共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
R.Z. Sabirov R.R. Azimov Y. Ando-Akatsuka T. Miyoshi Y. Okada 《The Journal of membrane biology》1999,172(1):67-76
To examine the extracellular Na+ sensitivity of a renal inwardly rectifying K+ channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K+ channel, ROMK1 (Kir1.1). When extracellular Na+ was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel
ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na+ from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na+ removal from the bath solution. A blocker of Na+/H+ antiporters, amiloride, largely inhibited the Na+ removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much
less sensitive to Na+ removal. Na+ removal was found to induce a significant decrease in intracellular pH in the oocytes using H+-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na+/H+ antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na+ in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular
Na+, and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na+ sensitivity of ROMK1 channel via regulating intracellular pH.
Received: 13 April 1999/Revised: 15 July 1999 相似文献
3.
C.H. Pedemonte T.A. Pressley M.F. Lokhandwala A.R. Cinelli 《The Journal of membrane biology》1997,155(3):219-227
Considerable evidence indicates that the renal Na+,K+-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones
and second messengers. Recently, it has been reported that amino acids close to the NH2-terminal end of the Na+,K+-ATPase α-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na+,K+-ATPase activity. To determine whether the α-subunit NH2-terminus is involved in the regulation of Na+,K+-ATPase activity by PKC, we have expressed the wild-type rodent Na+,K+-ATPase α-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH2-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of
rodent kidney cells. The presence of the α-subunit NH2-terminal segment was not necessary to express the maximal Na+,K+-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb+-transport in intact cells were the same in cells transfected with the wild-type rodent α1 and the NH2-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na+,K+-ATPase mediated Rb+-uptake and reduced the intracellular Na+ concentration of cells transfected with wild-type α1 cDNA. In contrast, these effects were not observed in cells expressing
the NH2-deletion mutant of the α-subunit. Treatment with phorbol ester appears to affect specifically the Na+,K+-ATPase activity and no evidence was observed that other proteins involved in Na+-transport were affected. These results indicate that amino acid(s) located at the α-subunit NH2-terminus participate in the regulation of the Na+,K+-ATPase activity by PKC.
Received: 10 July 1996/Revised: 19 September 1996 相似文献
4.
Renal A6 cells have been reported in which hyposmolality stimulates Na+ transport by increasing the number of conducting amiloride-sensitive 4-pS Na+ channels at the apical membrane. To study a possible role of protein tyrosine kinase (PTK) in the hyposmolality-induced signaling,
we investigated effects of PTK inhibitors on the hyposmolality-induced Na+ transport in A6 cells. Tyrphostin A23 (a PTK inhibitor) blocked the stimulatory action of hyposmolality on a number of the
conducting Na+ channels. Tyrphostin A23 also abolished macroscopic Na+ currents (amiloride-sensitive short-circuit current, I
Na
) by decreasing the elevating rate of the hyposmolality-increased I
Na
. Genistein (another type of PTK inhibitor) also showed an effect similar to tyrphostin A23. Brefeldin A (BFA), which is an
inhibitor of intracellular translocation of protein, blocked the action of hyposmolality on I
Na
by diminishing the elevating rate of the hyposmolality-increased I
Na
, mimicking the inhibitory action of PTK inhibitor. Further, hyposmolality increased the activity of PTK. These observations
suggest that hyposmolality would stimulate Na+ transport by translocating the Na+ channel protein (or regulatory protein) to the apical membrane via a PTK-dependent pathway. Further, hyposmolality also caused
an increase in the plasma (apical) membrane capacitance, which was remarkably blocked by treatment with tyrphostin A23 or
BFA. These observations also suggest that a PTK-dependent pathway would be involved in the hyposmolality-stimulated membrane
fusion in A6 cells.
Received: 6 October 1999/Revised: 4 February 2000 相似文献
5.
