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1.
W. -M. Weber B. Dannenmaier W. Clauss 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(2):153-159
The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm-2) with an initial short-circuit current of 29.0±2.9 A·cm-2. Removal of Na+ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l-1) added to the basolateral solution, depressed the short-circuit current completely. The Na+ current saturated at a concentration of 90 mmol Na+·l-1 in the apical solution (K
M=11.2±1.8 mmol·l-1). Amiloride (100 mol·l-1) on the apical side inhibited ca. 40% of the Na+ current and indicated the presence of Na+ channels. The dependence of Na+ current on the amiloride concentration followed Michaclis-Menten kinetics (K
i=2.9±0.4 mol·l-1). The amiloride analogue benzamil had a higher affinity to the Na+ channel (K
i=0.7±0.2 mol·l-1). Thus, Na+ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na+·l-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na+ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l-1) and isobutylmethylxanthine (1 mmol·l-1) nearly doubled short-circuit current and increased amiloride-sensitive Na+ currents by 50%. By current fluctuation analysis we estimated single Na+ channel current (2.7±0.9 pA) and Na+ channel density (3.6±0.6 channels·m-2) under control conditions. After cyclic adenosine monophosphate stimulation Na+ channel density increased to 5.4±1.1 channels·m-2, whereas single Na+ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na+ channel, and show the similarities and differences to vertebrate Na+ channels. Whereas the channel properties are different from the classical vertebrate Na+ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na+ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na+ channels.Abbreviations
slope of the background noise component
- ADH
antidiuretic hormone
- cAMP
cyclic adenosine monophosphate
-
f
frequency
-
f
c
coner frequency of the Lorentzian noise component
- Hepes
N-hydroxyethylpiperazine-N-ethanesulphonic acid
- BMX
isobutyl-methylxanthine
-
i
Na
single Na+ channel current
-
I
Na
max, maximal inhibitable Na+ current
-
I
SC
short circuit current
-
K
i
half maximal blocker concentration
-
K
M
Michaelis constandard error of the mean
-
S
(f)
power density of the Lorentzian noise component
-
S
0
plateau value of the Lorentzian noise component
- TMA
tetramethylammonium
- Trizma
TRIS-hydroxymethyl-amino-methane
-
V
max
maximal reaction velocity
-
V
T
transepithelial potential
-
K
half maximal blocker concentration 相似文献
2.
Bruce Cherksey Robert Durrie Peter E. Braun Victor S. Sapirstein 《Neurochemical research》1994,19(8):1101-1106
This study reports the analysis of K+ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K+ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K+ channel activity was regulated by Ca2+. Little or no change in activity was observed when Ca2+ was added to thecis side of the bilayer. Addition of 10 M total Ca2+ also resulted in little change in K+ channel activity. However, at 80 M total Ca2+ all K+ channel activity was suppressed along with the activation of a 100 pS Cl– channel. The K+ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K+ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G0, Gs, and Gi and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K+ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase
2–3 cyclic nucleotide phosphohydrolase
- EDTA
ethylenediamine N,N,N,N-tetraacetic acid
- G-protein
GTP(guanosine triphosphate) binding protein
- GTPS
guanosine 5-O-(3-thiotriphosphate)
- MAG
myelin associated glycoprotein
- Na+
K+ ATPase, Na+ and K+ stimulated adenosine triphosphatase
- PLP
myelin proteolipid protein
Special issue dedicated to Dr. Majorie B. Lees. 相似文献
3.
