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1.
Summary Previous experiments indicate that the apical membrane of the frog retinal pigment epithelium contains electrogenic NaK pumps. In the pressent experiments net potassium and rubidium transport across the epithelium was measured as a function of extracellular potassium (rubidium) concentration, [K]
o
([Rb]
o
). The net rate of retina-to-choroid42K(86Rb) transport increased monotonically as [K]
o
([Rb]
o
), increased from approximately 0.2 to 5mm on both sides of the tissue or on the apical (neural retinal) side of the tissue. No further increase was observed when [K]
o
([Rb]
o
) was elevated to 10mm. Net sodium transport was also stimulated by elevating [K]
o
. The net K transport was completely inhibited by 10–4
m ouabain in the solution bathing the apical membrane. Ouabain inhibited the unidirectional K flux in the direction of net flux but had not effect on the back-flux in the choroid-to-retina direction. The magnitude of the ouabain-inhibitable42K(86Rb) flux increased with [K]
o
([Rb]
o
). These results show that the apical membrane NaK pumps play an important role in the net active transport of potassium (rubidium) across the epithelium. The [K]
o
changes that modulate potassium transport coincide with the light-induced [K]
o
changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium. 相似文献
2.
Hiroshi Kitasato 《The Journal of general physiology》1973,62(5):535-549
Membrane current responses to sudden potential changes were recorded in solutions of various [K]o on 52 internodal cells of Nitella clavata. The membrane current after sudden depolarization had a component sensitive to [K]o which increased with time from 0.3 to 2.0 s and remained steady thereafter. This late current became zero at values of E and [K]o which suggests that the current was nearly all carried by K+. The potassium conductivity represented by this current increased with depolarization, with a half-maximum value at about -70 mV, and saturation at about -30 to -20 mV. The potassium conductance also increased with increasing [K]o, but less rapidly than predicted for constant potassium permeability. This failure of the conductance to increase with [K]o was relatively the same at all membrane potentials and may be explained by a model with a finite number of channels. No attempt was made to model the dependence of gK on time after depolarization or on membrane potential. However, the finding that the membrane potential did not affect the way in which the permeability depended on [K]o suggests that the membrane potential change does not affect the affinity of the sites, and that the increase in gK with time after depolarization is brought about by an increase in the number of channels with such sites. 相似文献
3.
Electropotential differences between the cytoplasm and external medium have been compared in the mature R. pipiens occyte and the ovulated unfertilized egg as a function of [Na]o, [K]o, [Ca]o and [Cl]o. In solutions containing 1.0 mM Ca++ the oocyte behaved as though it were predominantly permeable to K+ and Cl?, i.e., like a KCl electrode. However, the steady potential decreased with decreasing [Ca]o and in 5 × 10?4 mM [Ca]o the oocyte membrane behaved like a NaCl electrode. Studies on the steady potential as a function of [Na]o, [K]o and [Cl]o in 1.0 mM Ca++ or Ca-free solutions suggest that Ca++ controls the passive permeability of the oocyte membrane to Na+ and Cl?. In the ovulated unfertilized egg the K+ selectivity of the cell membrane disappeared and the system behaved like a NaCl electrode. No effect of external Ca++ or K+ concentration changes on the steady potential was observed. These results indicate that the ion permeability properties of the ovulated egg are similar to that of the ovarian oocyte in Ca-deficient medium, and suggests that the mechanism of ovulation may involve the removal of Ca++ regulation of ion permeability of the egg cell membrane. 相似文献
4.
A model is presented in which the potassium conductance (G
K
) changes responsible for the afterhyperpolarization (AHP) in motoneurones are postulated to follow a kinetics coherent with Hodgkin-Huxley membrane model. Such G
K
kinetics, which operates as a bistable system switched from one state to the other by the action potential, can be expressed by simple analytical expressions if the spike is approximated to a rectangular pulse. Accumulation of G
K
by repetitive activation of the model accurately describes the different features of AHP summation in motoneurones; moreover, this accumulation process enables a model for repetitive firing of motoneurones to display secondary range firing at steady state. 相似文献
5.