The Ca2+-activated maxi K+ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean
number of open channels (NP
o
) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the
stretch-activation of Ca2+-permeable cation channels, because the maxi K+ channel was not stretch-activated in both the cell-attached configuration using Ca2+-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K+ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K+ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K+ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV
t
) when luminal K+ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV
t
was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced
ΔV
t
to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV
t
to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV
t
was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV
t
=−7.2 ± 0.5 mV in control vs.ΔV
t
=−6.7 ± 0.6 mV in AA). These results suggest that the Ca2+-activated maxi K+ channel is responsible for flow-dependent K+ secretion by coupling with the stretch-activated Ca2+-permeable cation channel in the rabbit CNT.
Received: 21 August 1997/Revised: 20 March 1998 相似文献
6.
d-Aspartate (d-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means
of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. d-Asp influx is Na+-dependent with K
m
= 5 μm and V
max= 0.7 nmoles · min−1· mg protein−1. Influx is sigmoidal as f[Na+] with Na+
K
m
∼ 12 μm and Hill coefficient of 1.9. The cells establish steady-state d-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold
increase in V
max, with no change in K
m
. At initial [d-Asp] = 10 μm, RBA take up more than 90% of total d-Asp, and extracellular levels are reduced to levels below 1 μm. Ionophores that dissipate the ΔμNa+ inhibit gradient formation. Genistein (GEN, 100 μm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4α-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response
still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na+-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), l-Asp, and l-Glu, but not by d-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms.
mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are
expressed in these cells.
Received: 11 January 2001/Revised: 26 March 2001 相似文献
7.
Analysis of the mechanistic basis by which sodium-coupled transport systems respond to changes in membrane potential is inherently
complex. Algebraic expressions for the primary kinetic parameters (K
m
and V
max
) consist of multiple terms that encompass most rate constants in the transport cycle. Even for a relatively simple cotransport
system such as the Na+/alanine cotransporter in LLC-PK1 cells (1:1 Na+ to substrate coupling, and an ordered binding sequence), the algebraic expressions for K
m
for either substrate includes ten of the twelve rate constants necessary for modeling the full transport cycle. We show here
that the expression of K
m
of the first-bound substrate (Na+) simplifies markedly if the second-bound substrate (alanine) is held at a low concentration so that its' binding becomes
the rate limiting step. Under these conditions, the expression for the K
Na
m
includes rate constants for only two steps in the full cycle: (i) binding/dissociation of Na+, and (ii) conformational `translocation' of the substrate-free protein. The influence of imposed changes in membrane potential
on the apparent K
Na
m
for the LLC-PK1 alanine cotransporter at low alanine thus provides insight to potential dependence at these sites. The data show no potential
dependence for K
Na
m
at 5 μm alanine, despite marked potential dependence at 2 mm alanine when the full algebraic expression applies. The results suggest that neither translocation of the substrate-free
form of the transporter nor binding/dissociation of extracellular sodium are potential dependent events for this transport
system.
Received: 10 April 1998/Revised: 6 July 1998 相似文献
8.
D. Claveau I. Pellerin M. Leclerc M.G. Brunette 《The Journal of membrane biology》1998,165(3):265-274
In the rabbit as well as the rat, a Na+/H+ exchanger is expressed in the apical membrane of both the proximal and distal tubules of the renal cortex. Whereas the isoform
derived from the proximal tubule has been extensively studied, little information is available concerning the distal luminal
membrane isoform. To better characterize the latter isoform, we purified rabbit proximal and distal tubules, and examined
the ethylpropylamiloride (EIPA)-sensitive 22Na uptake by the luminal membrane vesicles from the two segments. The presence of 100 μm EIPA in the membrane suspension decreased the 15 sec Na+ uptake to 75.70 ± 4.70% and 50.30 ± 2.23% of the control values in vesicles from proximal and distal tubules, respectively.
The effect of EIPA on 35 mm Na+ uptake was concentration dependent, with a IC50 of 700 μm and 75 μm for the proximal and distal luminal membranes. Whereas the proximal tubule membrane isoform was insensitive to cimetidine
and clonidine up to a concentration of 2 mm, the 35 mm Na+ uptake by the distal membrane was strongly inhibited by cimetidine (IC50 700 μm) and modestly inhibited by clonidine (IC50 1.6 mm).
