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1.
Summary Stage V and VI (Dumont, J.N., 1972.J. Morphol.
136:153–180) oocytes ofXenopus laevis were treated with collagenase to remove follicular cells and were placed in K-free solution for 2 to 4 days to elevate internal [Na]. Na/K pump activity was studied by restoring the eggs to normal 3mm K Barth's solution and measuring membrane current-voltage (I–V) relationships before and after the addition of 10 m dihydroouabain (DHO) using a two-microelectrode voltage clamp. Two pulse protocols were used to measure membraneI–V relationships, both allowing membrane currents to be determined twice at each of a series of membrane potentials: (i) a down-up-down sequence of 5 mV, 1-sec stair steps and (ii) a similar sequence of 1-sec voltage pulses but with consecutive pulses separated by 4-sec recovery periods at the holding potential (–40 mV). The resulting membraneI–V relationships determined both before and during exposure to DHO showed significant hysteresis between the first and second current measurements at each voltage. DHO difference curves also usually showed hysteresis indicating that DHO caused a change in a component of current that varied with time. Since, by definition, the steady-state Na/K pumpI–V relationship must be free of hysteresis, the presence of hysteresis in DHO differenceI–V curves can be used as a criterion for excluding such data from consideration as a valid measure of the Na/K pumpI–V relationship. DHO differenceI–V relationships that did not show hysteresis were sigmoid functions of membrane potential when measured in normal (90mm) external Na solution. The Na/K pump current magnitude saturated near 0 mV at a value of 1.0–1.5 A cm–2, without evidence of negative slope conductance for potentials up to +55 mV. The Na/K pump current magnitude in Na-free external solution was approximately voltage independent. Since these forward-going Na/K pumpI–V relationships do not show a region of negative slope over the voltage range –110 to +55 mV, it is not necessary to postulate the existence of more than one voltage-dependent step in the reaction cycle of the forward-going Na/K pump. 相似文献
2.
H. J. Apell 《The Journal of membrane biology》1989,110(2):103-114
3.
David C. Gadsby Masakazu Nakao Anthony Bahinski 《Molecular and cellular biochemistry》1989,89(2):141-146
Experiments are reviewed here in which Na/K pump current was determined as strophanthidin-sensitive current in guinea-pig ventricular myocytes, voltage-clamped and internally-dialyzed via wide-tipped pipettes. In the presence of 150 mM extracellular [Na], both outward and inward pump current, during forward and reverse Na/K exchange respectively, were strongly voltage dependent. But reduction of external [Na] to 1.5 mM severely attenuated the voltage sensitivity of outward Na/K pump current. Voltage jumps elicited large transient pump currents during forward or reverse Na/K exchange, or when pump activity was restricted to Na translocation steps, but not when pumps were presumably engaged in K/K exchange. These findings indicate that Na translocation, but not K translocation, involves net charge movement through the membrane field, and that both forward and reverse Na/K transport cycles are rate-limited not by that voltage-sensitive step but by a subsequent voltage-insensitive step. 相似文献
4.
Summary Electrophysiological experiments were performed to analyze the Na+/K+-ATPase in full-grown prophase-arrested oocytes ofXenopus laevis. If the Na+/K+-ATPase is inhibited by dihydroouabain (DHO), the resting potential of the membrane of Na+-loaded oocytes may depolarize by nearly 50 mV. This hyperpolarizing contribution to the resting potential depends on the degree of activation of the Na+/K+-ATPase and varies with intra-cellular Na+ activity (a
Na
i
), and extracellular K+ (K
0
+
) It is concluded that variations ofa
Na
i
among different oocytes are primarily responsible for the variations of resting potentials measured in oocytes ofX. laevis. Under voltage-clamp conditions, the DHO-sensitive current also exhibits dependence ona
Na
i
that may be described by a Hill equation with a coefficient of 2. This current will be shown to be identical with the electrogenic current generated by the 3Na+/2K+ pump. The voltage dependence of the pump current was investigated at saturating values ofa
Na
i
(33 mmol/liter) and of K
0
+
(3 mmol/liter) in the range from –200 to +100 mV. The current was found to exhibit a characteristic maximum at about +20 mV. This is taken as evidence that in the physiological range at least two steps within the cycle of the pump are voltage dependent and are oppositely affected by the membrane potential. 相似文献
5.