The effects of anions on inorganicpyrophosphate-dependent H+-transport in isolated tonoplast vesicles from oat (Avena sativa L.) roots were determined. Both fluorescent and radioactive probes were used to measure formation of pH gradients and membrane potential in the vesicles. Pyrophosphate hydrolysis by the H+-translocating pyrophosphatase was unaffected by anions. Nonetheless, some anions (Cl-, Br- and NO3-) stimulated H+-transport while others (malate,
and iminodiacetate) did not. These differential effects were abolished when the membrane potential was clamped at zero mV using potassium and valinomycin. Stimulation of H+-transport by Cl- showed saturation kinetics whereas that by NO3- consisted of both a saturable component and a linear phase. For Cl- and NO3-, the saturable phase had a K
m
of about 2 mol·m-3. The anions that stimulated H+-transport also dissipated the membrane potential (.) generated by the pyrophosphatase. It is suggested that the stimulatory anions cross the tonoplast in response to the positive generated by the pyrophosphatase, causing dissipation of and stimulation of pH, as expected by the chemiosmotic hypothesis. The work is discussed in relation to recent studies of the effects of anions on ATP-dependent H+-transport at the tonoplast, and its relevance to anion accumulation in the vacuole in vivo is considered.Abbreviations and symools BTP
1,3-bis[tris(hydroxymethyl)-methylamino]-propane
- EGTA
ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid
- Hepes
4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid
- IDA
iminodiacetate
-
membrane potential
- pH
pH gradient
- PPase
inorganic pyrophosphatase
- PPi
morganic pyrophosphate 相似文献
4.
P. A. Dibrov R. L. Lazarova V. P. Skulachev M. L. Verkhovskaya 《Journal of bioenergetics and biomembranes》1989,21(3):347-357
Addition of Na+ to the K+-loadedVibrio alginolyticus cells, creating a 250-fold Na+ gradient, is shown to induce a transient increase in the intracellular ATP concentration, which is abolished by the Na+/H+ antiporter, monensin. The pNa-supported ATP synthesis requires an additional driving force supplied by endogenous respiration or, alternatively, by a K+ gradient (high [K+] inside). In the former case, ATP formation is resistant to the protonophorous uncoupler. Dicyclohexylcarbodiimide and diethylstilbestrol, but not vanadate, completely inhibit Na+ pulse-induced ATP formation. The data agree with the assumption that Na+-ATP-synthase is involved in oxidative phosphorylation inV. alginolyticus. Interrelation of H+ and Na+ cycles in bacteria is discussed.Abbreviations
and
electrochemical gradients of H+ and Na+, respectively
-
transmembrane electric potential difference
- pH, pNa, and pK
concentration gradients of H+, Na+, and K+, respectively
- CCCP
carbonyl cyanidem-chlorophenylhydrazone
- DCCD
N,N-dicyclohexylcarbodiimide
- DES
diesthylstilbestrol
- HQNO
2-heptyl-4-hydroxyquinolineN-oxide
- Tricine
N[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 相似文献
5.
Summary In this paper we describe current fluctuations in the mammalian epithelium, rabbit descending colon. Pieces of isolated colon epithelium bathed in Na+ or K+ Ringer's solutions were studied under short-circuit conditions with the current noise spectra recorded over the range of 1–200 Hz. When the epithelium was bathed on both sides with Na+ Ringer's solution (the mucosal solution contained 50 m amiloride), no Lorentzian components were found in the power spectrum. After imposition of a potassium gradient across the epithelium by replacement of the mucosal solution by K+ Ringer's (containing 50 m amiloride), a Lorentzian component appeared with an average corner frequency,f
c=15.6±0.91 Hz and a mean plateau valueS
o=(7.04±2.94)×10–20 A2 sec/cm2. The Lorentzian component was enhanced by voltage clamping the colon in a direction favorable for K+ entry across the apical membrane. Elimination of the K+ gradient by bathing the colon on both sides with K+ Ringer's solutions abolished the noise signal. The Lorentzian component was also depressed by mucosal addition of Cs+ or tetraethylammonium (TEA) and by serosal addition of Ba2+. The one-sided action of these K+ channel blockers suggests a cellular location for the fluctuating channels. Addition of nystatin to the mucosal solution abolished the Lorentzian component. Serosal nystatin did not affect the Lorentzian noise. This finding indicates an apical membrane location for the fluctuating channels. The data were similar in some respects to K+ channel fluctuations recorded from the apical membranes of amphibian epithelia such as the frog skin and toad gallbladder. The results are relevant to recent reports concerning transcellular potassium secretion in the colon and indicate that the colon possesses spontaneously fluctuating potassium channels in its apical membranes in parallel to the Na+ transport pathway. 相似文献
6.
