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1.
2.
L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral
membrane vesicles l-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective.
The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is
saturable with respect to external lactate with a K
m
of 39.2 ± 4.8 mm and a J
max of 8.9 ± 0.7 nmoles mg protein−1 sec−1. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains
a H+-lactate cotransporter, whereas in the apical membrane both H+-lactate and Na+-lactate cotransporters are present, even if they exhibit a low transport rate.
Received: 22 October 1996/Revised: 11 March 1997 相似文献
3.
I.C. Forster C.A. Wagner A.E. Busch F. Lang J. Biber N. Hernando H. Murer A. Werner 《The Journal of membrane biology》1997,160(1):9-25
The two electrode voltage clamp technique was used to investigate the steady-state and presteady-state kinetic properties
of the type II Na+/P
i
cotransporter NaPi-5, cloned from the kidney of winter flounder (Pseudopleuronectes americanus) and expressed in Xenopus laevis oocytes. Steady-state P
i
-induced currents had a voltage-independent apparent K
m
for P
i
of 0.03 mm and a Hill coefficient of 1.0 at neutral pH, when superfusing with 96 mm Na+. The apparent K
m
for Na+ at 1 mm P
i
was strongly voltage dependent (increasing from 32 mm at −70 mV to 77 mm at −30 mV) and the Hill coefficient was between 1 and 2, indicating cooperative binding of more than one Na+ ion. The maximum steady-state current was pH dependent, diminishing by 50% or more for a change from pH 7.8 to pH 6.3. Voltage
jumps elicited presteady-state relaxations in the presence of 96 mm Na+ which were suppressed at saturating P
i
(1 mm). Relaxations were absent in non-injected oocytes. Charge was balanced for equal positive and negative steps, saturated at
extremes of potential and reversed at the holding potential. Fitting the charge transfer to a Boltzmann relationship typically
gave a midpoint voltage (V
0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly
with the maximum P
i
-suppressed charge movement, indicating that the relaxations were NaPi-5-specific. The apparent transporter turnover was estimated
as 35 sec−1. The voltage dependence of the relaxations was P
i
-independent, whereas changes in Na+ shifted V
0.5 to −60 mV at 25 mm Na+. Protons suppressed relaxations but contributed to no detectable charge movement in zero external Na+. The voltage dependent presteady-state behavior of NaPi-5 could be described by a 3 state model in which the partial reactions
involving reorientation of the unloaded carrier and binding of Na+ contribute to transmembrane charge movement.
Received: 11 March 1997/Revised: 3 June 1997 相似文献
4.
The fumarate transport system of the bacterium Helicobacter pylori was investigated employing radioactive tracer analysis. The transport of fumarate at micromolar concentrations was saturable
with a K
M
of 220 ± 21 μm and V
max of 54 ± 2 nmole/min/mg protein at 20°C, depended on temperature between 4 and 40°C, and was susceptible to inhibitors, suggesting
the presence of one or more fumarate carriers. The release of fumarate from cells was also saturable with a K
M
of 464 ± 71 μm and V
max of 22 ± 2 nmol/min/mg protein at 20°C. The rates of fumarate influx at millomolar concentrations increased linearly with
permeant concentration, and depended on the age of the cells. The transport system was specific for dicarboxylic acids suggesting
that fumarate is taken up via dicarboxylate transporters. Succinate and fumarate appeared to form an antiport system. The
properties of fumarate transport were elucidated by investigating the effects of amino acids, monovalent cations, pH and potential
inhibitors. The results provided evidence that influx and efflux of fumarate at low concentrations from H. pylori cells was a carrier-mediated secondary transport with the driving force supplied by the chemical gradient of the anion. The
anaerobic C4-dicarboxylate transport protein identified in the genome of the bacterium appeared to be a good candidate for the fumarate
transporter.
Received: 11 December 1997/Revised: 7 May 1998 相似文献
5.