The incubation of proximal tubule suspensions with 1 mm (Bu2) cAMP decreased the 15-sec EIPA-sensitive Na+ uptake by the brush border membranes to 24.1 ± 2.38% of the control values. Unexpectedly, the same treatment of distal tubules
enhanced this uptake by 46.5 ± 10.3%. Finally, incubation of tubule suspensions with 100 nm phorbol 12-myristate 13-acetate (PMA) decreased the exchanger activity to 58.6 ± 3.04% and 79.7 ± 3.21% of the control values
in the proximal and distal luminal membranes, respectively. In conclusion, the high sensitivity of the distal luminal membrane
exchanger to various inhibitors, and its stimulation by cAMP-dependent protein kinase A, indicate that this isoform differs
from that of the proximal tubule and probably corresponds to isoform 1.
Received: 6 March 1998/Revised: 6 July 1998 相似文献
9.
M. Blot-Chabaud N. Coutry M. Laplace J.-P. Bonvalet N. Farman 《The Journal of membrane biology》1996,153(3):233-239
In the cortical collecting duct (CCD), arginin vasopressin (AVP) has been shown to increase the number and activity of basolateral
Na+-K+-ATPase by recruiting or activating a latent pool of pumps. However, the precise mechanism of this phenomenon is still unknown.
The aim of this study was to investigate whether this AVP-induced increase in basolateral Na+-K+-ATPase could depend on a dephosphorylation process. To this purpose, the effect of protein serine/threonine phosphatase (PP)
inhibitors was examined on both the specific 3H-ouabain binding (to evaluate the number of pumps in the basolateral membrane) and the ouabain-dependent 86Rb uptake (to evaluate pump functionality) in the presence or absence of AVP. In addition, the activity of two PP, PP1 and
PP2A, was measured and the influence of AVP was examined on both enzymes. Experiments have been performed on mouse CCD isolated
by microdissection. Results show that inhibition of PP2A prevents the AVP-induced increase in the number and activity of Na+-K+-ATPases, independent of an effect on the apical cell sodium entry. In addition, AVP rapidly increased the activity of PP2A
without effect on PP1. These data suggest that PP2A is implied in the regulation of Na+-K+-ATPase activity by AVP in the CCD and that the AVP-dependent increase in the number of Na+-K+-ATPases is mediated by a PP2A-dependent dephosphorylation process.
Received: 22 March 1996/Revised: 21 June 1996 相似文献
10.
Protection from Cell Death by mcl-1 Is Mediated by Membrane Hyperpolarization Induced By K+ Channel Activation 总被引:3,自引:0,他引:3
Mcl-1, a member of the Bcl-2 family, has been identified as an inhibitor of apoptosis induced by anticancer agents and radiation in myeloblastic leukemia
cells. The molecular mechanism underlying this phenomenon, however, is not yet understood. In the present study, we report
that hyperpolarization of the membrane potential is required for prevention of mcl-1 mediated cell death in murine myeloblastic FDC-P1 cells. In cells transfected with mcl-1, the membrane potential, measured by the whole-cell patch clamp, was hyperpolarized more than −30 mV compared with control
cells. The membrane potential was repolarized by increased extracellular K+ concentration (56 mV per 10-fold change in K+ concentration). Using the cell-attached patch-clamp technique, K+ channel activity was 1.7 times higher in mcl-1 transfected cells (NP
o
= 22.7 ± 3.3%) than control cells (NP
o
= 13.2 ± 1.9%). Viabilities of control and mcl-1 transfected cells after treatment with the cytotoxin etoposide (20 μg/ml), were 37.9 ± 3.9% and 78.2 ± 2.0%, respectively.
Suppression of K+ channel activity by 4-aminopyridine (4-AP) before etoposide treatment significantly reduced the viability of mcl-1 transfected cells to 49.0 ± 4.6%. These results indicate that as part of the prevention of cell death, mcl-1 causes a hyperpolarization of membrane potential through activation of K+ channel activity.
