CFTR-dependent and -independent swelling-activatedK currents in primary cultur - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:34 |只看该作者 |倒序浏览 |打印

作者：RadiaBelfodil, HervéBarrière, IsabelleRubera, MichelTauc, ChantalPoujeol, MichelBidet,  PhilippePoujeol作者单位：Unité Mixte de Recherche Centre National de la RechercheScientifique 6548 Université de Nice-Sophia Antipolis, O6108Nice Cedex  France $ q# G7 z" ?1 e- O0 T
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      【摘要】
   The role of CFTR inthe control of K currents was studied in mouse kidney.Whole cell clamp was used to identify K currents on thebasis of pharmacological sensitivities in primary cultures of proximal(PCT) and distal convoluted tubule (DCT) and cortical collecting tubule(CCT) from wild-type (WT) and CFTR knockout (KO) mice. In DCT and CCTcells, forskolin activated a 293B-sensitive K current inWT, but not in KO, mice. In these cells, a hypotonic shock inducedK currents blocked by charybdotoxin in WT, but not in KO,mice. In PCT cells from WT and KO mice, the hypotonicity-inducedK currents were insensitive to these toxins and wereactivated at extracellular pH 8.0 and inhibited at pH 6.0, suggestingthat the corresponding channel was TASK2. In conclusion, CFTR isimplicated in the control of KCNQ1 and Ca 2  -sensitiveswelling-activated K conductances in DCT and CCT, but notin proximal convoluted tubule, cells. In KO mice, impairment of theregulatory volume decrease process in DCT and CCT could be due to theloss of an autocrine mechanism, implicating ATP and adenosine, whichcontrols swelling-activated Cl and K channels. $ A0 y1 V" j# W: j
      【关键词】 kidney cystic fibrosis regulatory volume decrease cell volume calcium level
   INTRODUCTION

CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) is not only aCl channel sensitive to cAMP in the epithelia, but it isalso a modulator of other ion channels or transporters( 1 ). CFTR interacts with different K channels, such as ROMK ( 11, 19 ) or KCNQ1 ( 2 ),and probably also with K channels implicated in thecontrol of cell volume ( 30, 32 ). However, the nature ofthese interactions is often open to discussion. For instance, theinteraction could be direct by protein-protein interaction, as in thecase of ROMK2 ( 4, 19, 20 ), or indirect, by control of cAMPsensitivity, as for KCNQ1 ( 2 ). Concerning theswelling-activated K channel, modulation by CFTR couldoccur via control of the intracellular Ca 2 , because thisK channel could be a the Ca 2  -dependentK channel ( 32 ). It is clear that exactknowledge of the type of interactions that occur between CFTR and itspartners is important, inasmuch as it facilitates an understanding ofthe defects of ion handling in cystic fibrosis (CF) and,therefore, a possible treatment of the disease. The kidney is a usefultool for studying CFTR control of K conductance, inasmuchas there is a differential expression of K channels alongthe nephron, together with a differential expression of CFTR. In thecase of CFTR, despite the presence of cftr transcripts inproximal (PCT) and distal convoluted tubules (DCT), CFTR expression, along with cAMP-sensitive Cl conductance, was found inthe distal tubule only ( 21, 27 ). However, CFTR-dependentswelling-activated Cl channels were recorded in bothsegments. It was therefore interesting to study whether differentK channels are associated with CFTR-dependentCl channels in PCT and DCT. For this purpose, we carriedout patch-clamp experiments in primary cultures of PCT, DCT, andcortical collecting tubule (CCT) from wild-type and cftr / mice. The results demonstrate that, in DCT and CCTcells, CFTR is indispensable for the expression of a cAMP-sensitiveKCNQ1 conductance and for the activation of aCa 2  -dependent swelling-activated K conductance. However, regulation of these currents is quite different, because, in cftr / DCT or CCT cells, the cAMP-sensitivecurrent could not be restored by cAMP application, whereas theswelling-activated current had been restored by extracellular adenosineperfusion. This suggests that, in the distal tubule, KCNQ1 functiondepends on the integrity of CFTR, whereas the swelling-activatedK channel depends on the integrity of an upstreamregulatory mechanism implicating CFTR. As for the swelling-activatedCl conductance ( 25 ), such a mechanism couldinvolve an autocrine ATP release ( 3 ) followed byhydrolysis of ATP in adenosine and an adenosine-activatedCa 2  influx that finally activatesCa 2  -sensitive maxi and small K (BK and SK,respectively) channels. The absence of this cascade in cftr / epithelia explains why DCT and CCT cells areunable to undergo regulatory volume decrease (RVD) after a hypotonic shock. In contrast, in PCT, CFTR does not control swelling-activated K conductance, inasmuch as these channels belong to theTASK2 family, the expression of which is not related to CFTR. Thus, in cftr / PCT cells, the lack of RVD is mainly due to theabsence of regulation of the swelling-activated Cl currents.
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MATERIALS AND METHODS/ w% p0 j+ F2 S
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Animals
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CFTR knockout mice were generated by the gene-targetingmethodology described previously ( 28 ) at Centre deDéveloppement des Techniques Avancées pourl'Expérimentation Animale (Orléans, France). Thisstrain of mice was originally derived from ES129/Sv cells injected intoC57BL/6 embryos. Mice were backcrossed with C57BL/6 mice for threegenerations and then intercrossed. They were allowed free access tofood and water in a facility at 25 ± 1°C with a 12:12-hlight-dark cycle. The 4- to 6-wk-old wild-type cftr  /  miceand cftr / mice homozygous for the disrupted CFTR genewere killed by cervical dislocation, and the kidneys were removed. Allexperiments were performed in accordance with the guidelines of theFrench Agricultural Office and in compliance with the legislationgoverning animal studies., A3 g. [4 C7 p0 f) d; N
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Primary Cell Cultures7 V; U5 S5 i, X3 ~8 _

