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1.
45Ca 2+ fluxes across the plasma membrane of zygotes of the fucoid alga, Pelvetia fastagiata (J. Ag.) De Toni, were studied in artificial sea waters of various potassium concentrations. Except for two cases, hyperpolarization of the cell membrane (with low [K +]) increases, and depolarization (with high [K +]) decreases the influx of Ca 2+ over the range of [K +] studied (1–100 mM). The fractional increases of influx during hyperpolarization are close to the fractional increases in membrane potential but the decreases during depolarization are much smaller than those in membrane potential. In two anomalous cases, the influxes of 45Ca 2+ at a potassium concentration of 30 mM were about 20% higher than the control value instead of being 10% lower.The effluxes of 45Ca 2+ are increased by both hyperpolarization and by depolarization. On balance (and excepting the two anomalous cases) the net result of hyperpolarization should be to increase and that of depolarization to decrease intracellular [Ca 2+]. 相似文献
2.
The effect of external calcium and sodium ion concentrations on the calcium fluxes on the Pelvetia fastigiata De Toni egg was measured. Decreasing external [Ca 2+] greatly increased the permeability of the eggs to Ca 2+; at 1 mM external Ca 2+ this permeability was 60 times as great as it was at the normal [Ca 2+] of 10 mM. Lowering the external [Na +] also increased Ca 2+ influx; at 2 mM Na +, the Ca 2+ influx was 2–3 times as great as it was at the normal [Na +] if choline was used as a Na + substitute. Lithium was less effective as a Na + substitute in increasing Ca 2+ influx. The extra Ca 2+ influx in low [Na +] seemed to be dependent on internal [Na +]. The Ca 2+ efflux increased transiently and then declined in low Na + media. 相似文献
3.
Summary Net Cl – uptake as well as unidirectional 36Cl influx during regulatory volume increase (RVI) require external K +. Half-maximal rate of bumetanide-sensitive 36Cl uptake is attained at about 3.3 mm external K +. The bumetanide-sensitive K + influx found during RVI is strongly dependent on both Na + and Cl –. The bumetanide-sensitive unidirectional Na + influx during RVI is dependent on K + as well as on Cl –. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na +, K + and Cl –. In the presence of ouabain and Ba + the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na +, 0.8 K +, 2.0 Cl – or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K + and Cl – flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na +, K +, 2Cl – cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K + influx is activated. During hypotonic conditions a small bumetanide-sensitive K + influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca 2+ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K + influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na +, K +, Cl – cotransport involves both Ca 2+/calmodulin and protein kinase C. 相似文献
4.
Recent studies have provided evidence that depolarization in the absence of extracellular Ca 2+ can trigger Ca 2+ release from internal stores in a variety of neuron subtypes. Here we examine whether postganglionic sympathetic neurons are able to mobilize Ca 2+ from intracellular stores in response to depolarization, independent of Ca 2+ influx. We measured changes in cytosolic ΔF/F 0 in individual fluo-4 –loaded sympathetic ganglion neurons in response to maintained K + depolarization in the presence (2 mM) and absence of extracellular Ca 2+ ([Ca 2+] e). Progressive elevations in extracellular [K +] e caused increasing membrane depolarizations that were of similar magnitude in 0 and 2 mM [Ca 2+] e. Peak amplitude of ΔF/F 0 transients in 2 mM [Ca 2+] e increased in a linear fashion as the membrane become more depolarized. Peak elevations of ΔF/F 0 in 0 mM [Ca 2+] e were ~5–10% of those evoked at the same membrane potential in 2 mM [Ca 2+] e and exhibited an inverse U-shaped dependence on voltage. Both the rise and decay of ΔF/F 0 transients in 0 mM [Ca 2+] e were slower than those of ΔF/F 0 transients evoked in 2 mM [Ca 2+] e. Rises in ΔF/F 0 evoked by high [K +] e in the absence of extracellular Ca 2+ were blocked by thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+ ATPase, or the inositol 1,4,5-triphosphate (IP 3) receptor antagonists 2-aminoethoxydiphenyl borate and xestospongin C, but not by extracellular Cd 2+, the dihydropyridine antagonist nifedipine, or by ryanodine at concentrations that caused depletion of ryanodine-sensitive Ca 2+ stores. These results support the notion that postganglionic sympathetic neurons possess the ability to release Ca 2+ from IP 3-sensitive internal stores in response to membrane depolarization, independent of Ca 2+ influx. 相似文献
5.