Summary Unitary K+ currents in single cells isolated from ventricular muscle of newborn rat hearts were measured in response to different potentials and [K]
o
. TheI/V curves were linear for potentials more negative than the zero-current voltage: especially in high [K]
o
(150nm KCl), no clear outward currents could be detected indicating a drastic rectification in the inward direction. The channel is mainly selective to K+ but Na+ ions are also carried (P
Na/P
K=0.056). The channel conductance is proportional to the square root of [K]
o
but Na+ ions seem to have a facilitatory effect on K, the single-channel conductance. The channel activity, measured asP
o, i.e. the probability to find the channel in open state, decreased as the membrane was hyperpolarized. This behavior was tentatively explained by an inactivation process as the membrane becomes more negative. The rate constants of the transitions between the different states were calculated according to a C–O–C model. A control of the gating process by permeant ion K+ was postulated, based on the increase of one of the rate constants from the closed to the open state with [K]
o
. Finally, the macroscopicI/V curves calculated fromP
o and i, the unit current, were found to be characteristic of a ion-blocked inward rectifier. 相似文献
6.
Net uptake of potassium by low K, high Na cells of Neurospora at pH 5.8 is accompanied by net extrusion of sodium and hydrogen ions. The amount of potassium taken up by the cells is matched by the sum of sodium and hydrogen ions lost, under a variety of conditions: prolonged preincubation, partial respiratory inhibition (DNP), and lowered [K]o. All three fluxes are exponential with time and obey Michaelis kinetics as functions of [K]o. The V
max for net potassium uptake, 22.7 mmoles/kg cell water/min, is very close to that for K/K exchange reported previously (20 mmoles/kg cell water/min). However, the apparent Km for net potassium uptake, 11.8 mM [K]o, is an order of magnitude larger than the value (1 mM) for K/K exchange. It is suggested that a single transport system handles both net K uptake and K/K exchange, but that the affinity of the external site for potassium is influenced by the species of ion being extruded. 相似文献
7.
Corinne I. Rodgers John D. LaBrie R. Meldrum Robertson 《Journal of insect physiology》2009,55(7):599-3216
Stress-induced arrest of ventilatory motor pattern generation is tightly correlated with an abrupt increase in extracellular potassium concentration ([K+]o) within the metathoracic neuropil of the locust, Locusta migratoria. Na+/K+-ATPase inhibition with ouabain elicits repetitive surges of [K+]o that coincide with arrest and recovery of motor activity. Here we show that ouabain induces repetitive [K+]o events in a concentration-dependent manner. 10−5 M, 10−4 M, and 10−3 M ouabain was bath-applied in semi-intact locust preparations. 10−4 M and 10−3 M ouabain reliably induced repetitive [K+]o events whereas 10−5 M ouabain had no significant effect. In comparison to 10−4 M ouabain, 10−3 M ouabain increased the number and hastened the time to onset of repetitive [K+]o waves, prolonged [K+]o event duration, increased resting [K+]o, and diminished the absolute value of [K+]o waves. Recovery of motor patterning following [K+]o events was less likely in 10−3 M ouabain. In addition, we show that K+ channel inhibition using TEA suppressed the onset and decreased the amplitude of ouabain-induced repetitive [K+]o waves. Our results demonstrate that ventilatory circuit function in the locust CNS is dependent on the balance between mechanisms of [K+] accumulation and [K+] clearance. We suggest that with an imbalance in favour of accumulation the system tends towards a bistable state with transitions mediated by positive feedback involving voltage-dependent K+ channels. 相似文献
8.
