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1.
Accumulation of 2-aminoisobutyrate by mouse ascites tumour cells was studied in circumstances where nigericin reversed the normal direction of the Na+ concentration gradient. The membrane potential (delta psi) was assayed using oxonol V as a voltage-sensitive probe. The amino acid gradient (delta mu A) that formed did not significantly exceed the likely magnitude of the Na+ electrochemical gradient when this was in the range 2-6 kJ mol-1. When delta-Na mu increased up to 11 kJ mol-1, delta mu A was almost constant at 7-8 kJ mol-1. The observations indicate that when delta psi is large changes in cellular [Na+] in the range 16-80 mM scarcely affect delta mu A. 相似文献
2.
L Beaugé 《Biochimica et biophysica acta》1975,401(1):95-108
Unidirectional and net Na+ fluxes modified by changes in internal Na+ concentration ([Na+]i) were studied in human red blood cells incubated in K+-free solutions containing 10-minus 4 m ouabain. An increase in [Na+]i brought about (a) a reduction in net Na+ gain, (b) no change in Na+ influx, (c) a reduction in the rate constant for Na+ effux and (d) an increase in Na+ efflux. Similar reductions in net Na+ gain were observed when the changes in [Na+]i were carried out at constant [K+]i. In addition, the rate constant for 42K+ efflux was not affected by changes in [Na+]i. The electrical membrane potential (as determined from the chloride distribution ratio) was also constnat. Furosemide (10-minus 3 M) increased the net Na+ gain in concentration reduced Na+ efflux and increased Na+ influx: the magnitude of these effects was dependent onthe intracellular Na+. The reduction in the net Na+ gain as [Na+]i increased was unaffected by depletion of cellular ATP to values below 10 mumol/1 cells, and this effect was independent of the depletion method used 相似文献
3.
The influence of the transmembrane Na+ gradient on the intracellular free calcium concentration, [Ca2+]i, was studied in Sepharose gel-filtered platelets from healthy human subjects, using the Ca-sensitive fluorescent dye, fura-2. Raising the internal Na+ concentration, [Na+]i, by Na+ pump inhibition with 0.05 mM ouabain, without changing external Na+ did not cause a significant increase in [Ca2+]i. Substitution of extracellular Na+ by iso-osmolar sucrose induced a rapid (half-time about 2 min) and significant rise in [Ca2+]i; this effect was amplified in Na-loaded platelets. Partial restitution of external Na+ in these cells with increased [Ca2+]i promoted a significant and rapid Na+ concentration-dependent fall in [Ca2+]i; little decline in [Ca2+]i was observed if K+ was used instead of Na+. These observations represent in vitro evidence for the existence of a Na/Ca exchange mechanism in human platelets that may, in vivo, participate in the control of [Ca2+]i. 相似文献
4.
Effects of the K+ concentration in the bathing fluid ([K+]l) on the intracellular K+, Na+ and Cl- concentrations ([K+]i [Na+]i and [Cl-]i) as well as on the electrical potential were studied in rat duodenum. Changes in the mucosal K+ concentration ([K+]m), bringing the sum of Na+ and K+ concentrations to 147.2 mM constant, had little effect on the transmural potential difference (PDt), but did induce marked changes in the mucosal membrane potential (Vm). As [K+]m increased, Vm was depolarized gradually and obeyed the Nernst equation for a potassium electrode in the range of [K+]m greater than approx. 60 mM. Experiments of ion analyses were carried out on strips of duodenum to determine the effect of changing the external K+ concentrations on [K+] i, [Na+]i and [Cl-]i. An increase in [K+]o resulted in increases in [K+]i and [Cl-]i and a decrease in [Na+]i, [K+]i approaching its maximum at [K+]o greater than 70 mM. Such changes in [K+]i and [Na+]i seem to correlate quantitatively with the changes in [K+]o and [Na+]o. The values of the ratio of permeability coefficients, Pna+/PK+ were estimated using the Vm values and intracellular ion concentrations measured in these experiments. The results suggested that there appeared a rather abrupt increase in the PNa+/PK+ ratio from 0 to approx. 0.1, as [K+]m decreased. 相似文献
5.