Received: 30 March 1999/Revised: 20 July 1999 相似文献
11.
P.J. White 《The Journal of membrane biology》1996,152(1):89-99
Nitrogen is available to the plant in the form of NH+
4 in the soil solution. Here it is shown that a voltage-independent K+ channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to NH+
4. The channel was studied following its incorporation into planar 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine bilayers.
The unitary conductance of the channel was greater when assayed in the presence of 100 mm NH4Cl than 100 mm KCl. However, the probability of finding the channel open (P
o
) was lower in the presence of 100 mm NH4Cl (P
o
= 0.63) than in 100 mm KCl (P
o
= 0.8), suggesting that P
o
can be regulated by the (permeant) ions present in solution. When assayed in equimolar concentrations of NH4Cl (cis) and KCl (trans), the zero-current (reversal) potential for the channel (E
rev) exhibited a complex concentration dependence. At low cation concentrations, the apparent permeability of NH+
4 relative to K+ (PNH4/PK) was greater than 1.0. However, as the cation concentration was increased, PNH4/PK initially decreased to a minimum of 0.95 at 3 mm before increasing again to a maximum of 1.89 at 300 mm. At cation concentrations above 300 mm, PNH4/PK decreased slightly. This implies that the pore of the channel can be occupied by more than one cation simultaneously. Ammonium
permeation through the pore was simulated using a model which is composed of three energy barriers and two energy wells (the
ion-binding sites). The model (3B2S) allowed for single-file permeation, double cation occupancy, ion-ion repulsion within
the pore and surface potential effects. Results indicated that energy peaks and energy wells were situated asymmetrically
within the electrical distance of the pore, that cations repel each other within the pore and that the vestibules to the pore
contain negligible surface charge. The energy profile obtained for NH+
4 is compared with ones obtained for K+ and Na+. This information allows the fluxes through the K+ channel of the three major monovalent cations present in the soil solution to be predicted.
Received: 16 October 1995/Revised 12 March 1996 相似文献
12.
Two channels, distinguished by using single-channel patch-clamp, carry out potassium transport across the red cell membrane
of lamprey erythrocytes. A small-conductance, inwardly rectifying K+-selective channel was observed in both isotonic and hypotonic solutions (osmolarity decreased by 50%). The single-channel
conductance was 26 ± 3 pS in isotonic (132 mm K+) solutions and 24 ± 2 pS in hypotonic (63 mm K+) solutions. No outward conductance was found for this channel, and the channel activity was completely inhibited by barium.
Cell swelling activated another inwardly rectifying K+ channel with a larger inward conductance of 65 pS and outward conductance of 15 pS in the on-cell configuration. In this
channel, rectification was due to the block of outward currents by Mg2+ and Ca2+ ions, since when both ions were removed from the cytosolic side in inside-out patches the conductance of the channel was
nearly ohmic. In contrast to the small-conductance channel, the swelling-activated channel was observed also in the presence
of barium in the pipette. Neither type of channel was dependent on the presence of Ca2+ ions on the cytosolic side for activity.
Received: 18 July 1997/Revised: 30 January 1998 相似文献
13.
A detailed temperature dependence study of a well-defined plant ion channel, the Ca2+-activated K+ channel of Chara corallina, was performed over the temperature range of their habitats, 5–36°C, at 1°C resolution. The temperature dependence of the
channel unitary conductance at 50 mV shows discontinuities at 15 and 30°C. These temperatures limit the range within which
ion diffusion is characterized by the lowest activation energy (E
a
= 8.0 ± 1.6 kJ/mol) as compared to the regions below 15°C and above 30°C. Upon reversing membrane voltage polarity from 50
to −50 mV the pattern of temperature dependence switched from discontinuous to linear with E
a
= 13.6 ± 0.5 kJ/mol. The temperature dependence of the effective number of open channels at 50 mV showed a decrease with increasing
temperature, with a local minimum at 28°C. The mean open time exhibited a similar behavior. Changing the sign of membrane
potential from 50 to −50 mV abolished the minima in both temperature dependencies. These data are discussed in the light of
higher order phase transitions of the Characean membrane lipids and corresponding change in the lipid-protein interaction,
and their modulation by transmembrane voltage.