Insulin release, net fluxes of Ca 2+, and glucose metabolism were studied in a clonal cell line (RINmSF) established from a transplantable rat islet tumor. The insulin content amounted to only 0.03% of that of the total protein and decreased even further with subsequent passages. The insulin secretion was as high as 10 to 20% of the total hormone content per hour. Insulin release was stimulated by K + depolarization but not by exposure to glucose. In contrast to this secretory pattern, glucose but not K + stimulated the net uptake of Ca 2+ at micromolar concentrations of the ion. The glucose effect was not mimicked by 20 mM 3-O-methylglucose. It was as pronounced at 1 mM as at 20 mM of the sugar and corresponded to an uptake of 119 fmol cm –2 s –1. Glucose metabolism was typical for tumor cells with a high glycolytic flux and an oxidationtoutilization ratio as low as 0.05–0.15. Maximal oxidative degradation was attained already at l mM. This concentration was also equivalent to the K m for glucose utilization, indicating a substantial left-hand shift of the normal dose-response curve. It is suggested that glucose induces a depolarizationindependent net uptake of Ca 2+ by favouring intracellular buffering of the cation. 相似文献
6.
The influx of 45Ca into isolated guard cells of Commelina communis L. has been measured, using short uptake times, and washing in ice-cold La 3+-containing solutions to remove extracellular tracer after the loading period. Over 0.5–4 min the uptake was linear with time, through the origin. Over 20–200M external Ca 2+ the influx measured with 10–20 mM external KCl was in the range 0.3–2.3 pmol·cm -2·s -1 (on the basis of estimated guard-cell area); with only 1 mM KCl externally the 45Ca influx was significantly reduced, in the range 0.3–1.1 pmol·cm -2·s -1 for external Ca 2+ of 50–100 M. The results indicate that the Ca-channel is voltage-sensitive, opening with depolarisation. No consistent effect of the addition of abscisic acid could be found. In different experiments, on the addition of 0.1 mM abscisic acid the Ca 2+ influx was sometimes stimulated by 28–79%, was sometimes unaffected, and was sometimes inhibited by 16–29%. The results rule out a long-lasting stimulation of 45Ca influx by ABA, but they do not rule out a transient stimulation followed by inhibition, perphaps as a consequence of down-regulation of Ca 2+ influx by increasing cytoplasmic Ca 2+. The hypothesis that ABA may act via an action on Ca 2+ influx, increasing cytoplasmic Ca 2+, with consequent effects on voltage-dependent and Ca 2+-dependent ion channels in both plasmalemma and tonoplast, is neither proved nor disproved by these results.Abbreviations ABA
abscisic acid
- Ca o, K o
external Ca and K concentrations 相似文献
7.
Summary Kinetic properties of Na +–Ca 2+ exchange in a renal epithelial cell line (LLC-MK 2) were assessed by measuring cytosolic free Ca 2+ with fura-2 and 45Ca 2+ influx. Replacing external Na + with K + produced relatively small increases in free Ca 2+ and 45Ca 2+ uptake unless the cells were incubated with ouabain. Ouabain markedly increased cell Na + and strongly potentiated the effect of replacing external Na + with K + on free Ca 2+ and 45Ca 2+ uptake. 45Ca 2+ influx in 140 mm K + or N-methyl- d-glucamine minus influx in 140 mm Na + was used to quantify Na +–Ca 2+ exchange activity of Na +-loaded cells. The dependence of exchange on cell Na + was sigmoidal; the K
0.5 was 26±3 mmol/liter cell water space, and the Hill coefficient was 3.1±0.2. The kinetic features of the dependence of exchange on cell Na + partly account for the small increase in Ca 2+ influx when all external Na + is replaced by K +. Besides raising cell Na + ouabain appears to activate the exchanger. Magnesium competitively inhibited exchange activity. The potency of Mg 2+ was 8.2-fold lower with potassium instead of N-methyl- d-glucamine or choline as the replacement for external Na +. Potassium also increased the V
max of exchange by 86% and had no effect on the K
m for Ca 2+. The exchanger does not cause detectable 22Na +–Mg 2+ exchange and does not appear to require K + or transport 86Rb +. Although exchange activity was plentiful in the epithelial cells from monkey kidney, others from amphibian, canine, opossum, and porcine kidney had no detectable exchange activity. All of the measured kinetic properties of Na +–Ca 2+ exchange in the renal epithelial cells are very similar to those of the exchanger in rat aortic myocytes. 相似文献
8.