Preparations of plasma membrane vesicles were used as a tool to study the properties of the serotonin transporter in the central nervous system. The vesicles were obtained after hypotonic shock of synaptosomes purified from mouse cerebral cortex. Uptake of [3H]serotonin had a Na+-dependent and Na+-independent component. The Na+-dependent uptake was inhibited by classical blockers of serotonin uptake and had a Km of 63-180 nM, and a Vmax of 0.1-0.3 pmol mg-1 s-1 at 77 mM Na+. The uptake required the presence of external Na+ and internal K+. It required a Na+ gradient ([Na+]out greater than [Na+]in) and was stimulated by a gradient of K+ ([K+]in greater than [K+]out). Replacement of Cl- by other anions (NO2-, S2O3-(2-)) reduced uptake appreciably. Gramicidin prevented uptake. Although valinomycin increased uptake somewhat, the membrane potential per se could not drive uptake because no uptake was observed when a membrane potential was generated by the SCN- ion in the absence of internal K+ and with equal [Na+] inside and outside. The increase of uptake as a function of [Na+] indicated a Km for Na+ of 118 mM and a Hill number of 2.0, suggesting a requirement of two sodium ions for serotonin transport. The present results are accommodated very well by the model developed for porcine platelet serotonin transport (Nelson, P. J., and Rudnick, G. (1979) J. Biol. Chem. 254, 10084-10089), except for the number of sodium ions that are required for transport. 相似文献
6.
A proton gradient is the driving force for uphill transport of lactate in human placental brush-border membrane vesicles 总被引:2,自引:0,他引:2
D F Balkovetz F H Leibach V B Mahesh V Ganapathy 《The Journal of biological chemistry》1988,263(27):13823-13830
The characteristics of lactate transport in brush-border membrane vesicles isolated from normal human full-term placentas were investigated. Lactate transport in these vesicles was Na+-independent, but was greatly stimulated when the extravesicular pH was made acidic. In the presence of an inwardly directed H+ gradient ([H+]o greater than [H+]i), transient uphill transport of lactate could be demonstrated. This H+ gradient-dependent stimulation was not a result of a H+ diffusion potential. Transport of lactate in the presence of the H+ gradient was not inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or by furosemide, ruling out the participation of an anion exchanger in placental lactate transport. Many monocarboxylates strongly interacted with the lactate transport system, whereas, with the single exception of succinate, dicarboxylates did not. The monocarboxylates pyruvate and lactate, but not the dicarboxylate succinate, when present inside the vesicles, were able to exert a trans-stimulatory effect on the uptake of radiolabeled lactate. Kinetic analyses provided evidence for a single transport system with a Kt of 4.1 +/- 0.4 mM for lactate and a Vmax of 54.2 +/- 9.9 nmol/mg of protein/30 s. Pyruvate inhibited lactate transport competitively, by reducing the affinity of the system for lactate without altering the maximal velocity. It is concluded that human placental brush-border membranes possess a transport system specific for lactate and other monocarboxylates and that this transport system is Na+-independent and is energized by an inwardly directed H+ gradient. Lactate-H+ symport rather than lactate-OH- antiport appears to be the mechanism of the H+ gradient-dependent lactate transport in these membranes. 相似文献
7.
Triads and transverse tubules isolated from mammalian skeletal muscle actively accumulated Na+ in the presence of K+ and Mg-ATP. Active Na+ transport exhibited a fast single-exponential phase, lasting 2 min, followed by slower linear uptake that continued for 10 minutes. Valinomycin stimulated Na+ uptake, suggesting it decreased a pump-generated membrane potential gradient (Vm) that prevented further Na+ accumulation. At the end of the fast uptake phase transverse tubule vesicles incubated in 30 mM external [Na+] attained a ratio [Na+]in/[Na+]out=13.4. From this ratio and the transverse tubule volume of 0.35 microl/mg protein measured in this work, [Na+]in=400 mM was calculated. Determinations of active K+ transport in triads, using 86Rb+ as tracer, showed a 30% decrease in vesicular 86Rb+ content two minutes after initiating the reaction, followed by a slower uptake phase during which vesicles regained their initial 86Rb+ content after 10 minutes. Transverse tubule volume increase during active Na+ transport-as shown by light scattering changes of isolated vesicles--presumably accounted for the secondary Na+ and 86Rb+ uptake phases. These combined results indicate that isolated triads have highly sealed transverse tubules that can be polarized effectively by the Na+ pump through the generation of significant Na+ gradients. 相似文献
8.
