to 1.2% cholate and subsequent detergent removal by dialysis reorients almost all N-ethylmaleimide (NEM)-sensitive ATPases from the vesicle inside to the outside. ATP addition to cholate-pretreated, but not to intact, vesicles causes H+ uptake as visualized by the pH indicator, acridine organge. The reoriented H+-pump is electrogenic because permeant extravesicular anions or intravesicular K+ plus valinomycin enhance H+ transport. ATP stimulates H+ uptake with an apparentK m of 93 m. Support of H+ uptake andP i liberation by ATP>GTPITP> UTP indicates a preference for ATP and utilization of other nucleotides at lower efficiency. ADP is a potent, competitive inhibitor of ATP-driven H+ uptake,(K i , 24 m). Mg2+ and Mn2– support ATP-driven H+ uptake, but Ca2+, Ba2+ and Zn2+ do not. Imm Zn2+ inhibits MgATP-driven H+ transport completely. NEM-sensitiveP i liberation is stimulated by Mg2+ and Mg2– and, unlike H+ uptake, also by Ca2+ suggesting Ca2+-dependent ATP hydrolysis unrelated to H+ transport. The inside-out oriented H+-pump is relatively insensitive toward oligomycin, azide, N,N-dicyclohexylcarbodiimide (DCCD) and vanadate, but efficiently inhibited by NEM (apparentK i , 0.77 m), and 4-chloro-7-nitro-benzoxa-1,3-diazole (NBD-Cl; apparentK i , 0.39 m). Taken together, the H+-ATPase of proximal tubular brush-border membranes exhibits characteristics very similar to those of vacuolar type (V-type) H+-ATPases. Hence,V-type H+-ATPases occur not only in intracellular organelles but also in specialized plasma membrane areas.  相似文献   

2.
The Archaebacterium Haloferax volcanii concentrates K+ up to 3.6 M. This creates a very large K+ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K+ but the residual K+ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K+ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a K m of between 1 to 2 mM. The driving force for K+ transport has been explored. Internal K+ concentration is not in equilibrium with m suggesting that K+ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N-dicyclohexylcarbodiimide inhibits by 100% K+ transport even though membrane potential m is maintained under these conditions. By contrast, the necessity of a m for K+ accumulation has not yet been clearly demonstrated. K+ transport in H. volcanii can be compared with K+ transport via the Trk system in Escherichia coli.Abbreviations CCCP Carbonylcyanide m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - MES 2-[N-morpholino] ethane sulfonic acid - MOPS 3-[N-morpholino] propane sulfonic acid - TRIS Tris (hydroxymethyl) aminomethane - TPP tetraphenyl phosphonium  相似文献   

3.
The microsomal fraction isolated from dog mesenteric nerve fibres was found to contain ATPase activity stimulated by micromolar concentrations of Ca ions. Such a high-affinity Ca2+-ATPase (hereafter referred to as HA Ca-ATPase) followed a Michaelis-Menten kinetics with Km for Ca ions of 0.4 M and Vmax=12.5±2.4 mol Pi.mg–1h–1. The examination of the subcellular origin of HA Ca-ATPase revealed that this enzyme is associated with axonal plasma membranes as documented by its co-purification with several plasma membrane marker enzymes and with tetrodotoxin-sensitive3H-saxitoxin binding. The addition of exogenous magnesium ions (Mg) resulted in a non-competitive inhibition of HA Ca-ATPase with Ki=0.5 mM. The reaction velocity of HA Ca-ATPase was also inhibited by other divalent ions with the order of potency Mg>Mn >ZnCo>Ni. In contrast to low affinity (high Km) Mg- and Ca-ATPase, the HA Ca-ATPase was insensitive to the inhibition by sodium azide (10 mM) and sodium fluoride (10 mM). Similarly, the specific activity of HA Ca-ATPase was unaffected by vanadate (100 M) and N-ethylmaleinimide (100 M). It is concluded that axonal plasma membranes of dog mesenteric nerves contain HA Ca-ATPase which seems to be unrelated to calcium-transporting Mg-dependent, Ca-stimulated ATPase.Abbreviations used BSA bovine serum albumin - HA Ca-ATPase high-affinity Ca2+-ATPase - K-pNPPase onabain-sensitive, K+-stimulated p-nitrophenyl phosphatase - NEM N-ethylmaleinimide - SIM 250 mM sucrose, 10 mM imidazole-HCl pH 7.4 - TRIS tris (hydroxymethyl) aminomethane  相似文献   