The effect of veratridine on neurotransmitter release was studied using rat brain synaptosomes superfused at 37°C. Veratridine (5–75 M) caused a concentration-dependent release of [ 3H]GABA from prelabeled synaptosomes in the presence of 2.7 mM Ca 2+. In the whole range of veratridine concentrations, the release of [ 3H]GABA elicited by the drug was substantially increased rather than decreased in the absence of Ca 2+ or with Ca 2+ concentrations of 0.45 and 0.9 mM. The release of the amino acid was inhibited more by 5.4 mM than by 2.7 mM Ca 2+. The effect on endogenous (chemically measured) GABA was similar to that on [ 3H]GABA. The inhibitory effect of Ca 2+ on the veratridine-induced release of [ 3H]GABA was consistently seen in a variety of experimental conditions except one, namely when the experiment was run at room temperature (22–23°C) rather than at physiological temperature (37°C). In fact, at 22–23°C the release of GABA evoked by the alkaloid was somewhat potentiated by Ca 2+. At 37°C, glutamate appeared to behave similarly to GABA, whereas the veratridine-induced release of [ 3H]noradrenaline and [ 3H]dopamaine was largely Ca 2+-dependent. The mechanism of the release of transmitters elicited by veratridine is discussed. It is concluded that the evoked release of GABA and glutamate is due more to the veratridine-induced depolarization (Na + influx) than to the accompanying influx of Ca 2+, and it is suggested that the inhibitory effect of Ca 2+ on the overall release of amino acids is due to the antagonism exerted by the divalent cation on the veratridine action at the Na + channel. In contrast, in the case of catecholamines, the influx of Ca 2+ would have a prominent role in triggering exocytotic release, whereas the depolarization itself would have slight or no importance. 相似文献
9.
Summary The effect of cholecystokinin (CCK) and internal Ca 2+ on outward K + current in isolated pig pancreatic acinar cells has been investigated using the patch-clamp method for whole-cell current recording under voltage-clamp conditions. CCK (2 × 10 –10
M) applied to the bath evoked a marked increase in the outward K + current associated with depolarizing voltage steps, and this effect was fully reversible and acutely dependent on the presence of external Ca 2+. When strongly buffered Ca 2+-EGTA solutions were used inside the cells CCK failed to evoke an effect. Increasing the internal Ca 2+ concentration ([Ca 2+]
i
) from 5 × 10 –10
M to 10 –7 and 5 × 10 –7
M mimicked the effect of CCK. It would appear therefore that CCK controls K + conductance in the acinar cells via changes in the internal free ionized Ca 2+ concentration. 相似文献
10.
Summary K +, Cl –, and Ca 2+ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K +, K + channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K + channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K + channels showed a significant discrimination against Na + and Cl –. The Cl – channel opened at positive vacuolar membrane potentials for cytoplasmic Cl – influx had a high conductance of 110pS in symmetrical 100mM Cl –. When K + and Cl – channels were excluded from opening, no traces were found of Ca 2+ channel activity for vacuolar Ca 2+ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca 2+ channel which allowed influx of cytoplasmic Ca 2+ into the vacuole when the Ca 2+ concentration on the cytoplasmic side was high. When Ca 2+ was substituted by Ba 2+, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba 2+Sr 2+Ca 2+Mg 2+=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca 2+ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd 2+, but was insensitive to La 3+, Gd 3+, Ni 2+, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K +, Cl –, and Ca 2+ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells. 相似文献
11.