The tertiary sulphonium compound, -dimethylsulphoniopropionate (DMSP) and the quaternary ammonium compounds glycine betaine and homarine are important osmotica in Platymonas subcordiformis cells. Following hypersaline stresses the compounds were accumulated after a lag period of 3 h and equilibrium concentrations were reached 6 h later. In contrast to these organic solutes, mannitol was synthesised immediately and equilibrium concentrations were reached within 90 min. Hyposaline stresses induced losses of the organic solutes from the cells. The ions K+, Na+, Cl- and the above organic solutes can account for the osmotic balance of the cells.Abbreviations DMSP
-dimethylsulphoniopropionate
- i
intracellular osmolality
- o
extracellular osmolality 相似文献
7.
Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K+ solutions elicits a complex of Ca2+ and K+ currents. The tail current that accompanies a return to holding potential (–40 mV) contains two K+ components. The tail current elicited by a step to –110 mV of 50-msec duration contains fast-decaying (3.5 msec) and slow-decaying (20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K+], suggesting that they represent pure K+ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K+ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca2+ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K+ currents are inhibited by extracellular TEA+ in a concentration-dependent, noncooperative manner, whereas the fast K+ current alone is inhibited by 0.7mm quinidine. 相似文献
8.
Bent Vestergaard-Bogind Per Stampe Palle Christophersen 《The Journal of membrane biology》1987,95(2):121-130
Summary The conductance of the Ca2+-activated K+ channel (g
K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V
m
) and extracellular K+ concentration ([K+]ex). ATP-depleted cells, with fixed values of cellular K+ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K+ channels, to buffer-free salt solutions with given K+ concentrations. Outward-current conductances were calculated from initial net effluxes of K+, correspondingV
m
, monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E
K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of42K at (V
m
–E
K)0, using a single-file flux ratio exponent of 2.7. Within a [K+]ex range of 5.5 to 60mm and at (V
m
–E
K) 20 mV a basic conductance, which was independent of [K+]ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm2 between 0 and –100 mV. As (V
m
–E
K) decreased from 20 towards zero mVg
K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm2. The zero-current conductance was not significantly dependent on [K+]ex (30 to 156mm) corresponding toV
m
(–50 mV to 0). A further increase ing
K(Ca) symmetrically aroundE
K is suggested as (V
m
–E
K) becomes positive. Increasing the extracellular K+ concentration from zero and up to 3mm resulted in an increase ing
K(Ca) from 50 to 70 S/cm2. Since the driving force (V
m
–E
K) was larger than 20 mV within this range of [K+]ex this was probably a specific K+ activation ofg
K(Ca). In conclusion: The Ca2+-activated K+ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel. 相似文献
9.
Summary K+, Cl–, and Ca2+ 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 Ca2+ channel activity for vacuolar Ca2+ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca2+ channel which allowed influx of cytoplasmic Ca2+ into the vacuole when the Ca2+ concentration on the cytoplasmic side was high. When Ca2+ was substituted by Ba2+, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba2+Sr2+Ca2+Mg2+=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca2+ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd2+, but was insensitive to La3+, Gd3+, Ni2+, 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 Ca2+ 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
10.
Ba2+-Induced conductance fluctuations of spontaneously fluctuating K+ channels in the apical membrane of frog skin (Rana temporaria) 总被引:7,自引:0,他引:7
Summary We studied the influence of mucosal Ba2+ ions on the recently described (Zeiske & Van Driessche, 1979a, J. Membrane Biol.
