共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
Mo L Hellmich HL Fong P Wood T Embesi J Wills NK 《The Journal of membrane biology》1999,168(3):253-264
Loss of function mutations of the renal chloride channel, ClC-5, have been implicated in Dent's disease, a genetic disorder
characterized by low weight proteinuria, hypercalciuria, nephrolithasis and, in some cases, eventual renal failure. Recently,
our laboratory used an RT-PCR/RACE cloning strategy to isolate an amphibian cDNA from the renal epithelial cell line A6 that
had high homology to human ClC-5. We now report a full-length native ClC-5 clone (xClC-5, containing 5′ and 3′ untranslated
regions) isolated by screening a cDNA library from A6 cells that was successfully expressed in Xenopus oocytes. In addition, we compared the properties of xClC-5 and hClC-5 using isogenic constructs of xClC-5 and hClC-5 consisting
of the open reading frame subcloned into an optimized Xenopus expression vector. Expression of the full-length ``native'
xClC-5 clone resulted in large, strongly rectifying, outward currents that were not significantly affected by the chloride
channel blockers DIDS, DPC, and 9AC. The anion conductivity sequence was NO−
3 > Cl−= I− > HCO−
3 >> glutamate for xClC-5 and NO−
3 > Cl− > HCO−
3 > I− >> glutamate for hClC-5. Reduction of the extracellular pH (pH
o
) from 7.5 to 5.7 inhibited outward ClC-5 currents by 27 ± 9% for xClC-5 and 39 ± 7% for hClC-5. The results indicate that
amphibian and mammalian ClC-5 have highly similar functional properties. Unlike hClC-5 and most other ClC channels, expression
of xClC-5 in oocytes does not require the removal of its untranslated 5′ and 3′ regions. Acidic solutions inhibited both amphibian
and human ClC-5 currents, opposite to the stimulatory effects of low external pH on other ClC channels, suggesting a possibly
distinct regulatory mechanism for ClC-5 channels.
Received: 28 August 1998/Revised: 13 January 1999 相似文献
3.
T. Ishikawa 《The Journal of membrane biology》1996,153(2):147-159
A Ca2+-activated Cl− conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When
the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca2+ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of
the cells with pipette solutions containing 300 nm or less than 1 nm free Ca2+ strongly reduced the Cl− currents, indicating the currents were Ca2+-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent.
The anion permeability sequence of the Cl− channels was: NO−
3 (2.00) > I− (1.85) ≥ Br− (1.69) > Cl− (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette
solutions was increased from 0 to 10 mm, the Ca2+-dependency of the Cl− current amplitude shifted to lower free Ca2+ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca2+-activated Cl− currents. This suggests that Ca2+/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca2+-activated Cl− currents. The outward Cl− currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable
Cl− conductance controlled by cytosolic Ca2+ and ATP levels in rat submandibular acinar cells.
Received: 9 January 1996/Revised: 20 May 1996 相似文献
4.
Akihiro Inagaki Soichiro Yamaguchi Hiromi Takahashi-Iwanaga Toshihiko Iwanaga Toru Ishikawa 《The Journal of membrane biology》2010,235(1):27-41
ClC-2, a member of the voltage-gated Cl− channel family, is expressed in the distal colonic surface epithelial cells of various species, but its functional significance
remains unclear. Here, by means of electrophysiological and molecular biological techniques, we have identified and characterized
a ClC-2-like conductance naturally expressed by surface epithelial cells acutely dissociated from rectal colon of rats fed
a standard diet. Whole-cell patch-clamp experiments showed that the surface cells, whether an amiloride-sensitive Na+ conductance was present or not, displayed a strong hyperpolarization-activated, inwardly rectifying Cl− current. Analysis both by in situ hybridization and immunohistochemistry confirmed the expression of ClC-2 in the rectal
surface epithelium. The native Cl− current shared common electrophysiological properties including voltage-dependent activation, anion selectivity sequence,
and Zn2+ sensitivity with that recorded from HEK293 cells transfected with ClC-2 cloned from rat rectal colon (rClC-2). Cell-attached
patch recordings on the surface cells revealed that native ClC-2-like currents activated only at potentials at least 40 mV
more negative than resting membrane potentials. In Ussing chamber experiments with rat rectal mucosa, either basolateral or
apical application of Zn2+ (0.1 mM), which inhibited both native ClC-2-like currents and recombinant rClC-2 currents, had little, if any, effects on
basal amiloride-sensitive short-circuit current. Collectively, these results not only demonstrate that a functional ClC-2-type
Cl− channel is expressed in rat rectal surface epithelium, but also suggest that the channel activity may be negligible and thus
nonessential for controlling electrogenic Na+ transport in this surface epithelium under basal physiological conditions. 相似文献
5.