Calcium entry in squid axons during voltage clamp pulses 总被引:1,自引:0,他引:1
Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one. 相似文献
9.
The bacterium Vitreoscilla generates an electrical potential gradient due to sodium ion (delta psi Na+) across its membrane via respiratory-driven primary Na+ pump(s). The role of the delta psi Na+ as a driving force for ATP synthesis was, therefore, investigated. In respiring starved cells pulsed with 100 mM external Na+ [( Na+]o) there was a 167% net increase in cellular ATP concentration over basal levels compared with 0, 56, 78, and 78% for no addition, choline, Li+, and K+ controls, respectively. Doubling the [Na+]o to 200 mM boosted the net increase to 244% but a similar doubling of the choline caused only an increase to 78%. When the initial condition was intracellular Na+ ([Na+]i) = [Na+]o = 100 mM, there was a 94% net increase in cellular ATP compared with only 18 and 11% for Li+ and K+ controls, respectively, indicating that Nai+ may be the only cation tested that the cells extruded to generate the electrochemical gradient required to drive ATP synthesis. The Na(+)-dependent ATP synthesis was inhibited completely by monensin (12 microM), but only transiently by the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (100 microM), further evidence that the Na+ gradient and not a H+ gradient was driving the ATP synthesis. ATP synthesis in response to an artificially imposed H+ gradient (delta pH approximately 3) in the absence of an added cation, or in the presence of Li+, K+, or choline, yielded similar delta ATP/delta pH ratios of 0.98-1.22. In the presence of Na+, however, this ratio dropped to 0.23, indicating that Na+ inhibited H(+)-coupling to ATP synthesis and possibly that H+ and Na+ coupling to ATP synthesis share a common catalyst. The above evidence adds to previous findings that under normal growth conditions Na+ is probably the main coupling cation for ATP synthesis in Vitreoscilla. 相似文献
10.
V Dall'Asta O Bussolati G G Guidotti G C Gazzola 《The Journal of biological chemistry》1991,266(3):1591-1596
The energization of System A in cultured human fibroblasts has been studied by measuring the energy transfer from the electrochemical gradient of Na+ to the chemical gradient of the site A-specific substrate amino acid 2-methylaminoisobutyric acid. The co-transport Na+/amino acid, studied by kinetic analysis and radiochemical measurements, showed a coupling ratio of 1:1. The assessment of the Na+ electrochemical gradient in cultured adherent cells relied on the development of noninvasive procedures as follows: the membrane electrical potential was estimated from the accumulation of L-arginine at equilibrium (Bussolati, O., Laris, P. C., Nucci, F. A., Dall'Asta, V., Longo, N., Guidotti, G. G., and Gazzola, G. C. (1987) Am. J. Physiol. 253, C391-C397); the chemical gradient of Na+ was determined from spectrometric measurements of Na+. The accumulation of 2-methylaminoisobutyric acid was strongly sensitive to changes of Na+ gradient and of membrane electrical potential, indicating that the electrochemical gradient of Na+ contributed energy for the uphill transport of the amino acid through System A. Changes in the Na+ electrochemical gradient were obtained by: (i) alterations of extracellular concentration of Na+; (ii) changes of membrane electrical potential obtained by variation of extracellular [K+]; and (iii) changes of [Na+]in and membrane electrical potential upon incubation of the cells in serum-free saline solutions (Dall'Asta, V., Gazzola, G. C., Longo, N., Bussolati, O., Franchi-Gazzola, R., and Guidotti, G. G. (1986) Biochim. Biophys. Acta 860, 1-8). The correlation between the chemical gradient of 2-methylaminoisobutyric acid and the Na+ electrochemical potential followed a straight line with a yield close to the thermodynamic equilibrium, thus suggesting that the energy stored in the gradient of Na+ electrochemical potential is fully adequate to energize the intracellular accumulation of site A-reactive amino acids in human fibroblasts. 相似文献
11.