PCT, DCT, and CCT were microdissected under sterile conditions.Kidneys were perfused with Hanks' solution (GIBCO) containing 700 kU/lcollagenase (Worthington), cut into small pyramids that were incubatedfor 1 h at room temperature in the perfusion buffer (160 kU/lcollagenase, 1% Nuserum, and 1 mM CaCl 2 ), and continuously aerated. The pyramids were then rinsed thoroughly in the same bufferdevoid of collagenase. The individual nephrons were dissected by handin this buffer under binoculars using stainless steel needles mountedon Pasteur pipettes. The criteria used to identify the nephron segmentshave been described previously ( 6 ). Briefly, PCTcorresponded to the 1- to 1.5-mm segment of tissue located immediatelyafter the glomerulus. The DCT portion was the segment between themacula densa and the first branching with another tubule [connectingtubule (CNT)]. The CNT segment was discarded. CCT was identified asthe straight poorly branched portion that followed the CNT segment.After they were rinsed in the dissecting medium, tubules weretransferred to collagen-coated 35-mm petri dishes filled with culturemedium composed of equal quantities of DMEM and Ham's F-12 (GIBCO)containing 15 mM NaHCO 3, 20 mM HEPES, pH 7.4, 1% serum, 2 mM glutamine, 5 mg/l insulin, 50 nM dexamethasone, 10 µg/l epidermalgrowth factor, 5 mg/l transferrin, 30 nM sodium selenite, and 10 nMtriiodothyronine. Cultures were maintained at 37°C in a 5%CO 2 -95% air water-saturated atmosphere. The medium wasremoved 4 days after seeding and then every 2 days.4 l$ U7 Y; r, ^% R7 i' f
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Electrophysiological Studies" i& @0 C. R) ^! j; {8 k* Q! h
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Whole cell currents were recorded from 6- to 20-day-old culturedcells grown on collagen-coated supports maintained at 33°C for theduration of the experiments. The ruptured-patch whole cellconfiguration of the patch-clamp technique was used. Patch pipettes (2- to 3-M resistance) were made from borosilicate capillary tubes (1.5 mm OD, 1.1 mm ID; Propper Manufacturing) using a two-stage verticalpuller (model PP 83, Narishige, Tokyo, Japan) and filled with asolution containing (in mM) 105 potassium gluconate, 20 KCl, 1 MgCl 2, 5 or 20 EGTA, 5 MgATP, 5 glucose, and 10 HEPES, pH7.4. The bath solution contained (in mM) 135 sodium gluconate, 1 calcium gluconate, 5 potassium gluconate, 1 MgSO 4, 5 glucose, 60 mannitol, and 10 HEPES, pH 7.4. Cells were observed usingan inverted microscope, the stage of which was equipped with a water robot micromanipulator (model WR 89, Narishige). The patch pipette wasconnected via an Ag-AgCl wire to the head stage of a patch amplifier(model RK 400, Biologic). After formation of a gigaseal, thefast-compensation system of the amplifier was used to compensate forthe intrinsic input capacitance of the head stage and the pipettecapacitance. The membrane was ruptured by additional suction to achievethe conventional whole cell configuration. Settings available on theamplifier (model RK 400) were used to compensate for the cellcapacitance. No series resistance compensation was 20 M werediscarded. Solutions were perfused in the extracellular bath using afour-channel glass pipette, the tip of which was placed as close aspossible to the clamped cell.