4.
Summary The conductance of the Ca2+-activated K+ channel (g K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V m ) and extracellular K+ concentration ([K+]ex). ATP-depleted cells, with fixed values of cellular K+ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K+ channels, to buffer-free salt solutions with given K+ concentrations. Outward-current conductances were calculated from initial net effluxes of K+, correspondingV m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of42K at (V m –E K)0, using a single-file flux ratio exponent of 2.7. Within a [K+]ex range of 5.5 to 60mm and at (V m –E K) 20 mV a basic conductance, which was independent of [K+]ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm2 between 0 and –100 mV. As (V m –E K) decreased from 20 towards zero mVg K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm2. The zero-current conductance was not significantly dependent on [K+]ex (30 to 156mm) corresponding toV m (–50 mV to 0). A further increase ing K(Ca) symmetrically aroundE K is suggested as (V m –E K) becomes positive. Increasing the extracellular K+ concentration from zero and up to 3mm resulted in an increase ing K(Ca) from 50 to 70 S/cm2. Since the driving force (V m –E K) was larger than 20 mV within this range of [K+]ex this was probably a specific K+ activation ofg K(Ca). In conclusion: The Ca2+-activated K+ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.  相似文献   

5.
Summary This communication reports the kinetics of the Na+/ Ca2+ exchanger and of the plasma membrane (PM) Ca2+ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca2+ extrusion and its Na+ dependence for concentrations of cytoplasmic free Ca2+ ([Ca2+]cyt) in the 1–10-m range. The PM Ca2+ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147–163) for [Ca2+]cyt] 1.5 m with the fluorescent Ca2+ indicator quin2 (K d= 115 nm). That study determined that the PM Ca2+ pump in the basal state has a V max = 0.098 mm/min, a K m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca2+]cyt with the intracellular Ca2+ probe, rhod2 (K d= 500 nm), which has almost a fivefold lower affinity for Ca2+. An Appendix also describes the Mg2+ and pH dependence of the K dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca2+ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca2+ extrusion into a Ca2+-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca2+ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca2+ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca2+ with a K m= 0.97 ± 0.31 m and Vmax = 1.0 ± 0.6 mm/ min. (ii) At high [Ca2+]cyt, the exchanger works at a rate 10 times as large as the basal V max of the PM Ca2+ extrusion pump. (iii) The exchanger can work in reverse after Na+ loading of the cytoplasm by monensin. (iv) The PM Ca2+ extrusion pump is activated by exposure to [Ca2+]cyt 1.5 m for 20–50 sec. Activation raises the pump V max to 1.6 ± 0.6 mm/min and the K mto 0.55 ± 0.24 m. (v) The Ca2+ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca2+]cyt. In summary, the results show that the human platelet can extrude Ca2+ very rapidly at high [Ca2+]cyt. Both the Na+/Ca2+ exchanger and Ca2+ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5,-monophosphate - Ca-CAM calcium calmodulin; - DT dense tubules - B intrinsic cytoplasmic Ca2+ binding sites - R rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid - [Ca2+]cyt cytoplasmic Ca2+ activity - quin2 2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline - V or Vextrusion true rate of Ca2+ extrusion - fura-2 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid - AM acetoxymethyl ester - DMSO dimethylsulfoxide - CTC chlortetracycline - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - NMDG N-methyl-d-glucamine - PIPES 1,4-piperazine-bis-(ethanesulfonic acid) - HPLC high performance liquid chromatography - I fraction of high-affinity rhod2 complexed with Ca2+ - F the observed fluorescence - Fmin the minimal fluorescence observed in the absence of Ca2+ - Fmax the maximal fluorescence observed when the dye is saturated with Ca2+ - X1 the fraction of high-affinity dye - K d,1 dissociation constant of high-affinity dye - K d,2 dissociation constant of the low-affinity dye - -d1/dt rate of Ca2+ removal from the rhod2-Ca complex; - -dF/dt the slope representing the absolute rate of fluorescence decrease in a progress curve - Fmax (Fmax — Fmin)cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2 - F50 fluorescence of the high-affinity form ofrhod2for[Ca2+]cyt=50 nM - [Ca2+]0 external Ca2+concentration - K p proportionality constant between the total number of Ca2+ ions moved and the change in high-affinity rhod2 complexation to Ca2 - (d[Ca2+]cyt, T)/dt rate of Ca2+ influx obtained with maximal levels of ionomycin - kleak rate constant for passive inward Ca2+ leakage - kinno rate constant for ionomycin-mediated Ca2+ influx - T total - [rhod2]cyt,T total intracellular rhod2 concentration - [quin2]cyt,T total intracellular quin2 concentration - [B]T total cytoplasmic buffering capacity - A[Ca2+]cyt,T total number of Ca2+ ions moved into the cytoplasm - [rhod2-Ca]cyt, T change in concentration of total intracellular high-affinity rhod2 complexed to Ca2+ - [B-Ca]T change in concentration of total cytoplasmic binding sites complexed to Ca2+ - [quin2]cyt, T change in concentration of total intracellular quinl complexed to Ca2+ - change in the degree of intracellular quin2 saturation - 1 change in degree of saturation of cytoplasmic high-affinity rhod2 - 1-/t rate of change in degree of saturation of cytoplasmic high affinityrhod2 - Vobs observed rate of Ca2+ removal from the rhod2-Ca complex - V8.3 m the rate of Ca2+ removal from the high affinity rhod2-Ca complex at [Ca2+]cyt = 8.3 m - /t rate of change in of the degree of quin2 saturation - [Ca2+]cytT/t initial linear rate of ionomycin-mediated Ca2+ influx - EC50 effective concentration giving a half-maximal effect - [Na+]cyt cytoplasmic Na+ activity - CAM calmodulin - ACN acetonitrile - TFA trifuloroacetic acid  相似文献   