Many voltage-gated K + channels exhibit C-type inactivation. This typically slow process has been hypothesized to result from dilation of the outer-most ring of the carbonyls in the selectivity filter, destroying this ring’s ability to bind K + with high affinity. We report here strong enhancement of C-type inactivation upon extracellular addition of 10–40 mM Ca 2+ or 5–50 µM La 3+. These multivalent cations mildly increase the rate of C-type inactivation during depolarization and markedly promote inactivation and/or suppress recovery when membrane voltage (V m) is at resting levels (−80 to −100 mV). At −80 mV with 40 mM Ca 2+ and 0 mM K + externally, ShBΔN channels with the mutation T449A inactivate almost completely within 2 min or less with no pulsing. This behavior is observed only in those mutants that show C-type inactivation on depolarization and is distinct from the effects of Ca 2+ and La 3+ on activation (opening and closing of the V m-controlled gate), i.e., slower activation of K + channels and a positive shift of the mid-voltage of activation. The Ca 2+/La 3+ effects on C-type inactivation are antagonized by extracellular K + in the low millimolar range. This, together with the known ability of Ca 2+ and La 3+ to block inward current through K + channels at negative voltage, strongly suggests that Ca 2+/La 3+ acts at the outer mouth of the selectivity filter. We propose that at −80 mV, Ca 2+ or La 3+ ions compete effectively with K + at the channel’s outer mouth and prevent K + from stabilizing the filter’s outer carbonyl ring. 相似文献
12.
Summary The whole-cell patch-clamp method has been used to measure Ca 2+ influx through otherwise K +-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K + influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K + channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K + inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca 2+ is more negative than or equal to the K + equilibrium potential (–47 mV), indicating that the current is due to Ca 2+ influx through the K + channels prior to their closure. Decreasing internal [Ca 2+] (Ca
i
) from 200 to 2 n m or increasing the external [Ca 2+] (Ca
o
) from 1 to 10 m m increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca 2+ influx also decrease the amplitude of time-activated current, indicating that Ca 2+ permeation is slower than K + permeation, and so causes a partial block. Increasing Ca
o
also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. –160 mV, indicating that the Ca 2– block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K + channels in Zea mays guard cells also appear to have a Ca
i
-, and Ca
o
-dependent ability to mediate Ca 2+ influx. We suggest that the inwardly rectiying K + channels are part of a regulatory mechanism for Ca
i
. Changes in Ca
o
and (associated) changes in Ca
i
regulate a variety of intracellular processes and ion fluxes, including the K + and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments. 相似文献
13.
Influx of 45Ca into internodal cells of Chara corallina has been measured, using short uptake times, and a wash in ice-cold La 3+-containing pondwater after the labelling period to overcome the difficulty of distinguishing extracellular tracer from that in the cell. Over 5–15 min the uptake was linear with time, through the origin. The basal influx from 0.1 mM Ca 2+ externally was 0.25–0.5 pmol·cm -2·s -1, but some batches of cells showed higher fluxes. The influx was markedly stimulated by depolarisation in pondwater containing 20 mM K +. In cells in which the control flux was less than about 0.5 pmol·cm -2·s -1 there was no effect of 50 M nifedipine. In cells in which the control flux was greater than about 0.5 pmol·cm -2·s -1 (whether by natural variability, pretreatment, or by depolarisation in 20 mM K +), the flux was reduced by 50 M nifedipine to a value in the range 0.25–0.59 pmol·cm -2·s -1. It is suggested that two types of Ca-channel are probably involved, both opening on depolarisation, but only one sensitive to nifedipine. The flux was inhibited by 10 M BAY K 8644, which in animal cells more commonly opens Ca-channels. The apparent influx measured over long uptake times was much reduced, and the kinetics indicated filling a pool of apparent size about 1.45 nmol·cm -2 with a halftime of about 38 min, probably representing cytoplasmic stores. It is argued that in spite of the very small pool of (free+bound) cytoplasmic Ca 2+ the measured influx is a reasonable estimate of the influx at the plasmalemma.Abbreviations 0.4K-APW6
artificial pondwater, pH 6, containing 0.4 mM KCl
- 20 K-APW6
artificial pondwater, pH 6, containing 20 mM KCl
- Ca o
external Ca 2+ 相似文献
14.