Lisa C Mace Bradley M Palmer David A Brown Korinne N Jew Joshua M Lynch Jason M Glunt Todd A Parsons Joseph Y Cheung Russell L Moore 《Journal of applied physiology》2003,95(5):1994-2003
Effects of age and training on myocardial Na+/Ca2+ exchange were examined in young sedentary (YS; 14-15 mo), aged sedentary (AS; 27-31 mo), and aged trained (AT; 8- to 11-wk treadmill run training) male Fischer Brown Norway rats. Whole heart performance and isolated cardiocyte Na+/Ca2+ exchange characteristics were measured. At the whole heart level, a small but significant slowing of late isovolumic left ventricular (LV) relaxation, which may be indicative of altered Na+/Ca2+ exchange activity, was seen in hearts from AS rats. This subtle impairment in relaxation was not observed in hearts from AT rats. At the single-cardiocyte level, late action potential duration was prolonged, resting membrane potential was more positive, and overshoot potential was greater in cardiocytes from AS rats than from YS rats (P < 0.05). Training did not influence any of these age-related action potential characteristics. In electrically paced cardiocytes, neither shortening nor intracellular Ca2+ concentration ([Ca2+]i) dynamics was influenced by age or training. Similarly, neither age nor training influenced the rate of [Ca2+]i clearance via forward (Nain+ /Caout2+) Na+/Ca2+ exchange after caffeine-induced Ca2+ release from the sarcoplasmic reticulum or cardiac Na+/Ca2+ exchanger protein (NCX1) expression. However, when whole cell patch-clamp techniques combined with fluorescence microscopy were used to evaluate the ability of Na+/Ca2+ exchange to alter cytosolic [Ca2+] ([Ca2+]c) under conditions where membrane potential (Vm) and internal and external [Na+] and [Ca2+] could be controlled, we observed age-associated increases in forward Na+/Ca2+ exchange-mediated [Ca2+]c clearance (P < 0.05) that were not influenced by training. The age-related increase in forward Na+/Ca2+ exchange activity provides a hypothetical explanation for the late action potential prolongation observed in this study. 相似文献
12.
Sodium-calcium exchange and calcium-calcium exchange in internally dialyzed squid giant axons. 总被引:4,自引:0,他引:4
The influx and efflux of calcium (as 45Ca) and influx of sodium (as 24Na) were studied in internally dialyzed squid giant axons. The axons were poisoned with cyanide and ATP was omitted from the dialysis fluid. The internal ionized Ca2+ concentration ([Ca2+]i) was controlled with Ca-EGTA buffers. With [Ca2+]i greater than 0.5 muM, 45Ca efflux was largely dependent upon external Na and Ca. The Nao-dependent Ca efflux into Ca-free media appeared to saturate as [Ca2+]i was increased to 160 muM; the half-saturation concentration was about 8 muM Ca2+. In two experiments 24Na influx was measured; when [Ca2+]i was decreased from 160 muM to less than 0.5 muM, Na influx declined by about 5 pmoles/cm2 sec. The Nao-dependent Ca efflux averaged 1.6 pmoles/cm2 sec in axons with a [Ca2+]i of 160 muM, and was negligible in axons with a [Ca2+]i of less than 0.5 muM. Taken together, the Na influx and Ca efflux data may indicate that the fluxes are coupled with a stoichiometry of about 3 Na+-to-1 Ca2+. Ca efflux into Na-free media required the presence of both Ca and an alkali metal ion (but not Cs) in the external medium. Ca influx from Li-containing media was greatly reduced when [Ca2+]i was decreased from 160 to 0.23 muM, or when external Li was replaced by choline. These data provide evidence for a Ca-Ca exchange mechanism which is activated by certain alkali metal ions. The observations are consistent with a mobile carrier mechanism which can exchange Ca2+ ions from the axoplasm for either 3 Na+ ions, or one Ca2+ and an alkali metal ion (but not Cs) from the external medium. This mechanism may utilize energy from the Na electrochemical gradient to help extrude Ca against an electrochemical gradient. 相似文献
13.