Blasco T Aramayona JJ Alcalde AI Halaihel N Sarasa M Sorribas V 《The Journal of membrane biology》2000,178(2):127-135
Renal reabsorption appears to play a major role in d-mannose homeostasis. Here we show that in rat kidney, the transport of d-mannose by brush border membrane vesicles from tubular epithelial cells involves an uphill and rheogenic Na-dependent system,
which is fully inhibited by d-mannose itself, incompletely inhibited by d-glucose, d-fructose, phloridzin, and phloretin, and noninhibited by l-mannose or disaccharides. In addition, this system exhibits both low capacity (112.9 ± 15.6 pmol/mg/second) and high affinity
(0.18 ± 0.04 mm), with a 2:1 stoichiometry for the Na:d-mannose interaction, and low affinity for sodium (16.6 ± 3.67 mm).
We also show expression of d-mannose transport by Xenopus laevis oocytes injected with rat renal polyA+ RNA. Kinetic analysis of the expressed transport was performed after RNA enrichment by fractionation through a sucrose density
gradient and was shown to be identical to that measured in membrane vesicles. The RNA species encoding the expressed transport
has a small mean size, 1 kb approximately, and shows no homology with the SGLT family of Na-dependent d-glucose transporters, as shown by low stringent RT-PCR and northern analysis. The expressed transport is specific for d-mannose, since in spite of a significant inhibition by d-glucose and d-fructose, neither of these two substrates was transported above the level of the water-injected oocytes.
Received: 29 February 2000/Revised: 25 August 2000 相似文献
6.
When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1
is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino
and carboxylic groups are both deprotonated. The pH value mainly influences the V
max
in Na+ activation curves and l-leucine kinetics. The kinetic parameters are K
mNa
= 4.6 ± 2 mm, V
maxNa
= 14.8 ± 1.7 pmol/oocyte/5 min for pH 8.0 and K
mNa
= 2.8 ± 0.7 mm, V
maxNa
= 31.3 ± 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K
m
= 120.4 ± 24.2 μm, V
max
= 23.2 ± 1.4 pmol/oocyte/5 min at pH 8.0 and K
m
= 81.3 ± 24.2 μm, V
max
= 65.6 ± 3.9 pmol/oocyte/5 min at pH 10.0.
On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group,
(ii) an unsubstituted α-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of
length and ramification.
Received: 27 April 1999/Revised: 10 January 2000 相似文献
7.
To study vacuolar chloride (Cl−) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl− uptake into isolated tonoplast vesicles was measured using the Cl−-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm,
respectively, and showed a high sensitivity towards Cl−, with a Stern-Volmer constant of 173 m
−1 in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched,
if at all, by other anions. However, the fluorescence intensity and Cl−-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as
the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K
m
of 17.2 mm and a V
max
of 4.8 mm min−1. Vacuolar Cl− transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm
p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl− transport was actually significantly increased by 24%. To determine absolute fluxes of Cl− using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be
1.13 × 107 m−1. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl− transport of 31 nmol m−2 sec−1 was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques.
Received: 18 February 2000/Revised: 30 June 2000 相似文献
8.
GABAA channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline
and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches).
The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application
of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay
that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased
with GABA concentration from less than 10 pS (0.5 μm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC
50 value of 33 μm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of
1 μm diazepam, the GABA EC
50 decreased to 0.2 μm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA
antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 μm GABA the bicuculline IC
50 was 19 μm. The effect of GABA on channel conductance shows that the role of the ligand in GABAA receptor channel function is more complex than previously thought.
Received: 23 October 2000/Revised: 27 February 2001 相似文献
9.