6.
Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K+ solutions elicits a complex of Ca2+ and K+ currents. The tail current that accompanies a return to holding potential (–40 mV) contains two K+ components. The tail current elicited by a step to –110 mV of 50-msec duration contains fast-decaying (3.5 msec) and slow-decaying (20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K+], suggesting that they represent pure K+ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K+ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca2+ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K+ currents are inhibited by extracellular TEA+ in a concentration-dependent, noncooperative manner, whereas the fast K+ current alone is inhibited by 0.7mm quinidine.  相似文献   

7.
Summary The results of the accompanying electrophysiological study of the cloned Na+/glucose cotransporter from small intestine (Parent, L., Supplisson, S., Loo, D.D.F., Wright, E.M. (1992) J. Membrane Biol. 125:49–62) were evaluated in terms of a kinetic model. The steady-state and presteady-state cotransporter properties are described by a 6-state ordered kinetic model (mirror symmetry) with a Na+:MDG stoichiometry of 2. Carrier translocation in the membrane as well as Na+ and sugar binding and dissociation are treated as a function of their individual rate constants. Empty carrier translocation and Na+ binding/ dissociation are the only steps considered to be voltage dependent. Currents were associated with the translocation of the negatively charged carrier in the membrane. Negative membrane potential facilitates sugar transport. One numerical solution was found for the 14 rate constants that account quantitatively for our experiment observations: i.e., (i) sigmoidal shape of the sugar-specific current-voltage curves (absence of outward currents and inward current saturation at high negative potentials), (ii) Na+ and voltage dependence of K 0.5 sugar and i max sugar , (iii) sugar and voltage dependence of K 0.5 Na and i max Na , (iv) presteady-state currents and their dependence on external Na+, MDG and membrane potential, and (v) and carrier Na+ leak current. We conclude that the main voltage effect is on carrier translocation. Na+ ions that migrate from the extracellular medium to their binding sites sense 25 to 35% of the transmembrane voltage, whereas charges associated with the carrier translocation experiences 60 to 75% of the membrane electrical field. Internal Na+ ion binding is not voltage dependent. In our nonrapid equilibrium model, the rate-limiting step for sugar transport is a function of the membrane potential, [Na]0 and [MDG]0. At 0 mV and at saturating [Na]0 and [MDG]0, the rate-limiting step for sugar transport is the empty carrier translocation (5 sec–1). As the membrane potential is made more negative, the empty carrier translocation gets faster and the internal Na+ dissociation becomes increasingly rate limiting. However, as [Na]0 is decreased to less than 10 mm, the rate-limiting step is the external Na+ ions binding in the 0 to –150 mV potential range. At 0 mV, the external Na+ dissociation constant KNa is 80 mm and decreases to 24 mm at –150 mV. The external sugar dissociation constant KNaS is estimated to be 200 m and voltage independent. Finally, the internal leak pathway (CNa2 translocation) is insignificant. While we cannot rule out a more complex kinetic model, the electrical properties of the cloned Na+/glucose cotransporter are found to be adequately described by this 6-state kinetic model.We are grateful to Drs. A. Berteloot, S. Ciani, and J.-Y. Lapointe for stimulating discussions and thank our colleagues for comments. L.P. was recipient of a post-doctoral fellowship from the Medical Research Council of Canada. This work was supported by a grant from the U.S. Public Health Service DK 19567.  相似文献   