47:77) transepithelial, mucosa towards serosa directed K+ transport in the skin ofRana temporaria. The transport parametersG (conductance), PD (potential difference),I
sc (short-circuit current, K+ current), as well as the noise ofI
sc were recorded. Addition of millimolar concentrations of Ba2+ to the mucosal K+-containing solution resulted in a sudden but quickly reversible drop inI
sc.G andI
sc decreased continuously with increasing Ba2+ concentration, (Ba2+)
o
. The apparent Michaelis constant of the inhibition by Ba2+ lies within the range 40–80 m. The apical membrane seems to remain permselective for K+ up to 500 m (Ba2+)
o
. Higher (Ba2+)
o
, however, appears to induce a shunt (PD falls,G increases). This finding made an accurate determination of the nature of the inhibition difficult but our results tend to suggest a K+-channel block by K+–Ba2+ competition. In the presence of Ba2+, the power spectrum of the K+ current shows a second Lorentzian component in the low-frequency range, in addition to the high-frequency Lorentzian caused by spontaneous K+-channel fluctuations (Van Driessche & Zeiske, 1980). Both Lorentzian components are only present with mucosal K+ and can be depressed by addition of Cs+ ions, thus indicating that Ba2+ ions induce K+-channel fluctuations. The dependence of the parameters of the induced Lorentzian on (Ba2+)
o
, shows a rise in the plateau values to a maximum around 60 m (Ba2+)
o
, followed by a sharp and progressive decrease to very low values. The corner frequency which reflects the rate of the Ba2+-induced fluctuations, however, increases quasi-linearly up to 1mm (Ba2+)
o
with a tendency to saturate at higher (Ba2+)
o
. Based on a three-state model for the K+ channel (having one open state, one closed by the spontaneous fluctuation and one blocked by Ba2+) computer calculations compared favorably with our results. The effect of Ba2+ could be explained by assuming reversible binding at the outer side of the apical K+ channel, thereby blocking the open channel in competition with K+. The association-dissociation of Ba2+ at its receptor site is thought to cause a chopping of the K+ current, resulting in modulated current fluctuations. 相似文献
11.
Summary A highly enriched preparation of basolateral membrane vesicles was isolated from rabbit distal colon surface epithelial cells employing the method described by Wiener, Turnheim and van Os (Weiner, H., Turnheim, K., van Os, C.H. (1989)J. Membrane Biol.110:147–162) and incorporated into planar lipid bilayers. With very few exceptions, the channel activity observed was that of a high conductance, Ca2+-activated K+ channel. This channel is highly selective for K+ over Na+ and Cl–, displays voltage-gating similar to maxi K(Ca) channels found in other cell membranes, and kinetic analyses are consistent with the notion that K+ diffusion through the channel involves either the binding of a single K+ ion to a site within the channel or single-filling (multi-ion occupancy). Channel activity is inhibited by the venom from the scorpionLeiurus quinquestriatus, Ba2+, quinine, and trifluoperazine. The possible role of this channel in the function of these cells is discussed. 相似文献
12.
Summary Excised roots from axenically grown sunflower seedlings reduced or oxidized exogenously added 2,6-dichlorophenolindophenol (DCIP), DCIP-sulfonate (DCIP-S), and cytochromec, and affected simultaneous H+/K+ net fluxes. Experiments were performed with nonpretreated living and CN–-pretreated poisoned roots (control and CN–-roots). CN–-roots showed no H+/K+ net flux activity but still affected the redox state of the compounds tested. The hydrophobic electron acceptor DCIP decreased the rate of H+ efflux in control roots with extension of the maximum rate and optimal pH ranges, then the total net H+ efflux (H+) equalled that of the roots without DCIP. The simultaneously measured K+ influx rate was first inhibited, then inverted into efflux, and finally influx recovered to low rates. This effect could not be due to uptake of the negatively charged DCIP, but due to the lower H+ efflux and the transmembrane electron efflux caused by DCIP, which would depolarize the membrane and open outward K+ channels. The different H+ efflux kinetics characteristics, together with the small but significant DCIP reduction by CN–-roots were taken as evidence that an alternative CN–-resistant redox chain in the plasma membrane was involved in DCIP reduction. The hydrophilic electron acceptor DCIP-S enhanced both H+ and K+ flux rates by control roots. DCIP-S was not reduced, but slightly oxidized by control roots, after a lag, while CN–-roots did not significantly oxidize or reduce DCIP-S. Perhaps the hydrophobic DCIP could have access to and drain electrons from an intermediate carrier deep inside the membrane, to which the hydrophilic DCIP-S could not penetrate. Also cytochromec enhanced H+ and K+, consistent with the involvement of the CN–-resistant redox chain. Control roots did not reduce but oxidize cytochromec after a 15 min lag, and CN–-roots doubled the rate of cytochromec oxidation without any lag. NADH in the medium spontaneously reduced cytochromec, but control or CN–-roots oxidized cytochromec, despite of the presence of NADH. In this case CN–-roots were less efficient, while control roots doubled the rate of cytochromec oxidation by CN–-roots, after a 10 min lag in which cytochromec was reduced at the same rate as the medium plus NADH did. CN–-roots seemed to have a fully activated CN–-resistant branch. The described effects on K+ flux were consistent with the current hypothesis that redox compounds changed the electric membrane potential (de- or hyperpolarization), which induces the opening of voltage-gated in- or outward K+ channels.Abbreviations Cyt
c cytochromec
- DCIP
2,6-dichlorophenolindophenol
- DCIP-S
2,6-dichlorophenolindophenol 3-sulfonate
- HCF(III)
hexacyanoferrate (III)
- PM
plasma membrane
- SHAM
salicylhydroxamic acid
- VH+ and VK+ H+
efflux and K+ influx rates
- H+ and K+ total H+
efflux and K+ influx at the end of the experiment
- H+ and K+
buffering power of the titrated medium 相似文献
13.