Mucosal crude microsomes, prepared from proximal rat small intestine, exhibited significant Mg-dependent, Zn-ATPase activity;
V
max
= 23 μmoles Pi/mg protein/hr, K
m
= 160 nm, and Hill Coefficient, n= 1.5. Partial purification (∼10-fold) was achieved by detergent extraction, and centrifugation through 250 mm sucrose: V
max
= 268 units, K
m
= 1 nm, and n= 6. In partially purified preparations, the assay was linear with time to 60 min, and with protein concentration to 1 μg/300
μl. Activities at pH 8 and 8.5 were higher than at pH 7.2. The ATP K
m
was 0.7 mm, with an optimal ATP/Mg ratio of ∼2. Ca elicited ATPase activity but did not augment the Zn-dependent activity. In partially
purified preparations, the homologous salts of Co, Cd, Cu, and Mn exhibited no detectable activity. Vanadate inhibition studies
yielded two component kinetics with a K
i
of 12 μm for the first component, and 96 μm for the second component, in partially purified preparations. Tissue distribution analyses revealed gradients of activity.
In the proximal half of the small intestine, Mg/Zn activity increased progressively from crypt to villus tip. In long axis
studies, this activity decreased progressively from proximal to distal small bowel.
Received: 12 September 2000/Revised 6 January 2001 相似文献
10.
We here report on studies on the frog skin epithelium to identify the nature of its excretory H+ pump by comparing transport studies, using inhibitors highly specific for V-ATPases, with results from immunocytochemistry
using V-ATPase-directed antibodies. Bafilomycin A1 (10 μm) blocked H+ excretion (69 ± 8% inhibition) and therefore Na+ absorption (61 ± 17% inhibition after 60 min application, n= 6) in open-circuited skins bathed on their apical side with a 1 mm Na2SO4 solution, ``low-Na+ conditions' under which H+ and Na+ fluxes are coupled 1:1. The electrogenic outward H+ current measured in absence of Na+ transport (in the presence of 50 μm amiloride) was also blocked by 10 μm bafilomycin A1 or 5 μm concanamycin A. In contrast, no effects were found on the large and dominant Na+ transport (short-circuit current), which develops with apical solutions containing 115 mm Na+ (``high-Na+ conditions'), demonstrating a specific action on H+ transport. In immunocytochemistry, V-ATPase-like immunoreactivity to the monoclonal antibody E11 directed to the 31-kDa subunit
E of the bovine renal V-ATPase was localized only in mitochondria-rich cells (i) in their apical region which corresponds
to apical plasma membrane infoldings, and (ii) intracellularly in their neck region and apically around the nucleus. In membrane
extracts of the isolated frog skin epithelium, the selectivity of the antibody binding was tested with immunoblots. The antibody
labeled exclusively a band of about 31 kDa, very likely the corresponding subunit E of the frog V-ATPase. Our investigations
now deliver conclusive evidence that H+ excretion is mediated by a V-ATPase being the electrogenic H+ pump in frog skin.
Received: 21 May 1996/Revised: 24 December 1996 相似文献
11.
Haspel HC Stephenson KN Davies-Hill T El-Barbary A Lobo JF Croxen RL Mougrabi W Koehler-Stec EM Fenstermacher JD Simpson IA 《The Journal of membrane biology》1999,169(1):45-53
Barbiturates inhibit GLUT-1-mediated glucose transport across the blood-brain barrier, in cultured mammalian cells, and in
human erythrocytes. Barbiturates also interact directly with GLUT-1. The hypotheses that this inhibition of glucose transport
is (i) selective, preferring barbiturates over halogenated hydrocarbon inhalation anesthetics, and (ii) specific, favoring
some GLUT-# isoforms over others were tested. Several oxy- and thio-barbiturates inhibited [3H]-2-deoxyglucose uptake by GLUT-1 expressing murine fibroblasts with IC50s of 0.2–2.9 mm. Inhibition of GLUT-1 by barbiturates correlates with their overall lipid solubility and pharmacology, and requires hydrophobic
side chains on the core barbiturate structure. In contrast, several halogenated hydrocarbons and ethanol (all ≤10 mm) do not significantly inhibit glucose transport. The interaction of these three classes of anesthetics with purified GLUT-1
was evaluated by quenching of intrinsic protein fluorescence and displayed similar specificities and characteristics. The
ability of barbiturates to inhibit other facilitative glucose transporters was determined in cell types expressing predominantly
one isoform. Pentobarbital inhibits [3H]-2-deoxyglucose and [14C]-3-O-methyl-glucose uptake in cells expressing GLUT-1, GLUT-2, and GLUT-3 with IC50s of ∼1 mm. In contrast, GLUT-4 expressed in insulin-stimulated rat adipocytes was much less sensitive than the other isoforms to inhibition
by pentobarbital (IC50 of >10 mm). Thus, barbiturates selectively inhibit glucose transport by some, but not all, facilitative glucose transporter isoforms.