The steady-state voltage and [Na(+)](o) dependence of the electrogenic sodium pump was investigated in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, under conditions that promote the backward-running mode (K(+)-free seawater; ATP- and Na(+)-free internal solution containing ADP and orthophosphate). The ratio of pump-mediated (42)K(+) efflux to reverse pump current, I(pump) (both defined by sensitivity to dihydrodigitoxigenin, H(2)DTG), scaled by Faraday's constant, was -1.5 +/- 0.4 (n = 5; expected ratio for 2 K(+)/3 Na(+) stoichiometry is -2.0). Steady-state reverse pump current-voltage (I(pump)-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various V(m) to H(2)DTG or from the difference between membrane I-V relationships obtained by imposing V(m) staircases in the presence or absence of H(2)DTG. With the second method, we also investigated the influence of [Na(+)](o) (up to 800 mM, for which hypertonic solutions were used) on the steady-state reverse I(pump)-V relationship. The reverse I(pump)-V relationship is sigmoid, I(pump) saturating at large negative V(m), and each doubling of [Na(+)](o) causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltzmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state (22)Na(+) efflux during electroneutral Na(+)/Na(+) exchange, and follow without additional postulates from the same simple high field access channel model (Gadsby, D.C., R.F. Rakowski, and P. De Weer, 1993. Science. 260:100-103). This model predicts valence z = nlambda, where n (1.33 +/- 0.05) is the Hill coefficient of Na binding, and lambda (0.61 +/- 0.03) is the fraction of the membrane electric field traversed by Na ions reaching their binding site. More elaborate alternative models can accommodate all the steady-state features of the reverse pumping and electroneutral Na(+)/Na(+) exchange modes only with additional assumptions that render them less likely. 相似文献
6.
Time constants of slow inactivation were investigated in NH(2)-terminal deleted Shaker potassium channels using macro-patch recordings from Xenopus oocytes. Slow inactivation is voltage insensitive in physiological solutions or in simple experimental solutions such as K(+)(o)//K(+)(i) or Na(+)(o)//K(+)(i). However, when [Na(+)](i) is increased while [K(+)](i) is reduced, voltage sensitivity appears in the slow inactivation rates at positive potentials. In such solutions, the I-V curves show a region of negative slope conductance between approximately 0 and +60 mV, with strongly increased outward current at more positive voltages, yielding an N-shaped curvature. These changes in peak outward currents are associated with marked changes in the dominant slow inactivation time constant from approximately 1.5 s at potentials less than approximately +60 mV to approximately 30 ms at more than +150 mV. Since slow inactivation in Shaker channels is extremely sensitive to the concentrations and species of permeant ions, more rapid entry into slow inactivated state(s) might indicate decreased K(+) permeation and increased Na(+) permeation at positive potentials. However, the N-shaped I-V curve becomes fully developed before the onset of significant slow inactivation, indicating that this N-shaped I-V does not arise from permeability changes associated with entry into slow inactivated states. Thus, changes in the relative contributions of K(+) and Na(+) ions to outward currents could arise either: (a) from depletions of [K(+)](i) sufficient to permit increased Na(+) permeation, or (b) from voltage-dependent changes in K(+) and Na(+) permeabilities. Our results rule out the first of these mechanisms. Furthermore, effects of changing [K(+)](i) and [K(+)](o) on ramp I-V waveforms suggest that applied potential directly affects relative permeation by K(+) and Na(+) ions. Therefore, we conclude that the voltage sensitivity of slow inactivation rates arises indirectly as a result of voltage-dependent changes in the ion occupancy of these channels, and demonstrate that simple barrier models can predict such voltage-dependent changes in relative permeabilities. 相似文献
7.