8.
The effect of chemical modifiers of amino acid residues on the proton conductivity of H+-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H+ in ESMP as well as in particles deprived of F1 (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H+ exhibited two first-order phases, both an expression of the H+ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H+ conduction caused by respiratory H+ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H+ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H+ conduction. In USMP, H+ release followed simple first-order kinetics. Oligomycin depressed H+ release, enhanced respiratory H+, and restored the biphasicity of H+ release. Phenylglyoxal and tetranitromethane inhibited H+ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H+ release from USMP, introducing a very rapid phase of H+ release. Addition of soluble F1 to USMP also restored biphasicity of H+ release. A mechanism of proton conduction by F o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H+ conduction, and those modified by butanedione, which retard H+ diffusion.  相似文献   

9.
Vesicles derived from maize roots retain a membrane bound H+-ATPase that is able to pump H+ at the expense of ATP hydrolysis. In this work it is shown that heparin, fucose-branched chondroitin sulfate and dextran sulfate 8000 promote a shift of the H+-ATPase optimum pH from 6.0 to 7.0. This shift is a result of a dual effect of the sulfated polysaccharides, inhibition at pH 6.0 and activation at pH 7.O. At pH 6.0 dextran 8000 promotes an increase of the apparent Km for ATP from 0.28 to 0.95 mM and a decrease of the Vmax from 14.5 to 7.1 mol Pi/mg · 30 min–1. At pH 7.0 dextran 8000 promotes an increase in Vmax from 6.7 to 11.7 mol Pi/mg · 30 min–1. In the presence of lysophosphatidylcholine the inhibitory effect of the sulfated polysaccharides observed at pH 6.0 was not altered but the activation of pH 7.0 decreased. It was found that in the presence of sulfated polysaccharides the ATPase became highly sensitive to K+ and Na+. Both the inhibition at pH 6.0 and the activation promoted by the polysaccharide were antagonized by monovalent cations (K+>Na+Li+).Abbreviations Mops 4-morpholinopropanesulfonic acid - EDTA ethylenediaminetetraacetic acid - ACMA 9-amino-6-chloro-2-methoxyacridine - FCCP carbonyl cyanide p(trifluoromethoxy)-phenylhyrazone  相似文献   

10.
Summary We have investigated muscarinic receptor-operated Ca2+ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca2+ release and extracellular Ca2+ entry. The carbachol (Cch) dose response of intracellular Ca2+ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K 0.510 or 30 m, depending on the method for [Ca2+] i determination). However, similar data for Ca2+ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca2+ release and a second higher affinity site withK 0.52.5 m. In addition, the Ca2+ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K+ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca2+ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca2+] i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca2+ channel blocker La3+ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca2+ in these cells by increasing Ca2+ entry. Organic Ca2+ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca2+ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K+ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP3) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP3 formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca2+ entry in HSG-PA cells. One is associated with IP3 formation and intracellular Ca2+ release and is independent of membrane potential; the other is less dependent on IP3 formation and intracellular Ca2+ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.  相似文献   