Data acquisition and analysis. Voltage-clamp commands, data acquisition, and data analysis werecontrolled via a computer equipped with a Digidata 1200 interface (AxonInstruments). pCLAMP software (versions 5.51 and 6.0, Axon Instruments)was used to generate whole cell current-voltage ( I-V ) relationships, with the membrane currents resulting from voltage stimuli filtered at 1 kHz, sampled at 2.5 kHz, and stored directly onthe computer hard disk. Cells were held at 50 mV, and 400-ms pulsesfrom 100 to  120 mV were applied in 20-mV increments every 2 s.- d, y- `' J2 D: a; [

Expression in Cultured Cells
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The cDNA encoding CFTR was introduced into a polycistronicexpression vector derived from the pIRESneo plasmid (cytomegalovirus promoter; Clontech), in which the neomycin resistance gene had beenreplaced by cDNA encoding the chain of the human CD8 cell surfaceantigen. Distal cells were transfected using the DAC-30 methodaccording to the manufacturer's instructions (Eurogentec, Herstal,Belgium). The 6-day-old cultured cells grown on a 35-mm-diameter petridish were serum starved for 24 h before transfection. Transfected cells with 2 µg of CD8-CFTR coexpress CFTR and CD8 at their plasma membrane and can be visualized using anti-CD8 antibody-coated beads(Dynabeads M-450, Dynal, Oslo, Norway) (13a). Cells were electrophysiologically tested 48 h after transfection.! D% A( [* r# Y% }7 i0 g9 t
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Chemicals
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5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; Calbiochem)was prepared at 100 mM in DMSO and used at 0.1 mM in final solutions.Forskolin and ionomycin were prepared at 10 and 2 mM, respectively, inethanol and used at 10 and 2 µM, respectively, in bath medium.Tetraethylammonium (TEA), charybdotoxin (CTX), quinidine,6- N,N -diethyl- - -dibromomethylene- D -adenosine-5'-triphosphate trisodium (ARL-67156), apamin, forskolin, and ionomycin were obtained from Sigma (Saint Quentin Fallavier, France). 293B was prepared at 10 mM in DMSO and used at 10 µM in final solutions. Clofilium wasprepared at 10 mM in 50% DMSO-50% water and used at a final concentration of 10 µM. Clofilium and 293B were gifts from Dr. Barhanin (UMR CNRS 6097).1 ]7 G9 Q, Y( z. n