Summary A method was developed for the study of divalent cation transport events on the time scale of 20 msec or longer. Passive Ca 2+ equilibration across the membranes of the Ca 2+-ATPase rich fraction of sarcoplasmic reticulum (SR) was studied. The method makes use of the divalent cation sensitivity of the surface binding of the fluorescent probe 1-anilino-8-naphthalenesulfonate (ANS –). Binding to the inside and outside surfaces is distinguished in fluorescent stopped-flow experiments. The surface binding reactions of the probe are faster than the time resolution of the instrument (ca. 3 msec), while binding reactions requiring transport across the membrane could be resolved. In Ca 2+ influx experiments, the time course of fluorescent enhancement was monitored following a Ca 2+ jump. The kinetics of Ca 2+ efflux were studied by pre-equilibrating Ca 2+ across the membrane, removing the external Ca 2+ with an EGTA jump, and observing the time course of the fluorescence decrease. Rapid transport of ANS – (coupled to K +) was ensured by the addition of valinomycin. Two processes of Ca 2+ influx were observed: (i) a rapid process with small fluorescent amplitudes and a t
1/2 of 40–60 msec and (ii) a slow process with a large amplitude and a t
1/2 of 70–100 sec. The rates and extents of the two phases were quantitated in terms of the rates and extents of change in the Ca 2+ concentration in the SR lumen. The slow phase accounted for a larger change, in the internal free Ca 2+ concentration than did the first phase. For the influx of 10 mM Ca 2+, the rapid phase raises the internal Ca 2+ concentration to ca. 1 mM within its apparent t
1/2 of 20 msec. The slow phase brings about an increase of the internal Ca 2+ concentration to 4 mM within its apparent t
1/2 of 90 sec. The two phases have average rates of increase of internal free Ca 2+ concentration, [Ca]
i
/sec of ca. 50 mM/sec and ca. 0.02 mM/sec, respectively. The Ca 2+ influx rates increased with increasing KCl concentration and with increasing external Ca 2+ concentration.Two phases of Ca 2+ efflux were observed. The amplitudes and rates were analyzed and the fast phase was shown to account for more Ca 2+ movement than the slow phase. The rate of the fast phase was greatly increased by increasing the K + concentration. The rate of the slow phase efflux decreased with increasing Ca 2+ concentration in the external medium. The concentration for half-maximal inhibition was 4 M, a value close to the dissociation constant of the high affinity site on the Ca 2+-ATPase.The above constitutes a body of circumstantial evidence that the passive Ca 2+ permeability observed is mediated by the Ca 2+-ATPase, acting as a Ca 2+ for 2 K + exchanger. The fast phases are explained as a partial turnover of the pump in the steady state. The slow rate is explained by a preference of the ion binding translocator site of the carrier for an outward orientation.The ANS – technique was applied to the monovalent cation permeability of the Ca 2+-ATPase rich SR and the results of other studies were corroborated and extended. The interaction of valinomycin with intrinsic permeability mechanisms of the SR was considered. 相似文献
15.
In isolated synaptosomes from rat brain, 100 M antimycin A and 10 M oxamic acid inhibit the 32Pi-labeling of phosphatidylinositol-4,5-bisphosphate (PIP 2) and phosphatidylinositol-4-phosphate (PIP) by 90% and 95–99% respectively. 10 mM sodium fluoride inhibits the labeling by 50–60% and 10 mM A23187 inhibits the labeling by 63–70%. Phospholipase A 2 inhibits the labeling of PIP 2 and PIP by 93–94% and stimulates their degradation by 84–92%. Depolarization of synaptosomes with 75 mM K + or 100 M veratrine decreases the labeling of PIP 2 and PIP by 66–74%. The decreased labeling results in large part from the Ca 2+-dependent degradation of 32P-labeled PIP 2 and PIP as shown by pulse-chase experiments in which PIP 2 and PIP were prelabeled with 32Pi. Depolarization of synaptosomes results in the stimulation of 45Ca 2+ uptake with the concomitant hydrolysis of PIP and PIP 2. Addition of 1 mM Ca 2+ accounts for 25% of the enhanced degradation whereas depolarization with 75 mM K + accounts for 75% of the enhanced degradation of PIP 2 and PIP. Depolarization with 100 mM veratrine results in a 223% increase in inositol trisphosphate as evidenced by stimulation of 45Ca 2+ uptake. EGTA (10mM) and Mg 2+ (5–10 mM) inhibit the degradation of PIP and PIP 2 and counteract the action of 1 mM Ca 2+. Our data demonstrate that 45Ca 2+, Mg 2+, and membrane depolarization play an important role in the turnover of membrane phosphatidylinositols.Abbreviations ATP
adenosine triphosphate
- Pi
inorganic orthophosphate
- PIP
phosphatidylinositol-4-phosphate
- PIP 2
phosphatidylinositol-4,5,-bisphosphate
- IP 3
inositol-1,4,5-trisphosphate 相似文献
16.