We have measured the voltage dependence of the Na/K pump in isolated adult rat cardiac myocytes using the whole-cell patch-clamp technique. In the presence of 1–2 mM Ba and 0.1 mm Cd and nominally Ca-free, Na/K pump current (I
p) was measured as the change in current due to 1 mM ouabain. Voltage dependence of I
pwas measured between –140 and +40 or +60 mV using square voltage-pulse and voltage-ramp protocols, respectively. With 150 mM extracellular Na (Na
o
) and 5.4 mM extracellular K (K
o
), we found that the Na/K pump shows a strong positive voltage dependence between –140 and 0 mV and is voltage independent at positive potentials. Removing Na
o
reduced the voltage dependence at negative potentials with no effect at positive potentials. When K
o
was reduced, a negative slope appeared in the current-voltage (I-V) curve at positive potentials. We have investigated whether Na
i
(intracellular Na) might also affect the voltage dependence of I
pby varying Na in the patch pipette (Napip) between 20 and 85 mM. We found, as expected, that I
pincreased markedly as Napip was raised, saturating at about 70 mM Napip under these conditions. In contast, while I
psaturated near +20 mV and declined to about 40% of maximum at –120 mV, there was no effect of Napip under these conditions. In contrast, while I
psaturated near +20 mV and declined to about 40% of maximum at –120 mV, there was no effect of Napip on the voltage dependence of I
p. This suggests that neither Na
i
binding to the Na/K pump nor the conformational changes dependent on Na
i
binding are voltage dependent. These results are consistent with extracellular ion binding within the field of the membrane but do not rule out the possibility that other steps, such as Na translocation, are also voltage dependent.We wish to thank Ms. Melinda Price, Ms. Meei Liu and Mr. Randall Anderson for their technical assistance. This work was supported in part by National Institutes of Health grant HL44660. 相似文献
8.
The present study provides the first evidence that the abundance of catalytic alpha1-subunit of Na,K-ATPase increases in the course of T cell blast transformation. Immunodepressant cyclosporin A at anti-proliferative doses diminished the induction of alpha1 protein in activated lymphocytes. Furthermore, in competent T cells, IL-2 increases both the transport activity of Na/K pump and the content of Na,K-ATPase alpha1 protein in a time-dependent manner. A correlation was found between the long-term elevation in ouabain-sensitive Rb influxes and the increase in alpha1 protein content in late activated T cells. These results suggest that (1) the increased expression of Na,K-ATPase proteins underlie the cell cycle-dependent upregulation of ion pump during T cell transformation, and (2) IL-2 is involved in the regulated expression of Na,K-ATPase in human lymphocytes. 相似文献
9.
A large amount of experimental data on the characteristics of the cardiac Na+/K+ pump have been accumulated, but it remains difficult to predict the quantitative contribution of the pump in an intact cell because most measurements have been made under non-physiological conditions. To extrapolate the experimental findings to intact cells, we have developed a comprehensive Na+/K+ pump model based on the thermodynamic framework (Smith and Crampin, 2004) of the Post-Albers reaction cycle combined with access channel mechanisms. The new model explains a variety of experimental results for the Na+/K+ pump current (INaK), including the dependency on the concentrations of Na+ and K+, the membrane potential and the free energy of ATP hydrolysis. The model demonstrates that both the apparent affinity and the slope of the substrate-INaK relationship measured experimentally are affected by the composition of ions in the extra- and intracellular solutions, indirectly through alteration in the probability distribution of individual enzyme intermediates. By considering the voltage dependence in the Na+- and K+-binding steps, the experimental voltage-INaK relationship could be reconstructed with application of experimental ionic compositions in the model, and the view of voltage-dependent K+ binding was supported. Re-evaluation of charge movements accompanying Na+ and K+ translocations gave a reasonable number for the site density of the Na+/K+ pump on the membrane. The new model is relevant for simulation of cellular functions under various interventions, such as depression of energy metabolism. 相似文献
10.