RESULTS" y3 _' X  j9 O# Z6 n

K Currents Activated by Forskolin

Experiments were performed in a hyperosmotic extracellularsolution (350 mosmol/kgH 2 O) to characterize K currents activated by 10 µM forskolin in PCT, DCT, and CCT cells. Under these conditions, volume-activated K currents couldnot be detected. Moreover, to eliminate the Cl currents,experiments were performed in the presence of 0.1 mM NPPB in the bathsolution. In control cells not treated with forskolin, the voltage-stepprotocol elicited small currents that changed linearly with membranepotential in PCT, DCT, and CCT cells from kidneys of cftr  / and cftr / mice (data not shown). In cftr  /  mice, incubation of DCT and CCT cells with 10 µM forskolin for 10 minbefore the whole cell measurement induced a strong increase in membranecurrent amplitude (Fig. 1 A ).In DCT cells, the activated currents were linear over the duration ofthe onset pulse. They were outwardly rectifying, with a reversalpotential ( E rev ) of 81 ± 0.4 mV andslope conductances of 1.7 ± 0.2 nS at 60 mV and 15.8 ± 2 nS at 100 mV ( n = 19 cells from 5 different mice). InCCT cells, the currents shared identical characteristics with DCTcells. E rev was 80 ± 1.5 mV, andconductances were 1.4 ± 0.4 nS at 60 mV and 12.2 ± 1.2 nSat 100 mV ( n = 14 cells from 5 different mice). Incontrast to DCT and CCT cells, PCT cells did not present significantforskolin-activated K currents. In these cells, thecurrents reversed at 42.8 ± 2.2 mV with a slope conductance of2.6 ± 0.3 nS at 100 mV ( n = 19 cells from 5 different mice). In cftr / mice, incubation with forskolin did not increase K conductance in any of thecultured segments under study. Taken together, these observationsclearly indicated that, in DCT and CCT cells from cftr  / mice, the K conductance stimulated by forskolin wasassociated with CFTR.
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Fig. 1. A : forskolin-induced whole cell K currentsin proximal convoluted tubule (PCT), distal convoluted tubule (DCT),and cortical collecting tubule (CCT) cells in primary culture of cftr  /  and cftr / mice. Membrane voltage washeld at 50 mV and stepped to test potential of 100 to  120 mV in20-mV increments. Whole cell currents were recorded after 10 min ofincubation with 10 µM forskolin. B : effects of 10 µM293B, 1 mM tetraethylammonium (TEA), and 0.5 mM quinidine onforskolin-induced whole cell K currents measured at  100mV. Values are means ± SE; n, number of monolayersfrom 5 different mice.
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The forskolin-sensitive K currents measured at  100 mV arecompared in PCT, DCT, and CCT cells from cftr  /  and cftr / mice in Fig. 1 B. Only DCT and CCT fromwild-type mice exhibited forskolin-activated K conductance. In these cells, application of 10 µM 293B, 1 mM TEA, and0.5 mM quinidine blocked this current by 80 ± 2 ( n = 10), 55 ± 5 ( n = 18), and85 ± 4% ( n = 9), respectively.7 `% C- j1 u) Q- d  a# b7 i
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K Currents Induced by HypotonicShock
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To prevent development of Cl conductance, the effectof hypotonic swelling was tested in the presence of 0.1 mM NPPB in the bath solution. Control currents were recorded using an isotonic (290 mosmol/kgH 2 O) free Ca 2  (5 mM EGTA) solution inthe pipette and a hypertonic (350 mosmol/kgH 2 O) solutioncontaining 1 mM Ca 2  in the bath. In PCT, DCT, and CCTcells, the voltage-step protocol elicited small time-independentcurrents that changed linearly with the membrane voltage. The currentsat  100 mV were 119 ± 14 pA ( n = 24 cells from 5 different mice), 126 ± 16 pA ( n = 20 cells from 5 different mice), and 104 ± 20 pA ( n = 19 cellsfrom 5 different mice) in PCT, DCT, and CCT cells, respectively.Because of their small amplitude, the nature of these currents was not analyzed further. To produce a hypotonic shock, the monolayers wereperfused continuously with a 290 mosmol/kgH 2 O solution. In 75% of cftr  /  cells, an increase in whole cell current wasobserved in 3 min. In all epithelial cell types, the currents reached a maximum in 4-5 min. Currents recorded in PCT, DCT, and CCT cells from cftr  /  and cftr / mice during hypotonicshock are shown in Fig. 2 A. In cftr  /  cells, currents recorded in each cultured segmentshowed virtually no inactivation during the 400-ms pulse, and thechannels involved in this conductance were activated at depolarizedpotentials. The slope conductances measured at  100 mV were 12-16times the amplitude of those calculated at 60 mV. E rev was near the equilibrium potential forK in all segments: 73 ± 2 mV ( n = 9 cells from 5 mice), 80 ± 4 mV ( n = 13 cellsfrom 5 mice), and 72 ± 5 mV ( n = 9 cells from 5 mice) in PCT, DCT, and CCT, respectively. Regardless of the cell type,the swelling-activated K currents were strongly blockedwhen the cells were reexposed to hypertonic solution (Fig. 2 B ).
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Fig. 2. A : swelling-induced whole cell K currentsin PCT, DCT, and CCT cells in primary culture of cftr  /  and cftr / mice. Membrane voltage was held at 50 mV andstepped to test potential of 100 to  120 mV in 20-mV increments.Whole cell currents were recorded after 4-5 min of extracellularperfusion of a 30% hypotonic solution in the presence of 5 mM EGTA and5 mM MgATP in pipette solution and 1 mM calcium gluconate inextracellular bath. B : effects of hyperosmotic solution (350 mosmol/kgH 2 O) on swelling-induced whole cell K currents. Steady-state currents at  100 mV were measured 200 ms afteronset of pulse. Values are means ± SE; n, number ofmonolayers from 5 different mice.