Summary A divalent anionic dye, bis-[3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-pentamethine oxonol (WW 781) is a rapidly responding fluorescent indicator of KCl diffusion potentials induced in human red blood cells with valinomycin, gramicidin, and with the Ca ionophore A 23187 in the presence of external Ca. WW 781 has a sensitivity of 0.13% F/mV, a detection limit of 10 mV, a response time of less than 1 sec, and exhibits a decrease in fluorescence intensity upon hyperpolarization without detectable shifts in absorption or emission peaks. This dye does not perturb the normal resting potential, and unlike the slow permeant cyanine dyes, does not inhibit Ca-induced K conductance in human red blood cells. However, WW 781 does stimulate Ca-induced unidirectional Rb efflux. With Ca plus A 23187, the initial rapid change in dye fluorescence is sensitive to [Ca]
o
and to [A 23187], is reversible with excess EGTA, and is inhibited by quinine, oligomycin, and by trifluoperazine. A biphasic dependence of hyperpolarization on K
o
is evident at pH 6, where the ionic selectivity of activation is K, Rb>Cs>Na and that of conductance is K, Rb>Cs. Conditions were defined which permitted continuous monitoring ofE
m for at least 10 min, and the time dependence of the Ca-induced potentials was characterized. Since the properties of the Ca-induced changes in dye fluorescence correlate well with the known characteristics of Ca-induced K permeability, we conclude that WW 781 is a useful indicator of changes inE
m, provided that sufficient controls are employed to separate direct effects of Ca on dye fluorescence from the effects ofE
m on fluorescence. 相似文献
9.
Y. Péréon J. P. Louboutin J. Noireaud 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(3):203-211
Some contractile properties of small bundles (100–200 m diameter) of muscle fibres isolated from the extensor digitorum longus muscle of rats at different times of development were compared. An increase of resting potential was observed in these muscles from-26.9 mV at 1 day of age to-72.6 mV at 3 months. Twitch tension and duration of postnatal muscles 1–7 days were diminished by reducing [Ca]o (substituted by Mg2+) or adding inorganic cations (Ni2+, Cd2+, La3+), unlike in the oldest animals (14 days–3 months postnatal) where twitch responses were unaffected. In the latter, potentiation of the twitch tension was even recorded in the presence of Ni2+ (0.5–1 mmol·l-1) and Cd2+ (0.5–2 mmol·l-1). Properties of activation and inactivation of the developed tension following elevation of [K]o to 15–200 mmol·l-1 were analysed at the same stages of postnatal development. In contrast to the tension-membrane potential curves for activation, which presented an average negative shift of-17.6 mV between 1 day postnatal and 3 months of age, a voltage dependence of inactivation similar to that encountered in adult extensor digitorum longus muscles, was already reached at 7 days of age. These results suggest an asynchronism in the maturation of the potential-dependent characteristics of the depolarization-contraction coupling mechanism. Furthermore, during the first week postnatal, in relation with poorly developed membrane systems and low [Ca]i-recycling capability, [Ca]o plays a fundamental role in maintaining contraction by replenishing the intracellular calcium pool.Abbreviations ATPase
adenosine triphosphatase
- [Ca]o ([K]o)
extracellular calcium (potassium) concentration
- DC
depolarization-contraction
- EC
excitation-contraction
- e.d.l. muscle
extensor digitorum longus muscle
-
E
m
membrane potential
-
E
r
resting potential
- HEPES
N-2 hydroxyethylpiperazine-N-2 ethanesulphonic acid
-
I
fast
fast calcium current
- sr
sarcoplasmic reticulum
- T-tubules
transverse tubules 相似文献
10.
Jacqueline Bergman Mina Zaafrani Claude Bergman 《The Journal of membrane biology》1989,111(3):241-251
Summary The electrical responses induced by external applications of neutral amino acids were used to determine whether different carriers are expressed in the membrane of embryonic epithelial cells ofXenopus laevis. Competition experiments were performed under voltage-clamp conditions at constant membrane potential.Gly,l-Ala,l-Pro,l-Ser,l-Asn andl-Gln generate electrical responses with similar apparent kinetic constants and compete for the same carrier. They are [Na]
o
and voltage-dependent, insensitive to variations in [Cl]
o
and [HCO3]
o
, inhibited by pH
o
changes, by amiloride and, for a large fraction of the current, by MeAIB. The increase in [K]
o
at constant and negative membrane potential reduces the response, whereas lowering [K]
o
augments it.
l-Leu,l-Phe andl-Pro appear to compete for another carrier. They generate electrogenic responses insensitive to amiloride and MeAIB, as well as to alterations of membrane potential, [Na]
o
and [K]
o
. Lowering [Cl]
o
decreases their size, whereas increasing [HCO3]
o
at neutral pH
o
increases it.It is concluded that at least two and possibly three transport systems (A, ASC and L) are expressed in the membrane of the embryonic cells studied. An unexpected electrogenic character of the L system is revealed by the present study and seems to be indirectly linked to the transport function.
l-Pro seems to be transported by system A or ASC in the presence of Na and by system L in the absence of Na. MeAIB induces an inward current. 相似文献
11.