S. A. Lewis M. S. Ifshin D. D. F. Loo J. M. Diamond 《The Journal of membrane biology》1984,80(2):135-151
Summary Sodium channels in rabbit urinary bladder were studied by noise analysis. There are two components of short-circuit current (I
sc) and correspondingly two components of apical Na+ entry, one amiloride-sensitive (termedI
A and the A channel, respectively) and one amiloride-insensitive (I
L
and the leak pathway, respectively). The leak pathway gives rise tol/f noise, while the A channel in the presence of amiloride gives rise to Lorentzian noise. A two-state model of the A channel accounts well for how the corner frequency and plateau value of Lorentzian noise vary with amiloride concentration. The single-channel current is 0.64 pA, and the conducting channel density is on the order of 40 copies per cell. Triamterene blocks the A channel alone, and increasing external Na+ decreases the number but not the single-channel permeability of the A channel. Hydrostatic pressure pulses (punching) increase the number of both pathways. Repeated washing of the mucosal surface removes most of the leak pathway without affecting the A channel.Properties of the A channel revealed by noise analysis of various tight epithelia are compared, and the mechanism ofl/f noise is discussed. It is suggested that the A channel is synthesized intracellularly, stored in intracellular vesicles, transferred with or from vesicular membrane into apical membrane under the action of microfilaments, and degraded into the leak pathway, which is washed out into urine or destroyed. The A channel starts withP
Na/P
K30 and loses selectivity in stages untilP
Na/P
K reaches the free-solution mobility ratio (0.7) for the leak pathway. This turnover cycle functions as a mechanism of repair and regulation for Na+ channels, analogous to the repair and regulation of most intracellular proteins by turnover. Vesicular delivery of membrane channels may be operating in several other epithelia. 相似文献
14.
A ouabain sensitive inward current occurs in Xenopus oocytes in Na+ and K+ -free solutions. Several laboratories have investigated the properties of this current and suggested that acidic extracellular pH (pHo) produces a conducting pathway through the Na+/K+ pump that is permeable to H+ and blocked by [Na+]o. An alternative suggestion is that the current is mediated by an electrogenic H+-ATPase. Here we investigate the effect of pHo and [Na+]o on both transient and steady-state ouabain-sensitive current. At alkaline or neutral pHo the relaxation rate of pre-steady-state current is an exponential function of voltage. Its U-shaped voltage dependence becomes apparent at acidic pHo, as predicted by a model in which protonation of the Na+/K+ pump reduces the energy barrier between the internal solution and the Na+ occluded state. The model also predicts that acidic pHo increases steady-state current leak through the pump. The apparent pK of the titratable group(s) is 6, suggesting that histidine is involved in induction of the conductance pathway. 22Na efflux experiments in squid giant axon and current measurements in oocytes at acidic pHo suggest that both Na+ and H+ are permeant. The acid-induced inward current is reduced by high [Na+]o, consistent with block by Na+. A least squares analysis predicts that H+ is four orders of magnitude more permeant than Na+, and that block occurs when 3 Na+ ions occupy a low affinity binding site (K
0.5=130±30 mM) with a dielectric coefficient of 0.23±0.03. These data support the conclusion that the ouabain-sensitive conducting pathway is a result of passive leak of both Na+ and H+ through the Na+/K+ pump. 相似文献
15.