For the cftr / mice, the effect of hypotonic shock wasdifferent between PCT and DCT or CCT cells. Hypotonic shock always induced swelling-activated K currents in PCT cells( n = 9), whereas it did not significantly modify theK conductance in DCT ( n = 19) and CCT( n = 9) cells (Fig. 2 ). Current intensity measured at 100 mV for all types of cells from cftr  /  and cftr / mice is shown in Fig. 2 B. The inabilityof hypotonic shock to trigger swelling-activated K currents in DCT and CCT cells was observed in 100% of the 28 trials.Overall, the results indicate that CFTR protein could be implicated inthe control of swelling-activated K conductances interminal, but not in proximal, segments of the nephron.$ h' D( V& O, N- U, f* m$ g& w

The results reported on DCT and CCT cells show that swelling-activatedK conductances were roughly similar in these two celltypes. For this reason, no further distinction was made between DCT andCCT in the following experimental series.% T5 f& B6 Y- [# Y+ J- A( N

Pharmacology of Swelling-Activated K Channels: {( L" [, z8 ^% ]1 n  s$ g
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To further characterize the swelling-activated K currents, we tested the effect of various K channelblockers added separately to the bathing hypotonic solution. Theswelling-activated outward K current measured at  100 mVin PCT cells from cftr  /  mice is shown in Fig. 3. Perfusion of 1 mM TEA, 10 nM CTX, and10 µM 293B did not significantly modify the swelling-activatedK currents. In contrast, 0.5 mM quinidine and 10 µMclofilium decreased K current amplitude by 64 ± 6 ( n = 6) and 55 ± 5% ( n = 5),respectively (Fig. 3, A and B ). Similar resultswere obtained in PCT from cftr / mice, indicating thatthe swelling-activated K conductance was not related toCFTR in this segment.
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Fig. 3. Effect of K channel inhibitors on swelling-activatedK currents in PCT cells from cftr  /  mice.Membrane voltage was held at 50 mV and stepped to test potential of 100 to  120 mV in 20-mV increments. Whole cell currents were recordedafter 4-5 min of extracellular perfusion of a 30% hypotonicsolution containing 100 µM 5-nitro-2-(3-phenylpropylamino)-benzoicacid (NPPB) in the presence of 5 mM EGTA and 5 mM MgATP in pipettesolution. A : 0.5 mM quinidine perfused after development ofK currents. B : 10 µM clofilium perfused afterdevelopment of K currents. C : inhibition ofdifferent K channel inhibitors on swelling-activatedK currents in PCT cells from cftr  /  mice.Whole cell currents were recorded after 4-5 min of extracellularperfusion of a 30% hypotonic solution in the presence of 0.5 mMquinidine, 1 mM TEA, 10 nM charybdotoxin (CTX), 10 µM 293B, and 10 µM clofilium. Currents were measured 200 ms after onset of pulse at 100 mV. Values are means ± SE of 10 cells from 5 monolayers.8 H$ ~! z  S1 W* \* j; ]
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The effects of the same channel blockers in DCT and CCT cells from cftr  /  mice are illustrated in Fig. 4. The noticeable difference from the PCTcells was the strong reduction of swelling-activated K conductance in the presence of TEA and CTX (Fig. 4 A ):inhibition = 82 ± 7 ( n = 6) and 71 ± 3% ( n = 18) for TEA and CTX, respectively. Asexpected, quinidine was also very efficient in blocking the K currents (70 ± 5%, n = 5). Incontrast, 10 nM apamin was less efficient in blocking these currents(26 ± 3%, n = 18; Fig. 4, A and C ). Finally, as observed in PCT cells, 293B did notsignificantly decrease swelling-activated K currents inDCT and CCT cells." P! j4 n" J# `