A proton gradient, not a sodium gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles. 总被引:3,自引:1,他引:2
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C Tiruppathi D F Balkovetz V Ganapathy Y Miyamoto F H Leibach 《The Biochemical journal》1988,256(1):219-223
An inward-directed H+ gradient markedly stimulated lactate uptake in rabbit intestinal brush-border membrane vesicles, and uphill transport against a concentration gradient could be demonstrated under these conditions. Uptake of lactate was many-fold greater in the presence of a H+ gradient than in the presence of a Na+ gradient. Moreover, there was no evidence for uphill transport of lactate in the presence of a Na+ gradient. The H+-gradient-dependent stimulation of lactate uptake was not due to the effect of a H+-diffusion potential. The uptake process in the presence of a H+ gradient was saturable [Kt (concn. giving half-maximal transport) for lactate 12.7 +/- 4.5 mM] and was inhibited by many monocarboxylates. It is concluded that a H+ gradient, not a Na+ gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles. 相似文献
14.
A. A. Eddy 《The Biochemical journal》1968,108(3):489-498
1. Tumour cells were starved to deplete them of ATP and transferred to 0.9mm-glycine in Ringer solutions containing 2mm-sodium cyanide and various Na(+) and K(+) concentrations. The uptake of glycine then usually reached a peak by about 10min. 2. When cellular [Na(+)] and extracellular [Na(+)] were each about 30m-equiv./l., the maximum amount of glycine absorbed increased between 1.2- and 3.0-fold on lowering extracellular [K(+)] from 128 to 10m-equiv./l. 3. When extracellular [Na(+)] was 150m-equiv./l., the ratio, R, of the cellular to extracellular glycine concentrations increased progressively, from near 1 to about 9, when cellular [Na(+)] was lowered from 120 to 40m-equiv./l. 4. When cellular [Na(+)] was almost constant, either at 45 or 70m-equiv./l., R fell about 14-fold when extracellular [Na(+)] varied from 150 to 16m-equiv./l. 5. Values of R near 0.2 were found when cellular [Na(+)] was about four times as large as extracellular [Na(+)]. 6. R fell about threefold when the cells were put with 12mm- instead of 0.9mm-glycine. 7. The results were taken to imply that, under these conditions, the spontaneous movements of both Na(+) and K(+) across the cell membrane, down their respective concentration gradients, served to concentrate the glycine in the tumour cells (Christensen's hypothesis). 相似文献
15.
The accumulation of amino acids by mouse ascites-tumour cells. Dependence on but lack of equilibrium with the sodium-ion electrochemical gradient. 总被引:4,自引:3,他引:1
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1. The fluorescent dye 3,3'-dipropyloxadicarbocyanine was used to show that the tumour cells absorbed 2-aminoisobutyrate, glycine, L-leucine and L-isoleucine and certain other amino acids electrogenically. The Km values with respect to amino acid concentration ([A]o), obtained from the fluorescence assays, varied through the above series from 0.8 to 26 mM, with Vmax. fairly constant. 2. Similar Km values described the uptake of the 14C-labelled amino acids in five instances where this was measured. 3. Each amino acid lowered the membrane potential (E) by 10-20 mV when its cellular concentration ([A]i) had reached a steady value and [A]o was 10mM. In these experiments energy metabolism was maintained by glycolysis, 2,4-dinitrophenol was present and cellular respiration was inhibited. The corresponding net flow of amino acid through the Na+ symport was deduced by making use of the fact that the depolarization an amino acid initially caused was roughly proportional to the net influx of amino acid itself. 4. The steady-state depolarization was attributed to the presence of a leak pathway for the amino acid with a rate coefficient PA. As assayed in the absence of Na+, PA was about 5-fold larger for isoleucine than for glycine. 5. Direct estimates of Vmax./PA were similar to those inferred from the extent of depolarization in the steady state and [A]i. 6. A mathematical model was used to predict [A]i/[A]o in term of the measured values of [Na]o, [Na]i, E, Km and Vmax./PA. The predicted and observed values agreed fairly well when [A]o was 1 mM or 10 mM. 7. [A]i/[A]o varied from about 2.5 for 10 mM-isoleucine to 30 for 1 mM-2-aminoisobutyrate when delta microNa, expressed as a ratio, was ostensibly in the range 19-43. 8. The concentration of 2-aminoisobutyrate from a 0.1 mM solution in the presence or absence of ouabain was consistent with the model, whereas the concentration of isoleucine from a 0.1 mM solution exceeded the predicted values 2-5-fold. 9. The tumour cells concentrated 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid by a non-electrogenic mechanism, with which isoleucine may also interact. 相似文献
16.