Summary The reaction of abdominal skins of the frog speciesRana temporaria on mucosal K+-containing solutions was studied in an Ussing-type chamber by recording transepithelial potential difference (PD), short-circuit current (SCC) and conductance (G). With Na-Ringer's as serosal medium, a linear correlation between PD and the logarithm of the mucosal K+-concentration ([K]
o
) was obtained. The K+-dependent SCC saturated with increasing [K]
o
, and could quickly and reversibly be depressed by addition of Rb+, Cs+, and H+, Li+, Na+, and NH
4
+
did not influence K+ current. A large scatter was obtained for kinetic parameters like the slope of the PD-log [K]
o
-line (18–36.5 mV/decade), the apparent Michaelis constant (13–200mm), and the maximal current of the saturable SCC (6–50 A·cm–2), as well as for the degree of inhibition by Cs+ ions. This seemed to be caused by a time-dependent change during long time exposure to high [K]
o
(more than 30 sec), thereby inducing a selectivity loss of K+-transporting structures, together with an increase in SCC andG and a decrease in PD. Short time exposure to K+-containing solutions showed a competitive inhibition of K+ current by Cs+ ions, and a Michaelis constant of 6.6mm for the inhibitory action of Cs+. Proton titration resulted in a decrease of K+ current at pH<3. An acidic membrane component (apparent dissociation constant 2.5×10–3
m) is virtually controlling K+ transfer. Reducing the transepithelial K+-concentration gradient by raising the serosal potassium concentration was accompanied by the disappearance of SCC and PD. 相似文献
12.
Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the functional classes of Kv channels,
the Kv1 channels are the largest and the best studies and are known to play essential roles in excitable cell function, providing
an essential counterpoin to the various inward currents that trigger excitability. The serum potassium concentration [K
o
+
] is tightly regulated in mammals and disturbances can cause significant functional alterations in the electrical behavior
of excitable tissues in the nervous system and the heart. At least some of these changes may be mediated by Kv channels that
are regulated by changes in the extracellular K+ concentration. As well as changes in serum [K
o
+
], tissue acification is a frequent pathological condition known to inhibit Shaker and Kv1 voltage-gated potassium channels. In recent studies, it has become recognized that the acidification-induced inhibition
of some Kv1 channels is K
o
+
-dependent, and the suggestion has been made that pH and K
o
+
may regulate the channels via a common mechanism. Here we discuss P/C type inactivation as the common pathway by which some
Kv channels become unavailable at acid pH and lowered K
o
+
. It is suggested that binding of protons to a regulatory site in the outer pore mouth of some Kv channels favors transitions
to the inactivated state, whereas K+ ions exert countereffects. We suggest that modulation of the number of excitable voltage-gated K+ channels in the open vs inactivated states of the channels by physiological H+ and K+ concentrations represents an important pathway to control Kv channel function in health and disease. 相似文献
13.
At low extracellular pH (4–6), net uptake of potassium by Neurospora is a simple exponential process which obeys Michaelis kinetics as a function of [K]o. At high pH, however, potassium uptake becomes considerably more complex, and can be resolved into two distinct exponential components. The fast component (time constant = 1.2 min) is matched quantitatively by a rapid loss of sodium; it is attributed to ion exchange within the cell wall, since it is comparatively insensitive to low temperature and metabolic inhibitors. By contrast, the slower component (time constant = 10.9 min) is inhibited markedly at 0°C and by CN and deoxycorticosterone, and is thought to represent carrier-mediated transport of potassium across the cell membrane. This transport process exhibits sigmoid kinetics as a function of [K]o; the data can be fitted satisfactorily by two different two-site models (one involving a carrier site and a modifier site, the other an allosteric model). Either of these models could also accommodate the simple Michaelis kinetics at low pH. 相似文献
14.