Received: 10 November 1998/Revised: 3 February 1999 相似文献
12.
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
13.
M.-P. Blanchard N. Klauke S. Zitzmann H. Plattner 《The Journal of membrane biology》1999,169(3):155-165
We analyzed [Ca2+]
i
transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben
& Plattner, 1994. J. Cell Biol.
127:935–945; Plattner et al., 1994. J. Membrane Biol.
158:197–208). Wild-type cells (7S), nondischarge strain nd9–28°C and trichocyst-free strain ``trichless' (tl), respectively,
displayed similar, though somewhat diverging time course and plateau values of [Ca2+]
i
transients with moderate [Ca2+]
o
in the culture/assay fluid (50 μm or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca2+]
o
= 30 nm (c.f. [Ca2+]
rest
i
=∼50 to 100 nm), veratridine produced only a small cortical [Ca2+]
i
transient. This increased in size and spatial distribution at [Ca2+]
o
= 50 μm of 1 mm. Interestingly with unusually high yet nontoxic [Ca2+]
o
= 10 mm, [Ca2+]
i
transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With
[Ca2+]
o
= 30 nm, the restricted residual response observed is due to Ca2+ mobilization from subplasmalemmal stores. (ii) With moderate [Ca2+]
o
= 50 μm to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca2+
o influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl
cells, where free docking sites allow for rapid Ca2+ spread, and least in 7S cells, whose perfectly assembled docking sites may ``consume' a large part of the [Ca2+]
i
increase. (iii) With unusually high [Ca2+]
o
, mobilization of cortical stores and/or Ca2+
o
influx may be impeded by the known membrane stabilizing effect of Ca2+
o
counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and,
hence, not to be toxic side-effects, as confirmed by retention of injected calcein.
(v) Finally, Mn2+ entry during veratridine stimulation, documented by Fura-2 fluorescence quenching, may indicate activation of unspecific
Me2+ channels by veratridine. Our data have some bearing on analysis of other cells, notably neurons, whose response to veratridine
is of particular and continous interest.
Received: 8 December 1998/Revised: 2 March 1999 相似文献
14.
The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using
continuous short circuit current (I
SC
) measurements in HCO3
−/CO2 buffered solution. Steady state I
SC
averaged 73.8 ± 3.2 μA/cm2 (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na+ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y+ and a basal amino acid transport system y+L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic
amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I
SC
averaging 36.6 ± 5.4 μA/cm2 (n= 19) and 32.0 ± 7.2 μA/cm2 (n= 8), respectively. The response was preserved in the absence of bath Cl− (n= 4), but was abolished either in the absence of apical Na+ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I
SC
response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization
of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na+ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract
passive cationic amino acid leak into the CCD in the presence of electrogenic Na+ transport, consistent with reports of stimulated expression of Na+ and cationic amino acid transporters by aldosterone.
Received: 11 September 2000/Revised: 6 December 2000 相似文献
15.
Cobra venom cytotoxins (CTX) have been shown to disrupt cells as different as immunocytes, skeletal myocytes, erythrocytes
and tumor cells. Nevertheless, even subpopulations of tumor cells are differentially susceptible to CTX by an order of magnitude.