Washing corn ( Zea mays L.) root tissue in water causes loss of about one-third of the exchangeable Ca 2+ over the first 10 to 15 minutes. Upon transfer to K +-containing solutions, the tissue shows a short period of rapid K + influx which subsequently declines. Addition of 0.1 millimolar Ca 2+ decreases the initial rapid K + influx, but increases the sustained rate of K + and Cl − uptake. It was confirmed (Elzam and Hodges 1967 Plant Physiol 42: 1483-1488) that 0.1 millimolar Ca 2+ is more effective than higher concentrations for the initial inhibition, and that Mg 2+ will substitute. The inhibition arises from a mild shock affect of restoring Ca2+. With 0.1 millimolar Ca2+ net H+ efflux is blocked for 10 to 15 minutes and the cells are depolarized by about 30 millivolts. However, 1 millimolar Ca2+ rapidly produces increased K+ influx and blocks net H+ efflux for only a few minutes; blockage is preceded by a brief net H+ influx which may restore and increase ion transport by reactivating the plasmalemma H+-ATPase. Stimulation of electrogenic H+-pumping with fusicoccin eliminates the shock responses and minimizes Ca2+ effects on K+ influx. Fusicoccin also strongly decreases Ca2+ influx, but has no effect on Ca2+ efflux. Ice temperatures and high pH decreased Ca2+ efflux, but uncoupler and chlorpromazine did not. It is suggested that the inhibitory and promotive actions of Ca2+ are manifested through decreases or increases in the protonmotive force. 相似文献
17.
Summary The Ca 2+-activated K + channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na + outside, K + inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage ( I/V) relationship showed outward rectification and the null potential was more negative than –40 mV. In symmetrical K +-rich solutions the single-channel I/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca 2+-free solution containing 2 mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities ( P) above 0.1. Raising the free Ca 2+ concentration in contact with the membrane inside ([Ca 2+] i) to 1.5×10 –7
m had little effect on the relationship between membrane potential and P. When [Ca 2+] i was increased to 3×10 –7
m and 6×10 –7
m smaller potential changes were required to open the channels. Increasing [Ca 2+] i further to 8×10 –7
m again activated the channels, but the relationship between membrane potential and P was complex. Changing the membrane potential from –50 mV to +20 mV increased P from near 0 to 0.6 but further polarization to +50 mV decreased P to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca 2+] i=1 and 2 m. In this situation a membrane potential change from –70 to +20 mV increased P from near 0 to about 0.7 but further polarization to +80 mV reduced P to less than 0.1. The high-conductance K + channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca 2+] i within the range 0.1 to 1 m which suggests a physiological role for this channel in regulating the membrane potential and Ca 2+ influx through voltage-activated Ca 2+ channels. 相似文献
18.
Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K + and Cl – transport pathways. RVD is accelerated when a parallel K + transport pathway is provided by addition of gramicidin, indicating that the K + conductance is rate limiting. Addition of ionophore A23187 plus Ca 2+ also activates separate K + and Cl – transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K + and Cl – conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl – conductance is rate limiting. An A23187-induced activation of 42K and 36Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca 2+-activated K + channel. (ii) unaffected by substitution of NO
3
–
or SCN – for Cl –, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K + channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl – conductance. The Cl – conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl – transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca 2+-free media (probably by release of Ca 2+ from internal stores) is also transient, whereas the activation is persistent in Ca 2+-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl – transport pathway. These findings suggest that a transient increase in free cytosolic Ca 2+ may account for the transient activation of the Cl – transport pathway. The activated anion transport pathway is unselective, carrying both Cl –, Br –, NO
3
–
, and SCN –. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl – transport pathway and also blocks the activation of the K + transport pathway. This is demonstrated directly by 42K flux experiments and indirectly in media where the dominating anion (SCN –) has a high ground permeability. A comparison of the A23187-induced K + conductance estimated from 42K flux measurements at high external K +, and from net K – flux measurements suggests single-file behavior of the Ca 2+-activated K + channel. The number of Ca 2+-activated K + channels is estimated at about 100 per cell. 相似文献
19.
Summary 1. The purpose of this study was (a) to identify if astrocytes show a similar non-Nernstian depolarization in low K + or low Ca 2+ solutions as previously found in human glial and glioma cells, and (b) to analyze the influence of the K + conductance on the membrane potential of astrocytes.2. The membrane potential (E m) and the ionic conductance were studied with whole-cell patch-clamp technique in neonatal rat astrocytes (5–9 days in culture) and in human glioma cells (U-251MG).3. In 3.0 mM K +, E m was –75 ± 1.0 mV (mean ± SEM, n=39) in rat astrocytes and –79 ± 0.7 mV ( n=5) in U-251MG cells. In both cell types E m changed linearly to the logarithm of [K +] 0 between 3.0 and 160 mM K +. K + free medium caused astrocytes to hyperpolarize to –93 ± 2.7 mV ( n=21) and U-251MG cells to depolarize to –27 ± 2.1 mV ( n=3).4. The I-E curve did not show inward rectification in astrocytes at this developmental stage. The slope conductance (g) exhibited only a small decrease (–19%) in K + free solution and no significant change in 160 mM K +.5. Ba 2+ (1.0 mM) depolarized astrocytes to –45 ± 2.9 mV ( n=11), decreasing the slope conductance (g) by 42.4 ± 8.3% ( n=11). Ca 2+ free solution depolarized astrocytes to –53 ± 3.4 mV ( n=12) and resulted in a positive shift of the I-E curve, increasing g by 15.3 ± 8.2% ( n=8).6. Calculations indicated that a block of K + channels explains the depolarizing effect of Ba 2+. The effects of K + free or Ca 2+ free solutions on E m can be explained by a transformation of K + channels to non-specific leakage channels. That astrocytes show a different reaction to low K + than glioma cells can be related to the lack of inwardly rectifying K + channels in astrocytes at this developmental stage. 相似文献
20.
Summary The mechanism of salt tolerance was studied using isolated internodal cells of the charophyte Nitellopsis obtusa grown in fresh water. When 100 mM NaCl was added to artificial pond water (0.1 mM each of NaCl, KC1, CaCl 2), no cell survived for more than one day. Within the first 30 minutes, membrane potential (E m) depolarized and membrane resistance (R m) decreased markedly. Simultaneously, cytoplasmic Na + increased and K + decreased greatly. At steady state the increase in Na + content was roughly equal to the decrease in K + content. The Cl – content of the cytoplasm did not change. These results suggest that Na + enters the cytoplasm by exchange with cytoplasmic K +. Both the entry of Na + and the exit of K + are assumed to be passive and the latter being caused by membrane depolarization. Vacuolar K +, Na +, and Cl – remained virtually constant, suggesting that rapid influx of Na + from the cytoplasm did not occur.In 100 mM NaCl containing 10 mM CaCl 2, membrane depolarization, membrane resistance decrease and changes in cytoplasmic [Na +] and [K +] did not occur, and cells survived for many days. When cells treated with 100 mM NaCl were transferred within 1 hour to 100 mM NaCl containing 10 mM CaCl 2, Em decreased, Rm increased, cytoplasmic Na + and K + returned to their initial levels, and cells survived. Two possible mechanisms for the role of Ca 2+ in salt tolerance in Nitellopsis are discussed; one a reduction in plasmalemma permeability to Na + and the other a stimulation of active Na +-extrusion. 相似文献
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