Summary Loop diuretic-sensitive (Na+,K+,Cl–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na+,K+) gradien-driven36Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While86Rb uptake rates showed hyperbolic dependencies on Na+ and K+ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl– concentration, Hill coefficient 1.8, indicating a 1 Na+: 1 K+:2 Cl– stoichiometry of ion transport.The interaction of putative (Na+, K+, Cl–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [3H]-bumetanide. The requirement for the simulataneous presence of Na+,K+, and Cl–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na+, K+, Cl–)-cotransporter. [3H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K
D1=0.13 m;B
max1 =6.4 pmol/mg of protein) and low-affinity (K
D2=34 m;B
max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl– (K
1=60mm), whereas low-affinity sites seem to be unaffected by Cl–. We have shown that only high-affinity [3H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium. 相似文献
16.
This review summarizes our experiments on the significance of the -subunit in the functional expression of Na+/K+-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na+/K+-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH2-terminal domain to the extracellular COOH-terminus: the NH2-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na+/K+-ATPase by Na+/K+- and H+/K+-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na+/K+-ATPase -subunit and from Ile89 to the COOH–terminus of the H+/K+-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na+/K+-ATPase. 相似文献
17.
Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers 总被引:5,自引:0,他引:5
Plasma membrane was purified from roots of rye (Secale cereale L. cv. Rheidol) by aqueous-polymer two-phase partitioning and incorporated into planar bilayers of 1-palmitoyl-2-oleoyl phosphatidylethanolamine by stirring with an osmotic gradient. Since plasmamembrane vesicles were predominantly oriented with their cytoplasmic face internal, when fused to the bilayer the cytoplasmic side of channels faced the trans chamber. In asymmetrical (cis:trans) 280100 mM KCl, five distinct K+-selective channels were detected with mean chord-conductances (between +30 and -30 mV; volyages cis with respect to trans) of 500 pS, 194 pS, 49 pS, 21 pS and 10 pS. The frequencies of incorporation of these K+ channels into the bilayer were 48, 21, 50, 10 and 9%, in the order given (data from 159 bilayers). Only the 49 pS channel was characterized further in this paper, but the remarkable diversity of K+ channels found in this preparation is noteworthy and is the subject of further study. In symmetrical KCl solutions, the 49 pS channel exhibited non-ohmic unitary-current/voltage relationships. The chord-conductance (between +30 and-30 mV) of the channel in symmetrical 100 mM KCl was 39 pS. The unitary current was greater at positive voltages than at corresponding negative voltages and showed considerable rectification with increasing positive and negative voltages. This would represent inward rectification in vivo. Gating of the channel was not voltage-dependent and the channel was open for approx. 80% of the time. Presumably this is not the case in vivo, but we are at present uncertain of the in vivo controls of channel gating. The distribution of channel-open times could be approximated by the sum of two negative exponential functions, yielding two open-state time constants (o, the apparent mean lifetime of the channel-open state) of 1.0 ms and 5.7 s. The distribution of channel-closed times was best approximated by the sum of three negative exponential functions, yielding time constants (c, the apparent mean lifetime of the channel-closed state) of 1.1 ms, 51 ms and 11 s. This indicates at least a five-state kinetic model for the activity of the channel. The selectivity of the 49 pS channel, determined from both reversal potentials under biionic conditions (100 mM KCl100 mM cation chloride) and from conductance measurements in symmetrical 100 mM cation chloride, was Rb+ K+ > Cs+ > Na+ > Li+ > tetraethylammonium (TEA+). The 49 pS channel was reversibly inhibited by quinine (1 mM) but TEA+ (10 mM), Ba2+ (3 mM), Ca2+ (1 mM), 4-aminopyridine (1 mM) and charybdotoxin (3 M) were without effect when applied to the extracellular (cis) surface.Abbreviations and Symbols GHK
Goldman-Hodgkin-Katz
- I/V
current/voltage
- PEG
polyethyleneglycol
- Po
probability o f the channel being open
- TEA+
tetraethylammonium
- c
apparent mean lifetime of the channel-closed state
- o
apparent mean lifetime of the channel-open state
P.J.W. was supported by a grant from the Science and Engineering Research Council Membrane Initiative (GR/F 33971) to Professor E.A.C. MacRobbie and M.T. by the Glaxo Junior Research Fellowship at Churchill College, Cambridge. We thank Dr. D.T. Cooke (AFRC, Long Ashton Research Station, University of Bristol, UK) and Ms. J. Marshall (University of York, UK) for their advice and assistance with the aqueous-polymer two-phase partitioning of plasma membrane from rye roots, Mr. J. Banfield and Miss P. Parmar (University of Cambridge, UK) for technical assistance and Professor E.A.C. MacRobbie, Dr. G. Thiel (University of Cambridge, UK), Dr. M.R. Blatt (Wye College, University of London, UK), Dr. D. Sanders and Dr. E. Johannes (University of York, UK) for helpful discussions. 相似文献
18.