Received: 14 June 2000/Revised: 20 September 2000 相似文献
14.
T.-H. Hwang D.-J. Suh H.-R. Bae S.-H. Lee J.-S. Jung 《The Journal of membrane biology》1996,154(3):251-257
To study K+ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on
permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml)
in the symmetrical high K+ (145 mm) Ringer solution. During measurement of the macroscopic K+ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types
of K+ currents: one is an inwardly rectifying K+ current and the other is a slowly activating outwardly rectifying K+ current. The inwardly rectifying K+ current is inhibited by Ba2+. The slowly activating K+ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature.
This is consistent with the biophysical characteristics of I
SK channel. RT-PCR analysis revealed the presence of I
SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba2+ only had minimal affect on short-circuit current (I
sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I
SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia.
Received: 1 March 1996/Revised: 5 August 1996 相似文献
15.
We have previously reported that intralobular salivary duct cells contain an amiloride-sensitive Na+ conductance (probably located in the apical membranes). Since the amiloride-sensitive Na+ conductances in other tight epithelia have been reported to be controlled by extracellular (luminal) Na+, we decided to use whole-cell patch clamp techniques to investigate whether the Na+ conductance in salivary duct cells is also regulated by extracellular Na+. Using Na+-free pipette solutions, we observed that the whole-cell Na+ conductance increased when the extracellular Na+ was increased, whereas the whole-cell Na+ permeability, as defined in the Goldman equation, decreased. The dependency of the whole-cell Na+ conductance on extracellular Na+ could be described by the Michaelis-Menten equation with a K
m
of 47.3 mmol/1 and a maximum conductance (G
max) of 2.18 nS. To investigate whether this saturation of the Na+ conductance with increasing extracellular Na+ was due to a reduction in channel activity or to saturation of the single-channel current, we used fluctuation analysis of
the noise generated during the onset of blockade of the Na+ current with 200 μmol/l 6-chloro-3,5-diaminopyrazine-2-carboxamide. Using this technique, we estimated the single channel
conductance to be 4 pS when the channel was bathed symmetrically in 150 mmol/l Na+ solutions. We found that Na+ channel activity, defined as the open probability multiplied by the number of available channels, did not alter with increasing
extracellular Na+. On the other hand, the single-channel current saturated with increasing extracellular Na+ and, consequently, whole-cell Na+ permeability declined. In other words, the decline in Na+ permeability in salivary duct cells with increasing extracellular Na+ concentration is due simply to saturation of the single-channel Na+ conductance rather than to inactivation of channel activity.
Received: 27 July 1995/Revised: 7 December 1995 相似文献
16.
Outwardly oriented H+ gradients greatly enhanced thiamine transport rate in brush border membrane vesicles from duodenal and jejunal mucosa of
adult Wistar rats. At a gradient pHin5:pHout7.5, thiamine uptake showed an overshoot, which at 15 sec was three times as large as the uptake observed in the absence of
the gradient. Under the same conditions, the binding component of uptake accounted for only 10–13% of intravesicular transport.
At the same gradient, the K
m
and J
max values of the saturable component of the thiamine uptake curve after a 6 sec incubation time were 6.2 ± 1.4 μm and 14.9 ± 3 pmol · mg−1 protein · 6 sec−1 respectively. These values were about 3 and 5 times higher, respectively, than those recorded in the absence of H+ gradient. The saturable component of the thiamine antiport had a stoichiometric thiamine: H+ ratio of 1:1 and was inhibited by thiamine analogues, guanidine, guanidine derivatives, inhibitors of the guanidine/H+ antiport, and imipramine. Conversely, the guanidine/H+ antiport was inhibited by unlabeled thiamine and thiamine analogues; omeprazole caused an approximately fourfold increase
in thiamine transport rate. In the absence of H+ gradient, changes in transmembrane electrical potential did not affect thiamine uptake. At equilibrium, the percentage membrane-bound
thiamine taken up was positively correlated with the pH of the incubation medium, and increased from about 10% at pH 5 to
99% at pH 9.