P. K. Lauf 《Molecular and cellular biochemistry》1988,82(1-2):97-106
Summary The stimulatory effects of two thiol (SH) group oxidants, methylmethane thiosulfonate (MMTS) and diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide), on the kinetics of ouabain-resistant (OR) K:Cl [co]-transport in low K (LK) sheep red blood cells were compared with the effects of alkylating agents, notably N-ethylmaleimide (NEM). At low concentrations, both MMTS and diamide stimulated K:CI [co]-transportv and with a latency period, as measured by OR zero-trans K efflux and OR uptake of external Rb, Rbo, as K congener in Cl and NO3 media. At high concentrations the effect of diamide saturated, and that of MMTS disappeared. The stimulatory effect of MMTS was partially reversed by the reducing agent dithiothreitol (DTT) known to fully restore the diamide-activated K flux (Lauf, J. Memb. Biol. 101:179–188, 1988). In diamide pre-equilibrated LK sheep red cells, the Km of K:Cl [co]-transport for external Cl, Clo, was 84.3 mM, and 18.7 mM for Rbo, with nearly identical Vmax values around 4 mmol Rb/L cells × h for K (Rb) fluxes in Cl and after correction for the small Cl-independent component. Zero net K (Rb) flux existed at Kc (cell K)/Rbo concentration ratios, [K]c/[Rb]c, of 0.8 i.e. when the electrochemical driving forces across the membrane were about equal. The measured K efflux/Rb influx ratios were almost twice those predicted from [K]c/[Rb]o and the Cl equilibrium potential suggesting that the diamide-stimulated K (Rb) flux may occur through non-diffusional, carrier-mediated transport. The effects of NEM and of A23187 plus/minus Ca or chelators on K: [co]Cl-transport (Lauf, Am. J. Physiol. 249:C271–278, 1985) consisted primarily of Vmax changes. Thus, all chemical interventions resulted in an increase of the number of actively transporting K:Cl [co]-transport units or an augmented turnover number per existing site. 相似文献
15.
The mechanisms underlying the diverse responses to step current stimuli of models [Edman et al. (1987) J Physiol (Lond) 384:
649–669] of lobster slowly adapting stretch receptor organs (SAO) and fast-adapting stretch receptor organs (FAO) are analyzed.
In response to a step current, the models display three distinct types of firing reflecting the level of adaptation to the
stimulation. Low-amplitude currents evoke transient firing containing one to several action potentials before the system stabilizes
to a resting state. Conversely, high-amplitude stimulations induce a high frequency transient burst that can last several
seconds before the model returns to its quiescent state. In the SAO model, the transition between the two regimes is characterized
by a sustained pacemaker firing at an intermediate stimulation amplitude. The FAO model does not exhibit such a maintained
firing; rather, the duration of the transient firing increases at first with the stimulus intensity, goes through a maximum
and then decreases at larger intensities. Both models comprise seven variables representing the membrane potential, the sodium
fast activation, fast inactivation, slow inactivation, the potassium fast activation, slow inactivation gating variables,
and the intra cellular sodium concentration. To elucidate the mechanisms of the firing adaptations, the seven-variable model
for the lobster stretch receptor neuron is first reduced to a three-dimensional system by regrouping variables with similar
time scales. More precisely, we substituted the membrane potential V for the sodium fast activation equivalent potential V
m
, the potassium fast inactivation V
n
for the sodium fast inactivation V
h
, and the sodium slow inactivation V
l
for the potassium slow inactivation V
r
. Comparison of the responses of the reduced models to those of the original models revealed that the main behaviors of the
system were preserved in the reduction process. We classified the different types of responses of the reduced SAO and FAO
models to constant current stimulation. We analyzed the transient and stationary responses of the reduced models by constructing
bifurcation diagrams representing the qualitatively distinct dynamics of the models and the transitions between them. These
revealed that (1) the transient firings prior to reaching the stationary state can be accounted for by the sodium slow inactivation
evolving more slowly than the other two variables, so that the changes during the transient firings reflect the bifurcations
that the two-dimensional system undergoes when the sodium slow inactivation, considered as a parameter, is varied; and (2)
the stationary behaviors of the models are captured by the standard bifurcations of a two-dimensional system formed by the
membrane potential and the potassium fast inactivation. We found that each type of firing and the transitions between them
is due to the interplay between essentially three variables: two fast ones accounting for the action potential generation
and the post-discharge refractoriness, and a third slow one representing the adaptation.