Summary Voltage-clamped steps in the electric potential difference (PD) across the membrane in cells of the green alga,Chara inflata, cause voltage- and time-dependent current flows, interpreted to arise from opening and closing of various types of ion channel in the membrane. With cells in the light, these channels are normally closed, and the resting PD is probably determined by the operation of an H+ efflux pump. Positive steps in PD from the resting level often caused the opening of K+ channels with sigmoid kinetics. The channels began to show opening when the PD–120 mV for an external concentration of K+ of 1.0mm. Return of the PD to the resting level caused closing of the channels with complex kinetics. Various treatments of the cell could cause these K+ channels to open, and remain open continuously, with the PD then lying closer to the Nernst PD for K+. The K+ channels have been identified by the blocking effects of TEA+. Another group of channels, probably Cl– and Ca2+ associated with the action potential open when the PD is stepped to values less negative than –50 mV. Negative steps from the resting PD cause the slow opening, with a time course of seconds, of yet another type of channel, probably Cl–. 相似文献
19.
Summary Apical Na+ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N
Na)-membrane potential (mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K+ in order to determine whether theI
Na-mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K+ is considered inadvisable for determining theI
Na-mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI
Na-mc relationships under baseline conditions provided estimates of intracellular Na+ concentration and of apical Na+ permeability of 9 to 14mm and of 3 × 10–7 cm · sec–1, respectively, in reasonable agreement with estimates obtained by different techniques. 相似文献
20.
Plant roots accumulate K+ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K+ transporters which operate at millimolar and micromolar external [K+]: high-affinity K+ uptake is energised by symport with H+, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K+ uptake, a characterisation of the principal K+-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K+-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (PCl PK < 0.025) but less selective amongst monovalent cations (PNaPK0.17–0.3). Both the IRC and the ORC were blocked by Ba2+, Cs+ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (EK+), enabling ORCs to conduct mainly outward, but occasionally inward, K+ current. By contrast, gating of the 5-pS IRC was weakly voltageependent and IRC gating was invariably restricted to membrane potentials more negative than EK+, ensuring K+ transport was always inwardly directed. Studies on channel activity were conducted for a large number of root cells grown at two levels of external [K+], one where K+ uptake is likely to be principally through channels (6 mM K+) and one where it must be energised (100 M K+). Shifting growth conditions from high to low K+ did not affect single-channel properties such as conductance and selectivity, nor the manifestation of the ORC and 20-pS IRC, but led to enhanced activity of the 5-pS IRC. The enhanced activity of the 5-pS IRC was mirrored by a parallel increase in unidirectional 86Rb+ influx after low-K+ growth, clearly indicating a dominant role of this particular channel in K+ uptake at supra millimolar external [K+].Abbreviations EK+
potassium equilibrium potential
- Em
membrane potential
- HK
high [K+]
- IRC
inward rectifying channel
- LK
low [K+]
- ORC
outward rectifying channel
- TEA
tetra-ethyl-ammonium
Financial support was provided by the Biotechnology and Biological Sciences Research Council (Grant PG87/529) and by the European Union (Framework III, Biotechnology Programme). 相似文献