Received: 17 July 1997/Revised: 16 September 1997 相似文献
17.
The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K+ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K+ current was monitored under voltage clamp in 0.1–30 mm K+ and in equivalent concentrations of Rb+, Cs+ and Na+. From a conditioning voltage of −200 mV, clamp steps to voltages between −150 and +50 mV in 0.1 mm K+ activated current through outward-rectifying K+ channels (I
K,
out) at the plasma membrane in a voltage-dependent fashion. Increasing [K+]
o
shifted the voltage-sensitivity of I
K,
out in parallel with the equilibrium potential for K+ across the membrane. A similar effect of [K+]
o
was evident in the kinetics of I
K,
out activation and deactivation, as well as the steady-state conductance- (g
K
−) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near −130 mV in 0.1 mm K+, −80 mV in 1.0 mm K+, and −20 mV in 10 mm K+. Similar data were obtained with Rb+ and Cs+, but not with Na+, consistent with the relative efficacy of cation binding under equilibrium conditions (K+≥ Rb+ > Cs+ > > Na+). Changing Ca2+ or Mg2+ concentrations outside between 0.1 and 10 mm was without effect on the voltage-dependence of g
K
or on I
K,
out activation kinetics, although 10 mm [Ca2+]
o
accelerated current deactivation at voltages negative of −75 mV. At any one voltage, increasing [K+]
o
suppressed g
K
completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K+ was sensitive to voltage, varying from 0.5 to 20 mm with clamp voltages near −100 to 0 mV, respectively. These, and additional data indicate that extracellular K+ acts as a ligand and alters the voltage-dependence of I
K,
out gating; the results implicate K+-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the
channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of
two K+ ions outside affects the distribution between closed states of the channel.
Received: 27 November 1996/Revised: 4 March 1997 相似文献
18.
C. Bookstein M.W. Musch P.K. Dudeja R.L. McSwine Y. Xie T.A. Brasitus M.C. Rao E.B. Chang 《The Journal of membrane biology》1997,160(3):183-192
This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants
of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease
of ≈50% in the specific activities of both NHE1 and NHE3. Only V
max was affected; K
Na was unchanged. This effect was specific, as Na+, K+, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride,
5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely
unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues
in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular
transport by incubation at 4°C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect
does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity)
influences Na+/H+ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity.
Received: 23 January 1997/Revised: 1 August 1997 相似文献
19.
20.
We used 31P NMR to investigate the temperature-dependence of intracellular pH (pH
i
) in isolated frog skeletal muscles. We found that ln[H+
i
] is a linear function of 1/T
abs paralleling those of neutral water (i.e., H+= OH−) and of a solution containing the fixed pH buffers of frog muscle cytosol. This classical van't Hoff relationship was unaffected
by inhibition of glycolysis and was not dependent upon the pH or [Na+] in the bathing solution. Insulin stimulation of Na+-H+ exchange shifted the intercept in the alkaline direction but had no effect on the slope. Acid loading followed by washout
resulted in an amiloride-sensitive return to the (temperature dependent) basal pH
i
.
These results show that the temperature dependence of activation of Na+-H+ exchange is similar to that of the intracellular buffers, and suggest that constancy of [H+]/[OH−] with changing temperature is achieved in the short term by intracellular buffering and in the long term by the set-point
of the Na+-H+ exchanger. Proton activation of the exchanger has an apparent standard enthalpy change (ΔH°) under both control and insulin-stimulated
conditions that is similar to the ΔH° of the intracellular buffers and approximately half of the ΔH° for the dissociation
of water. Thus, the temperature-dependent component of the standard free-energy change (ΔF°) is unaffected by insulin stimulation,
suggesting that changes in Arrhenius activation energy (E
a
) may not be a part of the mechanism of hormone stimulation.
Received: 12 February 1997/Revised: 1 October 1997 相似文献