H Munakata K Schmid J H Collins A S Zot L K Lane A Schwartz 《Biochemical and biophysical research communications》1982,107(1):229-231
We have determined the carbohydrate compositions of the protein components of lamb kidney Na,K-ATPase. The α subunit contains a total of about 16 monosaccharide residues per mol of protein, while the β subunit contains about 36 residues per mol. The γ protein, a proteolipid associated with the Na,K-ATPase, contains only traces of carbohydrate. A comparison of our results with those of others shows considerable variability in the carbohydrate compositions of α and β subunits from different species. 相似文献
11.
The fluxes of 22Na+ and 86Rb+ in Arbacia sperm and oocytes were studied in order to determine how these cells carry out cation exchange with the sea environment. The uptake of these ions by serum followed a pattern of early rapid influx (initial 0.5 min) and subsequent efflux (1–3 min) followed by a gradual uptake (after 3 min). Neither the uptake nor the efflux of these cations by Arbacia sperm were affected by ouabain, suggesting that influx and efflux of 22Na+ and 86Rb+ in Arbacia sperm occur predominantly by passive transport. The 22Na+ uptake by Arbacia oocytes showed a steady increase after an initial rapid uptake. A slight but significant inhibition of 22Na+ uptake was observed with ouabain. However, 86Rb+ uptake by the oocytes reached an early equilibrium and was not affected by ouabain. The uptake of Rb+ by Arbacia oocyte is by passive transport while that of Na+ is both by passive and active transport. 相似文献
12.
13.
Quaternary organic amines inhibit Na,K pump current in a voltage-dependent manner: direct evidence of an extracellular access channel in the Na,K-ATPase
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The effects of organic quaternary amines, tetraethylammonium (TEA) chloride and benzyltriethylammonium (BTEA) chloride, on Na,K pump current were examined in rat cardiac myocytes superfused in extracellular Na(+)-free solutions and whole-cell voltage-clamped with patch electrodes containing a high Na(+)-salt solution. Extracellular application of these quaternary amines competitively inhibited extracellular K(+) (K(+)(o)) activation of Na,K pump current; however, the concentration for half maximal inhibition of Na,K pump current at 0 mV (K(0)(Q)) by BTEA, 4.0 +/- 0.3 mM, was much lower than the K(0)(Q) for TEA, 26.6 +/- 0.7 mM. Even so, the fraction of the membrane electric field dissipated during K(+)(o) activation of Na,K pump current (lambda(K)), 39 +/- 1%, was similar to lambda(K) determined in the presence of TEA (37 +/- 2%) and BTEA (35 +/- 2%), an indication that the membrane potential (V(M)) dependence for K(+)(o) activation of the Na,K pump current was unaffected by TEA and BTEA. TEA was found to inhibit the Na,K pump current in a V(M)-independent manner, i.e., inhibition of current dissipated 4 +/- 2% of the membrane electric field. In contrast, BTEA dissipated 40 +/- 5% of the membrane electric field during inhibition of Na,K pump current. Thus, BTEA inhibition of the Na,K-ATPase is V(M)-dependent. The competitive nature of inhibition as well as the similar fractions of the membrane electric field dissipated during K(+)(o)-dependent activation and BTEA-dependent inhibition of Na,K pump current suggest that BTEA inhibits the Na,K-ATPase at or very near the enzyme's K(+)(o) binding site(s) located in the membrane electric field. Given previous findings that organic quaternary amines are not occluded by the Na,K-ATPase, these data clearly demonstrate that an ion channel-like structure provides access to K(+)(o) binding sites in the enzyme. 相似文献
14.