Received: 28 February 2000 / Accepted in revised form: 4 October 2000 相似文献
16.
Travis J. Mitchell Camila Zugarramurdi J. Fernando Olivera Craig Gatto Pablo Artigas 《Biophysical journal》2014
The Na/K pump hydrolyzes ATP to export three intracellular Na (Nai) as it imports two extracellular K (Ko) across animal plasma membranes. Within the protein, two ion-binding sites (sites I and II) can reciprocally bind Na or K, but a third site (site III) exclusively binds Na in a voltage-dependent fashion. In the absence of Nao and Ko, the pump passively imports protons, generating an inward current (IH). To elucidate the mechanisms of IH, we used voltage-clamp techniques to investigate the [H]o, [Na]o, and voltage dependence of IH in Na/K pumps from ventricular myocytes and in ouabain-resistant pumps expressed in Xenopus oocytes. Lowering pHo revealed that Ho both activates IH (in a voltage-dependent manner) and inhibits it (in a voltage-independent manner) by binding to different sites. Nao effects depend on pHo; at pHo where no Ho inhibition is observed, Nao inhibits IH at all concentrations, but when applied at pHo that inhibits pump-mediated current, low [Na]o activates IH and high [Na]o inhibits it. Our results demonstrate that IH is a property inherent to Na/K pumps, not linked to the oocyte expression environment, explains differences in the characteristics of IH previously reported in the literature, and supports a model in which 1), protons leak through site III; 2), binding of two Na or two protons to sites I and II inhibits proton transport; and 3), pumps with mixed Na/proton occupancy of sites I and II remain permeable to protons. 相似文献
17.
Transient potassium currents distinctively affect firing properties, particularly in regulating the latency before repetitive
firing. Pyramidal cells of the dorsal cochlear nucleus (DCN) have two transient potassium currents, I
Kif and I
Kis, fast and slowly inactivating, respectively, and they exhibit firing patterns with dramatically variable latencies. They
show immediate repetitive firing, or only after a long latency with or without a leading spike, the so-called pauser and buildup
patterns. We consider a conductance-based, ten-variable, single-compartment model for the DCN pyramidal cells (Kanold and
Manis 2001). We develop and analyze a reduced three-variable integrate-and-fire model (KM-LIF) which captures the qualitative
firing features. We apply dynamical systems methods to explain the underlying biophysical and mathematical mechanisms for
the firing behaviors, including the characteristic firing patterns, the latency phase, the onset of repetitive firing, and
some discontinuities in the timing of latency duration (e.i. first spike latency and first inter spike interval). Moreover,
we obtain new insights associated with the leading spike by phase plane analysis. We further demonstrate the effects of possible
heterogeneity of I
Kis. The latency before repetitive firing can be controlled to cover a large range by tuning of the relative amounts of I
Kif and I
Kis. Finally, we find for the full system robust bistability when enough I
Kis is present. 相似文献
18.
D. D. Wheeler 《Neurochemical research》1984,9(5):649-660
Initial velocity of synaptosomal GABA uptake and steady state distribution ratio (DR) have been measured as a function of medium potassium concentration ([K]o) and in the presence of veratridine. Previous studies of the effect of [K]o and veratridine on membrane potential have been utilized to relate GABA uptake to membrane potential. Both initial velocity of uptake and steady state DR are functions of membrane potential; initial velocity and ln DR are linearly related to membrane potential. Final DR is determined by the electrochemical potential gradient for the transported ions. Previous studies of the relation of [Na]o to uptake have shown that either one or two sodium ions may be translocated with each GABA molecule. Present results are consistent with this assumption if it is also assumed that an anion is co-transported with each GABA molecule. 相似文献
19.