11.
We have examined the effect of the Ca2+ (Mg2+)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent 45Ca2+ uptake into IP3-sensitive Ca2+ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca2+ uptake was inhibited by TG up to 10 nm (apparent Ki4.2 nm, Ca2+ pool I). An additional increase of inhibition up to 85–90% of total Ca2+ uptake could be achieved at 15 to 20 nm of TG (apparent Ki12.1 nm, Ca2+ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent Ki10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca2+ uptake. About 30–40% of total Ca2+ uptake was inhibited by 100 m of vanadate (apparent Ki18 m, Ca2+ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent Ki300 m) or by TG up to 10 nm (Ca2+ pool I). The amount of IP3-induced Ca2+ release was constant at 25% over a wide range of Ca2+ filling. About 10–20% remained unreleasable by IP3. Reduction of IP3 releasable Ca2+ in the presence of inhibitors showed similar dose-response curves as Ca2+ uptake (apparent Ki 3.0 nm for IP3-induced Ca2+ release as compared to 4.2 nm for Ca2+ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca2+ pump fills the IP3-sensitive Ca2+ pool I. At TG concentrations >10 nm which blocked Ca2+ pool II the apparent Ki values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent Ki values were 18 m for Ca2+ uptake and 7 m for Ca2+ release (Ca2+ pool II). At vanadate concentrations up to 1 mm the apparent Ki values were 300 and 200 m, respectively (Ca2+ pool I). Both Ca2+ pools I and II also showed different sensitivities to IP3. Dose-response curves for IP3 in the absence of inhibitors (control) showed an apparent Km value for IP3 at 0.6 m. In the presence of TG (inhibition of Ca2+ pool I) the curve was shifted to the left with an apparent Km for IP3 at 0.08 m. In the presence of vanadate (inhibition of Ca2+ pool II), the apparent Km for IP3 was 2.1 m. These data allow the conclusion that there are at least three different Ca2+ uptake mechanisms present in pancreatic acinar cells: TG- and IP3 insensitive but highly vanadate-sensitive Ca2+ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca2+ pools with different TG-, vanadate- and IP3-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.  相似文献   

12.
Elementary K+ currents were recorded at 19 °C in cell-attached and in inside-out patches excised from neonatal rat heart myocytes. An outwardly rectifying K+ channel which prevented Na+ ions from permeating could be detected in about 10% of the patches attaining (at 5 mmol/l external K+ and between – 20 mV and + 20 mV) a unitary conductance of 66 +- 3.9 pS. K (outw.-rect.) + channels have one open and at least two closed states. Open probability and open rose steeply on shifting the membrane potential in the positive direction, thereby tending to saturate. Open probability (at –7 mV) was as low as 3 ± 1% but increased several-fold on exposing the cytoplasmic surface to Mg-ATP (100 mol/l) without a concomitant change of open. No channel activation occurred in response to ATP in the absence of cytoplasmic Mg–+. The cytoplasmic administration of the catalytic subunit of protein kinase A (120–150 /ml) or GTP--S (100 mol/l) caused a similar channel activation. GDP--S (100 mol/l) was also tested and found to be ineffective in this respect. This suggests that cardiac K (outw.-rect.) + channels are metabolically modulated by both cAMP-dependent phosphorylation and a G-protein. Offprint requests to: M. Kohlhardt  相似文献   

13.
Summary Human red cells were prepared with various cellular Na+ and K+ concentrations at a constant sum of 156mm. At maximal activation of the K+ conductance,g K(Ca), the net efflux of K+ was determined as a function of the cellular Na+ and K+ concentrations and the membrane potential,V m , at a fixed [K+]ex of 3.5mm.V m was only varied from (V m E K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g K(Ca) as a function of cellular Na+ and K+ concentrations atV m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na+. Since the present Ca2+-activated K+ channels have been shown to be of the multi-ion type, the experimental data from each set of Na+ and K+ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na+ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na+ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca2+ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg K(Ca) for outward currents at (V m E K)>25 25 mV reflects a voltage-dependent Na+ block of the Ca2+-activated K+ channels.  相似文献   