The neuropeptide somatostatin causes membrane hyperpolarization and reduces the intracellular free calcium ion concentration ([Ca2+]i) in GH pituitary cells. In this study, we have used the fluorescent dyes bisoxonol (bis,-(1,3-diethylthiobarbiturate)-trimethineoxonol) and quin2 to elucidate the mechanisms by which these ionic effects are triggered. Addition of 100 nM somatostatin to GH4C1 cells caused a 3.4 mV hyperpolarization and a 26% decrease in [Ca2+]i within 30 s. These effects were not accompanied by changes in intracellular cAMP concentrations and occurred in cells containing either basal or maximally elevated cAMP levels. To determine which of the major permeant ions were involved in these actions of somatostatin, we examined its ability to elicit changes in the membrane potential and the [Ca2+]i when the transmembrane concentration gradients for Na+, Cl-, Ca2+, and K+ were individually altered. Substitution of impermeant organic ions for Na+ or Cl- did not block either the hyperpolarization or the decrease in [Ca2+]i induced by somatostatin. Decreasing extracellular Ca2+ from 1 mM to 250 nM abolished the reduction in [Ca2+]i but did not prevent the hyperpolarization response. These results show that hyperpolarization was not primarily due to changes in the conductances of Na+, Cl-, or Ca2+. Although the somatostatin-induced decrease in [Ca2+]i did require Ca2+ influx, it was independent of changes in Na+ or Cl- conductance. In contrast, elevating the extracellular [K+] from 4.6 to 50 mM completely blocked both the somatostatin-induced hyperpolarization and the reduction in [Ca2+]i. Furthermore, hyperpolarization of the cells with gramicidin mimicked the effect of somatostatin to decrease the [Ca2+]i and prevented any additional effect by the hormone. These results indicate that somatostatin increases a K+ conductance, which hyperpolarizes GH4C1 cells, and thereby secondarily decreases Ca2+ influx. Since the somatostatin-induced decrease in [Ca2+]i is independent of changes in intracellular cAMP levels, it may be responsible for somatostatin inhibition of hormone secretion by its cAMP-independent mechanism. 相似文献
17.
Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers 总被引:1,自引:0,他引:1
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《The Journal of general physiology》1995,106(3):445-466
Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride- sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12- 13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (< 30 mM) and 10-100 microM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans. Buffering of the cis compartment [Ca2+] to nearly zero (< 1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 +/- 0.02 vs 0.02 +/- 0.01 in control), but markedly reduced the influence of both cis- and trans-[Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCa2+] of 10-100 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels. 相似文献
18.
To investigate whether the Na permeability of the resting membrane is determined predominantly by the excitable Na channel, we examined the effects of tetrodotoxin (TTX) and the complete removal of external Na+ on the resting potential. In the intact squid axon bathed in K-free artificial seawater, both TTX and the removal of Na+ produced small hyperpolarizations. The effect of Na removal, however, was larger than that of TTX. In the perfused squid axon, the hyperpolarization produced by the removal of external Na+ was greatly enhanced when the internal K concentration ([K+]i) was reduced. The effect of TTX, on the other hand, was not sensitive to the [K+]i or to the membrane potential. For [K+]i = 50 mM and [K+]o = 0, the average hyperpolarization produced by TTX was 1.2 mV, while the hyperpolarization produced by Na removal was approximately 21 mV. The difference between these two effects suggests that the majority of the resting Na current passes through pathways other than the excitable Na channel. 相似文献
19.
P C Laris M Bootman H A Pershadsingh R M Johnstone 《Biochimica et biophysica acta》1978,512(2):397-414
The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na+ :K+ pump. The membrane potential (monitored by the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na+ but not cellular ATP. Na+ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na+ and amino acids. Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na+ (approx. 30mM), the Na+ :K+ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na+ was raised to 60mM, the activity of the pump changed the membrane potential from the range -25 to -30 mV to -44 to -63 mV. This hyperpolarization required external K+ and was inhibited by ouabain. 相似文献
20.
In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential. 相似文献