Fig. 4. Effect of K channel inhibitors on swelling-activatedK currents in DCT cells from cftr  /  mice.Membrane voltage was held at 50 mV and stepped to test potential of 100 to  120 mV in 20-mV increments. Whole cell currents were recordedafter 4-5 min of extracellular perfusion of a 30% hypotonicsolution containing 100 µM NPPB in the presence of 5 mM EGTA and 5 mMMgATP in pipette solution. A : 10 nM CTX and 10 nM apaminperfused after development of K currents. B : 10 nM CTX 10 nM apamin perfused after development of K currents. C : inhibition of K channel inhibitorson swelling-activated K currents in PCT cells from cftr  /  mice. Whole cell currents were recorded after4-5 min of extracellular perfusion of a 30% hypotonic solution inthe presence of 0.5 mM quinidine, 1 mM TEA, 10 nM CTX, 10 µM 293B, 10 nM apamin, and 10 nM CTX apamin. Currents were measured 200 msafter onset of pulse at  100 mV. Values are means ± SE of 15 cells from 5 monolayers.5 Q2 O1 k, H# c$ {1 J% b

The relative insensitivity to a number of known K channelinhibitors of the swelling-activated K current of PCTcells led us to carry out a further experiment to obtain moreinformation concerning the nature of this channel.' {( N3 O9 w% I( u9 S) @

Regulation of the K ConductanceInduced by Hypotonic Shock incftr / PCT Cells* W' R& o/ N9 Q! X1 M& P7 w

Role of extracellular pH. To study the modulation of the swelling-activated K currents by extracellular pH (pH e ), PCT cells were swollenin hypotonic solutions (270 mosmol/kgH 2 O) adjusted topH e 6.0-8.0. Trace recordings at three different pHvalues are shown in Fig. 5 A.Compared with the control K currents measured atpH e 7.4, K currents at pH e 6.0 were reduced by 53 ± 3% ( n = 9), whereas currents at pH e 8.0 were increased by 44 ± 10%( n = 9). The corresponding I-V curves arereported in Fig. 5 B. Change in pH e did notsignificantly modify E rev : 73 ± 8, 73 ± 2, and 74 ± 4 mV at pH e 6.0, 7.4, and8.0, respectively ( n = 9). Inhibition of theK current at acidic pH became significant for membranepotential of  20 mV [K current = 254 ± 53 ( n = 9) and 436 ± 60 pA ( n = 10)at pH e 6.0 and 7.4, respectively (unpaired t -test = 2.27, P stimulation of currents at alkaline pH became significant for membranepotential of 20 mV [K current = 358 ± 75 ( n = 6) and 174 ± 37 pA ( n = 10)at pH e 8.0 and 7.4, respectively (unpaired t -test = 2.19, P/ d, J$ b/ e# v
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Fig. 5. Effect of extracellular pH on development of hypotonicity-inducedK currents in cultured PCT cells from cftr  / mice. Membrane voltage was held at 50 mV and stepped to testpotential of 100 to  120 mV in 20-mV increments. A : wholecell currents recorded after 4-5 min of extracellular perfusion ofa 30% hypotonic solution at pH 6.0, 7.4, and 8.0 in the presence of 5 mM EGTA and 5 mM MgATP in pipette solution and 100 µM NPPB inextracellular solution. B : average current-voltage( I-V ) relationships measured 200 ms after onset of pulse inthe same cell at rest ( A ) during hypotonic stimulation at pH6.0, 7.4, and 8.0. Values are means ± SE of 9 cells from 5 monolayers.