In the present study, our objective was to compare CTX-specific binding with cytolytic potency for two disparate cell types
in vitro. We investigated the lytic activity of cytotoxin-III from Naja naja atra (NNA, fraction D) using heart cells and human leukemic T-cells (CEM cells). For both cell types, 50% cytolysis, assessed
by tetrazolium dye conversion, occurred with μm concentrations of toxin (EC50= 2.2 μm). We examined the binding of radiolabeled CTX III to both heart cells and CEM cells and found the apparent dissociation constant
(K
Dapp) to be 0.69 μm and 0.75 μm, for CEM and heart cells respectively. The B
max for the CEM cells was 1.0 fmoles/cell and that for heart cells was 5.2 fmoles/cell, both exhibiting positive cooperativity
between the sites (Hill coefficients 1.4, T-cells; 1.6, heart). Relatively modest dissociation constants plus high numbers
of binding sites per cell are consistent with a model of CTX binding to plasma membranes by interaction with phospholipids
in the bilayer. Our results suggest that the lytic activity of this cytotoxin follows its binding to a population of sites
on the cells in a cooperative fashion.
Received: 8 May 1995/Revised: 17 November 1995 相似文献
16.
R. Fernández J.R. Bosqueiro A.C. Cassola G. Malnic 《The Journal of membrane biology》1997,157(2):193-201
The presence of an electrogenic H+-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies
the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting
duct. Transepithelial PD (V
t
) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion.
The tubules were perfused with electrolyte solutions to which 2 × 10−7
m bafilomycin or 4.6 × 10−8
m concanamycin were added. No significant increase in lumen-negative V
t
upon perfusion with these inhibitors as compared to control, was observed as well as when 10−3
m amiloride, 10−5
m benzamil or 3 mm Ba2+ were perfused alone or in combination. The effect of an inhibition of electrogenic H+ secretion, i.e., increase in lumen-negative V
t
by 2–4 mV, was observed only when Cl− channels were blocked by 10−5
m 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this
segment from 1.21 ± 0.14 (n= 8) to 0.62 ± 0.03 (8) nmol.cm−2.sec−1 as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate
that: (i) the participation of the vacuolar H+ ATPase in the establishment of cortical late distal tubule V
t
is minor in physiological conditions, but can be demonstrated after blocking Cl− channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H+ secretion in this segment is reduced by a Cl− channel blocker, supporting coupling of H+-ATPase with Cl− transport.
Received: 6 July 1996/Revised: 27 December 1996 相似文献
17.
Rectification of the Olfactory Cyclic Nucleotide-Gated Channel by Intracellular Polyamines 总被引:5,自引:0,他引:5
J.W. Lynch 《The Journal of membrane biology》1999,170(3):213-227
Polyamine-induced inward rectification of cyclic nucleotide-gated channels was studied in inside-out patches from rat olfactory
neurons. The polyamines, spermine, spermidine and putrescine, induced an `instantaneous' voltage-dependent inhibition with
K
d
values at 0 mV of 39, 121 μm and 2.7 mm, respectively. Hill coefficients for inhibition were significantly < 1, suggesting an allosteric inhibitory mechanism. The
Woodhull model for voltage-dependent block predicted that all 3 polyamines bound to a site 1/3 of the electrical distance
through the membrane from the internal side. Instantaneous inhibition was relieved at positive potentials, implying significant
polyamine permeation. Spermine also induced exponential current relaxations to a `steady-state' impermeant level. This inhibition
was also mediated by a binding site 1/3 of the electrical distance through the pore, but with a K
d
of 2.6 mm. Spermine inhibition was explained by postulating two spermine binding sites at a similar depth. Occupation of the first
site occurs rapidly and with high affinity, but once a spermine molecule has bound, it inhibits spermine occupation of the
second binding site via electrostatic repulsion. This repulsion is overcome at higher membrane potentials, but results in
a lower apparent binding affinity for the second spermine molecule. The on-rate constant for the second spermine binding saturated
at a low rate (∼200 sec−1 at +120 mV), providing further evidence for an allosteric mechanism. Polyamine-induced inward rectification was significant
at physiological concentrations.