Phosphorylation-dependent events have been shown to modulate the activity of several members of the mammalian CLC Cl− channel gene family, including the inward rectifier ClC-2. In the present study we investigated the regulation of rat ClC-2
expressed in the TSA-201 cell line (a transformed HEK293 cell line that stably expresses the SV40 T-antigen) by protein kinases.
Protein kinase A activation phosphorylated ClC-2 in vivo, whereas stimulation of protein kinase C with phorbol 12-myristate
13-acetate did not. In vitro labeling studies confirmed that protein kinase A could directly phosphorylate ClC-2, and that
protein kinase C and Ca2+/calmodulin-dependent protein kinase II did not. Nevertheless, protein kinase A-dependent phosphorylation of CLC-2 failed
to regulate either the magnitude or the kinetics of the hyperpolarization-activated Cl− currents. Considered together, we demonstrate that protein kinase A activation results in the phosphorylation of rat ClC-2
in vivo, but this event is independent of Cl− channel activity.
Received: 20 November 2000/Revised: 28 March 2001 相似文献
6.
Shenghao Liu Xiaohang Huang Chenlin Liu Bailin Cong Guang Han 《Journal of applied phycology》2008,20(1):29-34
Whole-cell patch-clamp techniques were used to study ion channels of a marine alga. High quality protoplasts suitable for
electrophysiological studies were isolated from the green marine alga, Ulva pertusa, using enzyme mixtures consisting of cellulase and abalone power and identified by calcofluor fluorescence. The vitality
of protoplasts varied depending on the alga growth stage, and those isolated from younger tissue in March maintained a high
vitality with high sealing success rate compared with protoplasts isolated from mature or non-growing plants in August or
November. In the whole-cell configuration, large inward currents were elicited by negative voltage pulses. The voltage-dependent
component was predominantly carried by Cl−, as confirmed by the use of the Cl− channel inhibitor DIDS and reversal potential of current-voltage plots. This evidence suggests that hyperpolarization-activated
Cl− permeable channels are responsible for the influx of Cl− into U. pertusa cells. Voltage-dependent outward currents were also recorded in several protoplasts, and their properties need further investigation. 相似文献
7.
Using the whole-cell patch-clamp technique, we examined Cl−-selective currents manifested by strial marginal cells isolated from the inner ear of gerbils. A large Cl−-selective conductance of ∼18 nS/pF was found from nonswollen cells in isotonic buffer containing 150 mm Cl−. Under a quasi-symmetrical Cl− condition, the `instantaneous' current-voltage relation was close to linear, while the current-voltage relation obtained
at the end of command pulses of duration 400 msec showed weak outward rectification. The permeability sequence for anionic
currents was as SCN− > Br−≅ Cl− > F− > NO−
3≅ I− > gluconate−, corresponding to Eisenmann's sequence V. When whole-cell voltage clamped in isotonic bathing solutions, the cells exhibited
volume changes that were accounted for by the Cl− currents driven by the imposed electrochemical potential gradients. The volume change was elicited by lowered extracellular
Cl− concentration, anion substitution and altered holding potentials. The Cl− conductance varied in parallel with cell volume when challenged by bath anisotonicity. The whole-cell Cl− current was only partially blocked by both 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 0.5 mm) and diphenylamine-2-carboxylic acid (DPC, 1.0 mm), but 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS, 0.5 mm) was without effect. The properties of the present whole-cell Cl− current resembled those of the single Cl− channel previously found in the basolateral membrane of the marginal cell (Takeuchi et al., Hearing Res.
83:89–100, 1995), suggesting that the volume-correlated Cl− conductance could be ascribed predominantly to the basolateral membrane. This Cl− conductance may function not only in cell volume regulation but also for the transport of Cl− and the setting of membrane potential in marginal cells under physiological conditions.
Received: 15 August 1995/Revised: 3 November 1995 相似文献
8.
9.
10.