S. J. D. Karlish 《Journal of bioenergetics and biomembranes》1980,12(3-4):111-136
Conformational changes have been studied in (Na,K) ATPase labeled at or near the ATP binding region with fluorescein following incubation with fluorescein isothiocyanate (FITC). One or two fluorescein groups are bound per ATPase molecule. (Na,K) ATPase activity, phosphorylation from ATP, and nucleotide binding are abolished in labeled enzyme, but phosphorylation from inorganic phosphate or K-phosphatase activity are only partially inactivated. The fluorescein groups are incorporated only into the 96 KD catalytic chain of the (Na,K) ATPase, and presence of ATP during the incubation with FITC protects against the incorporation and inhibition of enzymic activity. Upon trypsin treatment of labeled membranes the fluorescein appears first in a 58 KD fragment and eventually is released into the medium. The fluorescein-labeled (Na,K) ATPase shows a large quenching of fluorescence (15–20%) on conversion of the E1 or E1 · Na conformation in cation-free or Na+-rich media to the E2 · (K) form in K+ (or congeners Tl+, Rb+, Cs+, NH
4
+
) rich media. Cation titrations suggest that K+ and Na+ ions compete at a single binding site and stabilize E1 · Na or E2 · (K) respectively;K
K0.23 mM,K
Na1.2 mM. The rate of the conformational transition E2 · (K) E1 · Na is slow,k=0.3 sec–1, but contrary to previous experience [7, 8] ATP does not stimulate this rate. The rate of the transitions E1 + K+ E2 · (K) rises sharply with K+ concentration and shows saturation behavior, from which ak
max286 sec–1 andK
k74 mM are deduced. The data support and extend the previous suggestion that K+ ions bound initially at a low-affinity (probably cytoplasm oriented) site in state E1 are trapped in the occluded form E2 · (K) by the conformational change poised far (K
c1000) in the direction of E2 · (K). It is proposed in addition that at least two binding sites for K+ exist at the cytoplasmic surface of isolated (Na,K) ATPase in state E1 but a large difference in affinities precludes detection in fluorescence titrations of more than one site. A variety of ligands in addition to K+ produce fluorescence-quenched or E2 forms of the labeled (Na,K) ATPase. These include Mg2+ plus inorganic phosphate, without or with K+ ions (E2P or E2P · K) or with ouabain (E2-ouabain or E2P · ouabain). Na+ ions antagonize these effects. The collected data support the notion that there may be many subspecies of the E1 and E2 forms (either phosphorylated or nonphosphorylated) with different numbers of Na+ and/or K+ ions bound or occluded, each subspecies having a characteristic ability to catalyze reactions and/or transport cations. The relationship between the conformational changes in fluorescein-labeled enzyme and the subunit structure of the (Na,K) ATPase is discussed with particular reference to half of the site models for ATP hydrolysis. 相似文献
20.
The maintenance of cellular ion homeostasis is crucial for optimal neural function and thus it is of great importance to understand its regulation. Glial cells are extensively coupled by gap junctions forming a network that is suggested to serve as a spatial buffer for potassium (K+) ions. We have investigated the role of glial spatial buffering in the regulation of extracellular K+ concentration ([K+]o) within the locust metathoracic ganglion by pharmacologically inhibiting gap junctions. Using K+-sensitive microelectrodes, we measured [K+]o near the ventilatory neuropile while simultaneously recording the ventilatory rhythm as a model of neural circuit function. We found that blockade of gap junctions with either carbenoxolone (CBX), 18β-glycyrrhetinic acid (18β-GA) or meclofenamic acid (MFA) reliably induced repetitive [K+]o surges and caused a progressive impairment in the ability to maintain baseline [K+]o levels throughout the treatment period. We also show that a low dose of CBX that did not induce surging activity increased the vulnerability of locust neural tissue to spreading depression (SD) induced by Na+/K+-ATPase inhibition with ouabain. CBX pre-treatment increased the number of SD events induced by ouabain and hindered the recovery of [K+]o back to baseline levels between events. Our results suggest that glial spatial buffering through gap junctions plays an essential role in the regulation of [K+]o under normal conditions and also contributes to a component of [K+]o clearance following physiologically elevated levels of [K+]o. 相似文献