The behavior of Na/K pump currents when exposed to an oscillating electric field is studied by computer simulation. The pump
current from a single pump molecule was sketched based on previous experimental results. The oscillating electric field is
designed as a symmetric, dichotomous waveform varying the membrane potential from −30 to −150 mV around the membrane resting
potential of −90 mV. Based on experimental results from skeletal muscle fibers, the energy needed to overcome the electrochemical
potentials for the Na and K-transports are calculated in response to the field’s two half-cycles. We found that a specially
designed oscillating electric field can eventually synchronize the pump molecules so that all the individual pumps run at
the same pumping rate and phase as the field oscillation. They extrude Na ions during the positive half-cycle and pump in
K ions during the negative half-cycle. The field can force the two ion-transports into the corresponding half-cycles, respectively,
but cannot determine their detailed positions. In other words, the oscillating electric field can synchronize pumps in terms
of their pumping loops but not at a specific step in the loop. These results are consistent with our experimental results
in measurement of the pump currents. 相似文献
15.
Isolation and characterization of (Na,K)-ATPase proteolipid 总被引:5,自引:0,他引:5
A S Reeves J H Collins A Schwartz 《Biochemical and biophysical research communications》1980,95(4):1591-1598
16.
Palytoxin binds to Na/K pumps to generate nonselective cation channels whose pore likely comprises at least part of the pump's ion translocation pathway. We systematically analyzed palytoxin's interactions with native human Na/K pumps in outside-out patches from HEK293 cells over a broad range of ionic and nucleotide conditions, and with or without cardiotonic steroids. With 5 mM internal (pipette) [MgATP], palytoxin activated the conductance with an apparent affinity that was highest for Na(+)-containing (K(+)-free) external and internal solutions, lowest for K(+)-containing (Na(+)-free) external and internal solutions, and intermediate for the mixed external Na(+)/internal K(+), and external K(+)/internal Na(+) conditions; with Na(+) solutions and MgATP, the mean dwell time of palytoxin on the Na/K pump was about one day. With Na(+) solutions, the apparent affinity for palytoxin action was low after equilibration of patches with nucleotide-free pipette solution. That apparent affinity was increased in two phases as the equilibrating [MgATP] was raised over the submicromolar, and submillimolar, ranges, but was increased by pipette MgAMPPNP in a single phase, over the submillimolar range; the apparent affinity at saturating [MgAMPPNP] remained approximately 30-fold lower than at saturating [MgATP]. After palytoxin washout, the conductance decay that reflects palytoxin unbinding was accelerated by cardiotonic steroid. When Na/K pumps were preincubated with cardiotonic steroid, subsequent activation of palytoxin-induced conductance was greatly slowed, even after washout of the cardiotonic steroid, but activation could still be accelerated by increasing palytoxin concentration. These results indicate that palytoxin and a cardiotonic steroid can simultaneously occupy the same Na/K pump, each destabilizing the other. The palytoxin-induced channels were permeable to several large organic cations, including N-methyl-d-glucamine(+), suggesting that the narrowest section of the pore must be approximately 7.5 A wide. Enhanced understanding of palytoxin action now allows its use for examining the structures and mechanisms of the gates that occlude/deocclude transported ions during the normal Na/K pump cycle. 相似文献
17.
The Na+-dependent respiratory chain NADH: quinone oxidoreductase of the marine bacterium, Vibrio alginolyticus, was extracted from membrane by a detergent, Liponox DCH, and was purified by chromatography on QAE-Sephadex and Bio-Gel HTP. The activity of NADH oxidation was separated into two fractions. The one fraction could react with several artificial electron acceptors including Q-1, but could not reduce ubiquinone and menaquinone such as Q-5 and menaquinone-4, which was called NADH dehydrogenase. The other fraction could reduce Q-5 and menaquinone-4 in addition to the NADH dehydrogenase activity, which was called quinone reductase. The purified NADH dehydrogenase consumed NADH in excess of the amount of Q-1 and the reduced Q-1 (quinol) was not produced at all due to an oxidation-reduction cycle of semiquinone radicals. The quinone reductase, however, consumed NADH with the quantitative formation of quinol on account of a dismutation reaction of semiquinone radicals. Identical to the membrane-bound NADH: quinone oxidoreductase, the quinone reductase specifically required Na+ for the activity and was inhibited by 2-heptyl-4-hydroxyquinoline N-oxide. The electron transfer in the quinone reductase was formulated in a form of quinone cycle and the dismutation reaction of semiquinone radicals was assigned to be coupled to the Na+ pump in the respiratory chain of this organism. 相似文献
18.