14.
Outside-out configuration of the patch clamp technique was used to test whether an intracellular application of G protein activator (GTPS) affects ATP-activated Ca2+-permeable channels in rat macrophages without any agonist in the bath solution. With 145 mm K+ (pCa 8.0) in the pipette solution, activity of channels permeable to a variety of divalent cations and Na+ was observed and general channel characteristics were found to be identical to those of ATP-activated ones. Absence of extracellular ATP makes it possible to avoid the influence of ATP receptor desensitization and to study the channel selectivity using a number of divalent cations (105 mm) and Na+ (145 mm) as the charge carriers. Permeability sequence estimated by extrapolated reversal potential measurements was: Ca2+ Ba2+ Mn2+ Sr2+ Na+ K+ = 68 30 26 10 3.5 1. Slope conductances (in pS) for permeant ions rank as follows: Ca2+ Sr2+ Na+ Mn2+ Ba2+ = 19 18 14 12 10. Unitary Ca2+ currents display a tendency to saturate with the Ca2+ concentration increase with apparent dissociation constant (K d ) of 10 mm. No block of Na+ permeation by extracellular Ca2+ in millimolar range was found. The data obtained suggest that (i) activation of some G protein is sufficient to gate the channels without the ATP receptor being occupied, (ii) the ATP receptor activation results in the gating of a special channel with the properties that differ markedly from those of the receptoroperated or voltage-gated Ca2+-permeable channels on the other cell types.DeceasedThe authors are grateful to K. Kiselyov and A. Mamin for technical assistance. The work was supported by the Russian Basic Research Foundation, Grant N 93-04-21722 and was made possible in part by Grant N R4A000 from the International Science Foundation.  相似文献   

15.
Summary The whole-cell voltage-clamp technique was employed to study the -adrenergic modulation of voltage-gated K+ currents in CD8+ human peripheral blood lymphocytes. The -receptor agonist, isoproterenol, decreased the peak current amplitude and increased the rate of inactivation of the delayed rectifier K+ current. In addition, isoproterenol decreased the voltage dependence of steady-state inactivation and shifted the steady-state inactivation curve to the left. Isoproterenol, on the other hand, had no significant effect on the steady-state parameters of current activation. The isoproterenol-induced decrease in peak current amplitude was inhibited by the -blocker propranolol. Bath application of dibutyryl cAMP (1mm) mimicked the effects of isoproterenol on both K+ current amplitude and time course of inactivation. Furthermore, the reduction in the peak current amplitude in response to isoproterenol was attenuated when PKI5–24 (2–5 m), a synthetic peptide inhibitor of cAMP-dependent protein kinase, was present in the pipette solution. The increase in the rate of inactivation of the K+ currents in response to isoproterenol was mimicked by the internal application of GTP--S (300 m) and by exposure of the cell to cholera toxin (1 g/ml), suggesting the involvement of a G protein. These results demonstrate that the voltage-dependent K+ conductance in T lymphocytes can be modulated by -adrenergic stimulation. The effects of -agonists, i.e., isoproterenol, appear to be receptor mediated and could involve cAMP-dependent protein kinase as well as G proteins. Since inhibition of the delayed rectifier K+ current has been found to decrease the proliferative response in T lymphocytes, the -adrenergic modulation of K+ current may well serve as a feedback control mechanism limiting the extent of cellular proliferation.  相似文献   

16.
Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 M. At steady state conditions, methionine is concentrated 30–50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 M. We recently reported that methionine uptake is decreased by elevations in extracellular K+. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5M, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K+ equal to 6.9 mM, the apparentV max of methionine transport is 182 pmol/min/mg protein, and theK m is 1.3 M. Where K+ is shifted to 11.9 mM, theK m remains unchanged, and theV max is reduced by half.  相似文献   