Recent studies have suggested a role for a chloride current in the modulation of pacemaker potentials generated by interstitial
cells of Cajal. Patch-clamp recordings were made from inside–out patches of cultured interstitial cells of Cajal from the
murine small intestine. The majority of patches were quiescent immediately after excision, but in some patches currents activated
spontaneously after a period of 10 min to 1 h. Currents could also be activated by strongly polarizing the patch. It was found
that the currents activated in both cases included a chloride channel. This channel could also be activated by ATP and the
catalytic subunit of protein kinase A. The channel had conductance states (±SD) of 53 ± 25.35, 126 ± 21.44, 180 ± 12.57 and
211 ± 8.86 pS. It was outwardly rectifying (as a function of open probability) and deactivated (i.e., gave a tail current)
but showed no inactivation. The permeability sequence of the channel was I−>>Br−≥Cl−>Asp−. It was unaffected in magnitude or rectification by changing the free Ca2+ concentration of the bath between <10 nm, 100 nm (control) and 2 mm. 相似文献
11.
Single inward rectifier K+ channels were studied in Xenopus laevis embryonic myocytes. We have characterized in detail the channel which is most frequently observed (Kir) although we routinely
observe three other smaller current levels with the properties of inward rectifier K+ channels (Kir(0.3), Kir(0.5) and Kir(0.7)). For Kir, slope conductances of inward currents were 10.3, 20.3, and 27.9 pS, in 60, 120 and 200 mM [K+]
o
respectively. Extracellular Ba2+ blocked the normally high channel activity in a concentration-dependent manner (K
A
= 7.8 μm, −90 mV). In whole-cell recordings of inward rectifier K+ current, marked voltage dependence of Ba2+ block over the physiological range of potentials was observed. We also examined current rectification. Following step depolarizations
to voltages positive to E
K
, outward currents through Kir channels were not observed even when the cytoplasmic face of excised patches were exposed to
Mg2+-free solution at pH 9.1. This was probably also true for Kir(0.3), Kir(0.5) and Kir(0.7) channels. We then examined the possibility of modulation of Kir channel activity and found neither ATP nor GTP-γS had any
effect on Kir channel activity when added to the solution perfusing the cytoplasmic face of a patch. Kinetic analysis revealed
Kir channels with a single open state (mean dwell time 72 msec) and two closed states (time constants 1.4, 79 msec). These
results suggest that the native Kir channels of Xenopus myocytes have similar properties to the cloned strong inward rectifier K+ channels, in terms of conductance, kinetics and barium block but does show some differences in the effects of modulators
of channel activity. Furthermore, skeletal muscle may contain either different inward rectifier channels or a single-channel
type which can exist in stable subconductance states.
Received: 16 September 1996/Revised: 14 March 1997 相似文献
12.
Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through
the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to
investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles.
Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex.
These currents are essentially mediated by K+ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage
and inhibited by extracellular Ca2+ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca2+-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various
models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As
a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive
states via independent first order reactions: a fast inactivation/activation by Ca2+-binding and -release, respectively (rate constants >>103 sec−1) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the
range of 103 sec−1).
With 10 mm K+ and 1 mm Ca2+ in the external solution, intact coleoptile cells have a membrane voltage (V) of −105 ± 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K+ uptake required for cell elongation. Extracellular TEA or Ca2+, which inhibit the K+ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects
of Ca2+ and TEA on both processes and the similar Ca2+ concentration required for half maximal inhibition of growth (4.3 mm Ca2+) and for conductance (1.2 mm Ca2+) suggest that K+ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles.
Received: 6 June 1995/Revised: 12 September 1995 相似文献
13.
Swelling-activated Cl− currents (I
Cl,swell
) have been characterized in a mouse renal inner medullary collecting duct cell line (mIMCD-K2). Currents activated by exposing
the cells to hypotonicity exhibited characteristic outward rectification and time- and voltage-dependent inactivation at positive
potentials and showed an anion selectivity of I− > Br− > Cl− > Asp−. NPPB (100 μm) inhibited the current in a voltage independent manner, as did exposure to 10 μm tamoxifen and 500 μm niflumic acid (NFA). In contrast, DIDS (100 μm) blocked the current with a characteristic voltage dependency. These characteristics of I
Cl,swell
in mIMCD-K2 cells are essentially identical to those of heterologously expressed cardiac CLC-3.