Synchronization of the Na/K pump molecules in a cell membrane was studied in frog skeletal muscle fibers using double Vaseline-gap
voltage-clamp techniques. We found that the pumping rate of naturally random-paced pump molecules can be artificially synchronized
by a pulsed, symmetric, oscillating membrane potential with a frequency comparable to the physiological turnover rate. The
synchronized pump currents show separated outward and inward components, where the magnitude of the outward component is about
three times the randomly-paced pump currents, and the magnitude-ratio of the outward to inward pump currents is close to 3:2,
which reflects the stoichiometric ratio of the pump molecules. Once synchronized, the pumping rate is restricted to the field
frequency, and the pump currents are mainly dependent on the field frequency, but not the field strength. In contrast to previous
work, which by restraining the pumps at a presteady state succeeded in triggering the steps of the pump cycle only individually
and between interruptions, here we synchronize the pumps running continuously and in a normal running mode. 相似文献
19.
We experimentally studied the Na/K pump currents evoked by a train of squared pulses whose pulse-duration is about the time course of Na-extrusion at physiological conditions. The magnitude of the measured pump current can be as much as three-fold of that induced by the traditional single pulse measurement. The increase in the pump current is directly dependent on the number of pre-pulses. The larger the number of the pre-pulses is, the higher the current magnitude can be obtained. At a particular number of pre-pulses, the pump current becomes saturated. These results suggest that a large number of pre-pulses may synchronize the pump molecules to work at the same pace. As a result, the pump molecules may extrude Na ions at the same time corresponding to the stimulation pulses, and pump in K ions at the same time during the pulse intervals. Therefore, the measured pump current is three-fold of that measured by a single pulse where the outward and inward pump currents are canceled each other. 相似文献
20.
Summary Knowledge of the voltage dependencies of apical and basolateral conductances is important in determining the factors that regulate transcellular transport. To gain this knowledge it is necessary to distinguish between cellular and paracellular currents and conductances. This is generally done by sequentially measuring transepithelial current/voltage (I
t
/V
t
) and conductance/voltage (g
t
/V
t
) relationships before and after the abolition of cellular sodium transport with amiloride. Often, however, there are variable time-dependent and voltage-dependent responses to voltage perturbation both in the absence and presence of amiloride, pointing to effects on the paracellular pathway. We have here investigated these phenomena systematically and found that the difficulties were significantly lessened by the use of an intermittent technique, measuringI
t
andg
t
before and after brief (<10 sec) exposure to amiloride at each setting ofV
t
.I/V relationships were characterized by these means in frog skins (Rana pipiens, Northern variety, andRana temporaria). Cellular current,I
c
, decreased with hyperpolarization (larger serosa positive clamps) ofV
t
. DerivedI
c
/V
t
relationships betweenV
t
=0 and 175 mV (serosa positive) were slightly concave upwards. Because values of cell conductance,g
c
, remained finite, it was possible to demonstrate reversal ofI
c
. Values of the reversal potentialV' averaged 156±14 (sd,n=18) mV. Simultaneous microelectrode measurements permitted also the calculation of apical and basolateral conductances,g
a
andg
b
. The apical conductance decreased monotonically with increasing positivity ofV
t
(andV
a
). In contrast, in the range in which the basolateral conductance could be evaluated adequately (V
t
<125 mV),g
b
increased with more positive values ofV
t
(andV
b
). That is, there was an inverse relation betweeng
b and cellular current at the quasi-steady state, 10–30 sec after the transepithelial voltage step. 相似文献