Received: 17 February 1999/Revised: 27 April 1999 相似文献
18.
Isoform-Specific Function and Distribution of Na/K Pumps in the Frog Lens Epithelium 总被引:3,自引:1,他引:2
Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies
of the Na/K ATPase. RNase protection assays showed that all cells express α1- and α2-isoforms of the Na/K pump but not the α3-isoform, however the α2-isoform dominates in anterior cells whereas the α1-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the
Na/K pump current (I
P
), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I
P
blockade data indicate the α1-isoform has a dissociation constant of 100 μm DHO whereas for the α2-isoform it is 0.75 μm DHO. Both α1- and α2-isoforms are half maximally activated at an intracellular Na+-concentration of 9 mm. The α1-isoform is half maximally activated at an extracellular K+-concentration of 3.9 mm whereas for the α2-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the α1-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the α2-isoform. Under normal physiological conditions, I
P
in equatorial cells is approximately 0.23 μA/μF, and in anterior cells it is about 0.14 μA/μF. These current densities refer
to the area of cell membrane assuming a capacitance of around 1 μF/cm2. Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 μA/cm2 at the equator vs. 0.5 μA/cm2 at the anterior pole.
Received: 17 May 2000/Revised: 11 August 2000 相似文献
19.
Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells 总被引:5,自引:0,他引:5
The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings
of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of
channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K
Ca
) channel and (iii) a small K+ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear
current-voltage (I-V) relationship (27 pS) and was equally conductive for Na+ and K+, but not permeable to Cl− or N-methyl-d-glucamine. Channel activity required the presence of Ca2+ at the cytosolic face, but was detected at Ca2+ concentrations as low as 10−7
m (open probability (P
o
) = 0.11 ± 0.03, n= 8). Gadolinium decreased P
o
of the NSC channel from both the external and cytosolic side (IC50∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K+ could be fitted to the I-V relationship in asymmetrical K+ and Na+ solutions. The channel was impermeable to Cl− and N-methyl-d-glucamine. P
o
of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca2+. TEA (20 mm), charybdotoxin (100 nm) and Ba2+ (1 mm) but not amiloride (1 mm) reduced P
o
from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P
o
and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward
current. We conclude that the NSC channel is Ca2+ activated, voltage-insensitive and involved in both constitutive K+ and Na+ reabsorption from endolymph while the BK channel might participate in the K+ pathway under stimulated conditions that produce an elevated intracellular Ca2+ or depolarized membrane potential.
Received: 14 October 1999/Revised: 10 December 1999 相似文献
20.
The relationships between currents generated by the rabbit Na+/glucose cotransporter (SGLT1) and the fluxes of Na+ and sugar were investigated using Xenopus laevis oocytes expressing SGLT1. In individual voltage-clamped oocytes we measured: (i) the current evoked by 10 mmαMG and the 22Na+ uptake at 10 mm Na+; (ii) the currents evoked by 50 to 500 μm [14C]αMG and the [14C]αMG uptakes at 100 mm Na+; and (iii) phlorizin-sensitive leak currents in the absence of sugar and 22Na+ uptakes at 10 mm Na+. We demonstrate that the SGLT1 leak currents are Na+ currents, and that the sugar-evoked currents are directly proportional to both αMG and Na+ uptakes. The Na+/αMG coupling coefficients were estimated to be 1.6 at −70 mV and 1.9 at −110 mV. This suggests that the rabbit SGLT1 Na+/αMG stoichiometry for sugar uptake is 2 under fully saturating, zero-trans conditions. Coupling coefficients of less than 2 are expected under nonsaturating conditions due to uncoupled Na+ fluxes (slippage). The similarity between the Na+ Hill coefficients and the coupling coefficients suggests strong cooperativity between the two Na+ binding sites.
Received: 6 October 1997/Revised: 5 December 1997 相似文献