A defining feature of CLC-3 is that activation of PKC by PDBu inhibits the conductance. In mIMCD-K2 cells preincubation with
PDBu (100 nm) prevented the activation of I
Cl,swell
by hypotonicity. However, PDBu inhibition of I
Cl,swell
was reversed after PDBu withdrawal, but this was refractory to subsequent PDBu inhibition. Activation of either the cystic
fibrosis transmembrane conductance regulator (CFTR) or Ca2+ activated Cl− conductance (CaCC), which are coexpressed in mIMCD-K2 cells prior to PDBu treatment, abolished the PDBu inhibition of I
Cl,swell
. Control of I
Cl,swell
by PKC therefore depends on the physiological status of the cell.
In intact mIMCD-K2 layers in Ussing chambers, forskolin stimulation of an inward short-circuit current (due to transepithelial
Cl− secretion via apical CFTR) was inhibited by cell swelling upon hypotonic exposure at the basolateral surface. Activation
of I
Cl,swell
is therefore capable of regulating transepithelial Cl− secretion and suggests that I
Cl,swell
is located at the basolateral membrane. PDBu exposure prior to or during hypotonic challenge was ineffective in reversing
the swelling-activated inhibition of Cl− secretion, but tamoxifen (100 μm) abolished the hypotonic inhibition of forskolin-stimulated short-circuit current (I
sc
).
RT-PCR analysis confirmed expression of mRNA for members of the CLC family, including both CLC-2 and 3, in the mIMCD-K2 cell
line.
Received: 24 February 2000/Revised: 26 May 2000 相似文献
14.
15.
Melanoma cells are transformed melanocytes of neural crest origin. K+ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize
ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify
voltage-gated (KV), Ca2+-activated (KCa), and inwardly rectifying (KIR) K+ channels; swelling-sensitive Cl− channels (Clswell); voltage-gated Ca2+ channels (CaV) and Ca2+ channels activated by depletion of intracellular Ca2+ stores (CRAC); and voltage-gated Na+ channels (NaV). The presence of Ca2+ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca2+]
i
. The expression of K+ channels varied widely between different cell lines and was also influenced by culture conditions. KIR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for KIR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). KCa channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). KCa channels in C8146 and SK28 cells were sensitive to CTX (K
d
= 4 nm), but were unaffected by apamin. KV channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed
CRAC channels and a novel Clswell channel. Clswell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1
in symmetrical Cl−). We conclude that different melanoma cell lines express a diversity of ion channel types.
Received: 2 April 1996/Revised: 22 August 1996 相似文献
16.
Acid-sensitive outwardly rectifying anion channels (ASOR) have been described in several mammalian cell types. The present
whole-cell patch-clamp study elucidated whether those channels are expressed in erythrocytes. To this end whole-cell recordings
were made in human erythrocytes from healthy donors treated with low pH and high osmotic pressure. When the pipette solution
had a reduced Cl− concentration, treatment of the cells with Cl−-containing normal and hyperosmotic (addition of sucrose and polyethelene glycol 1000 [PEG-1000] to the Ringer) media with
low pH significantly increased the conductance of the cells at positive voltages. Channel activity was highest in the PEG-1000
media (95 and 300 mM PEG-1000, pH 4.5 and 4.3, respectively) where the current–voltage curves demonstrated strong outward
rectification and reversed at −40 mV. Substitution of the Cl−-containing medium with Cl−-free medium resulted in a decrease of the conductance at hyperpolarizing voltages, a shift in reversal potential (to 0 mV) and loss of outward rectification. The chloride currents were inhibited by chloride channels blockers DIDS and NPPB (IC50 for both was ~1 mM) but not with niflumic acid and amiloride. The observations reveal expression of ASOR in erythrocytes. 相似文献
17.
Inward rectification by hyperpolarization-activated Na current in the marine ciliate Euplotes vannus
Thomas Krüppel 《The Journal of membrane biology》1993,133(3):263-270
Summary The ionic mechanisms underlying inward or anomalous rectification have been studied in the marine hypotrichous ciliate Euplotes vannus. Inward-current pulses of moderate amplitude elicited time-dependent rectification that started from a hyperpolarization peak and was expressed as a depolarizing sag towards rest. Voltage-clamp analysis showed that this depolarization is caused by the activation of a complex inward current that does not inactivate with time. The current is carried by a major Na and a minor K component. The Na-current component has been identified by its concentration-dependent reduction in low extra-cellular Na solutions and the capability of Li2+ as Na substitute to carry the current, though with a slightly reduced amplitude. The K-current component has been isolated from the total current after the replacement of Na2+ within the experimental solution. It was blocked in media that contained 10 mmol/liter TEA, a well-known blocker for K inwardly rectifying currents. TEA was only effective at membrane potentials close to or negative to the potassium equilibrium potential. The inward current was reduced after the injection of the Ca chelator EGTA into the cell. Also the elimination of the ciliary membrane, by deciliating cells with ethanol, reduced the amplitude of the inwardly rectifying currents. Both experiments indicate a regulatory function of Ca22+ in inward rectification.The author is grateful to Harald Mikoleit for technical assistance and preparing the figures and to Prof. W. Lueken for his critical comments. This work was supported by Deutsche Forschungsgemeinschaft, SFB 171, C7. 相似文献
18.