17.
Summary Previous studies indicate a particular sensitivity of red blood cell Na+-Li+ countertransport activity to small variations in the fatty acid composition of membrane phospholipids. To assess whether the interindividual variability of Na+-Li+ countertransport is related to differences in the species pattern of erythrocyte phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vivo, the molecular species composition of PC and PE as well as the kinetics of Na+-Li+ countertransport were analyzed in parallel in normo- and hyperlipidemic donors. Both in diacyl PC and in diacyl-PE the species 160/204 and 160/182 were, respectively, positively and negatively related to the apparent maximal velocity of Na+-Li+ countertransport. The sum of all species with 204 at sn2 of diacyl-PE exhibited a strong positive (r = 0.82, 2p < 0.001), and those containing 182 a negative correlation (r = –0.63, 2p < 0.01) to the transport activity. Essentially similar connections were observed between these species and the apparent affinity of the transport system for intracellular Na+. To evaluate whether the associations between molecular species of membrane phospholipids and Na+-Li+ countertransport activity were indicative of a causal relationship, the species 160/204-PC and 160/182-PC were selectively introduced into the erythrocyte membrane by means of the PC-specific transfer protein. Replacement of 11% of native PC by 160/182-PC inhibited the transport rate by about 25%. Exchange of 6 and 9% of PC with 160/204-PC, in contrast, accelerated the transport rate by 30 and 60%, respectively. The accordance between the in vivo relations and the results of the in vitro modification strongly suggests that elevations and reductions in the arachidonic acid and linoleic acid content of membrane PC and PE contribute to the interindividual variability of red blood cell Na+-Li+ counter-transport activity and its acceleration in hyperlipidemias.The authors wish to thank Dr. W.O. Richter (II. Medizinische Klinik, Klinikum Großhadern, Universität München) for selection of the patients and Dr. T. Brosche (Universität ErlangenNürnberg) for gaschromatographic analyses. This study was supported in part by a grant of the Wilhelm-Sander-Stiftung to B.E.  相似文献   

18.
This review summarizes our experiments on the significance of the -subunit in the functional expression of Na+/K+-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na+/K+-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH2-terminal domain to the extracellular COOH-terminus: the NH2-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na+/K+-ATPase by Na+/K+- and H+/K+-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na+/K+-ATPase -subunit and from Ile89 to the COOH–terminus of the H+/K+-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na+/K+-ATPase.  相似文献   

19.
Subfamilies of voltage-activated K+ channels (Kv1-4) contribute to controlling neuron excitability and the underlying functional parameters. Genes encoding the multiple subunits from each of these protein groups have been cloned, expressed and the resultant distinct K+ currents characterized. The predicted amino acid sequences showed that each subunit contains six putative membrane-spanning -helical segments (S1-6), with one (S4) being deemed responsible for the channels' voltage sensing. Additionally, there is an H5 region, of incompletely defined structure, that traverses the membrane and forms the ion pore; residues therein responsible for K+ selectivity have been identified. Susceptibility of certain K+ currents produced by the Shaker-related subfamily (Kv1) to inhibition by -dendrotoxin has allowed purification of authentic K+ channels from mammalian brain. These are large (Mr 400 kD), octomeric sialoglycoproteins composed of and subunits in a stoichiometry of ()4()4, with subtypes being created by combinations of subunit isoforms. Subsequent cloning of the genes for 1, 2 and 3 subunits revealed novel sequences for these hydrophilic proteins that are postulated to be associated with the subunits on the inner side of the membrane. Coexpression of 1 and Kv1.4 subunits demonstrated that this auxiliary protein accelerates the inactivation of the K+ current, a striking effect mediated by an N-terminal moiety. Models are presented that indicate the functional domains pinpointed in the channel proteins.  相似文献   

20.
Addition of Na+ to the K+-loadedVibrio alginolyticus cells, creating a 250-fold Na+ gradient, is shown to induce a transient increase in the intracellular ATP concentration, which is abolished by the Na+/H+ antiporter, monensin. The pNa-supported ATP synthesis requires an additional driving force supplied by endogenous respiration or, alternatively, by a K+ gradient (high [K+] inside). In the former case, ATP formation is resistant to the protonophorous uncoupler. Dicyclohexylcarbodiimide and diethylstilbestrol, but not vanadate, completely inhibit Na+ pulse-induced ATP formation. The data agree with the assumption that Na+-ATP-synthase is involved in oxidative phosphorylation inV. alginolyticus. Interrelation of H+ and Na+ cycles in bacteria is discussed.Abbreviations and electrochemical gradients of H+ and Na+, respectively - transmembrane electric potential difference - pH, pNa, and pK concentration gradients of H+, Na+, and K+, respectively - CCCP carbonyl cyanidem-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diesthylstilbestrol - HQNO 2-heptyl-4-hydroxyquinolineN-oxide - Tricine N[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine  相似文献   

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