The rat primary cultured-airway monolayer had been an excellent model for deciphering the ion channel after nystatin permeabilization
of its basolateral or apical membrane (Hwang et al., 1996). After apical membrane permeabilization of rat primary cultured-airway
monolayer, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive outwardly rectifying depolarization-induced
Cl− (BORDIC) currents were observed across the basolateral membrane in symmetrical NMG-Cl solution in this study. No significant
Cl− current induced by the application of voltage clamping was observed across the apical membrane in symmetrical NMG-Cl solution
after basolateral membrane permeabilization. The halide permeability sequence for BORDIC current was Br−≒ I− > Cl−. BORDIC current was not affected by basolaterally applied bumetanide (0.5 mm). Basolateral DIDS (0.2 mm) but not apical DIDS inhibited CFTR mediated short-circuit current (I
sc
) in an intact monolayer of rat airway epithelia, a T84 human colonal epithelial cell line, and a Calu-3 human airway epithelial
cell line. This is the first report showing that depolarization induced Cl− current is present on the basolateral membrane of airway epithelia.
Received: 7 October 1999/Revised: 24 April 2000 相似文献
19.
Sitsapesan R 《The Journal of membrane biology》1999,168(2):159-168
Tonoplast K+ channels of Chara corallina are well characterized but only a few reports mention anion channels, which are likely to play an important role in the tonoplast
action potential and osmoregulation of this plant. For experiments internodal cells were isolated. Cytoplasmic droplets were
formed in an iso-osmotic bath solution according to a modified procedure. Ion channels with conductances of 48 pS and 170
pS were detected by the patch-clamp technique. In the absence of K+ in the bath solution the 170 pS channel was not observed at negative pipette potential values. When Cl− on either the vacuolar side or the cytoplasmic side was partly replaced with F−, the reversal potential of the 48 pS channel shifted conform to the Cl− equilibrium potential with similar behavior in droplet-attached and excised patch mode. These results showed that the 48
pS channel was a Cl− channel. In droplet-attached mode the channel rectified outward current flow, and the slope conductance was smaller. When
Chara droplets were formed in a bath solution containing low (10−8
m) Ca2+, then no Cl− channels could be detected either in droplet-attached or in inside-out patch mode. Channel activity was restored if Ca2+ was applied to the cytoplasmic side of inside-out patches. Rectification properties in the inside-out patch configuration
could be controlled by the holding pipette potential. Holding potential values negative or positive to the calculated reversal
potential for Cl− ions induced opposite rectification properties. Our results show Ca2+-activated Cl− channels in the tonoplast of Chara with holding potential dependent rectification.
Received: 30 March 1999/Revised: 10 August 1999 相似文献
20.
Chloride (Cl−) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole
cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl2, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl− current activated by 10−5
m forskolin, 10−3
m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10−6
m phorbol 12-myristate 13-acetate (PMA), 10−3
m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10−7
m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br− > Cl−≫ I− > glutamate. This current was inhibited by 10−3
m diphenylamine-2-carboxylate (DPC) and 10−4
m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10−3
m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic
fibrosis transmembrane conductance regulator (CFTR) Cl− conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl− current blocked by DIDS. To determine the exact location of the Cl− conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded
on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl− removal from the apical solution induced a Cl− efflux which was stimulated by 10−5
m forskolin, 10−7 calcitonin and inhibited by 10−5
m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br− influx through the Cl− pathway. Forskolin and calcitonin had no effect on the basolateral Cl− permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl− conductance in the apical membrane through a cAMP-dependent mechanism.
Received: 5 July 1995/Revised: 21 December 1995 相似文献