Role of extracellular Ca 2  . To eliminate the possibility that cytosolic Ca 2  isinvolved in the development of hypotonicity-induced K currents, experiments were generally performed using pipette solutionscontaining 5 mM EGTA without additional Ca 2 . The effectsof extracellular Ca 2  on the development ofhypotonicity-induced K currents were also tested in cftr  /  PCT cells. When the hypotonic shock was carried outin the absence of bath Ca 2 , development of theK current was not significantly modified (Fig. 6, A and B ). The channel implicated in this response was clearly blocked byhypertonicity and 0.5 mM quinidine (Fig. 6, C-E ). Theseresults, which were also obtained using cftr / PCT cells(data not shown), indicate that Ca 2  is not involved incontrol of the swelling-activated K conductance measuredin PCT cells in primary culture.8 T" c" o* P0 ^
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Fig. 6. Effect of extracellular Ca 2  on development ofhypotonicity-induced K currents in cultured PCT cells from cftr  /  mice. Membrane voltage was held at 50 mV andstepped to test potential of 100 to  120 mV in 20-mV increments.Whole cell currents were recorded in the absence of extracellularCa 2  and in the presence of 100 µM NPPB in extracellularsolution. A : control cells. B : after 4-5 minof extracellular perfusion of a 30% hypotonic solution. C :after replacement of extracellular solution with a hypertonic solution. D : 0.5 mM quinidine. E : currents measured 200 msafter onset of pulse at  100 mV in the same cell at rest. Values aremeans ± SE of 6 cells from 6 monolayers.

Regulation of the K ConductanceInduced by Hypotonic Shock incftr / andcftr / DCT Cells
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Role of external Ca 2  . As reported above, the experiments were carried out in the presence of5 mM EGTA in the pipette solutions. In DCT cells from cftr  /  mice, the absence of bath Ca 2  completely prevented hypotonicity from inducing K currents(Fig. 7, A and B ).Conversely, perfusion of a solution containing 1 mM freeCa 2  restored the response to hypotonicity (Fig. 7 C ). As expected, this swelling-activated K conductance was blocked by hypertonicity and by CTX apamin (10 nM; Fig. 7, D and E ). As previously described forswelling-activated Cl currents in DCT cells( 1 ), it appears that an influx of Ca 2  isrequired to permit the development of swelling-activated K currents in cftr  /  DCT cells. It was therefore interestingto study the role of extracellular Ca 2  in cftr / DCT cells. Whole cell currents were recorded inthe presence of 20 mM EGTA in the pipette solution and 1 mM freeCa 2  in the bath (Fig. 8 ). Inthe absence of ionomycin in the bath solution, the hypotonic shockremained inefficient for triggering K currents in cftr / cells (Fig. 8 A ). However, when thehypotonic shock was performed in the presence of 2 µM ionomycin,K currents were activated within 5 min (Fig. 8 B ). These currents were clearly due to K movements, because they were blocked by CTX apamin and TEA (Fig. 8, C and D ). Moreover, analysis of the I-V curves (Fig. 8 E ) indicated that theinstantaneous outwardly rectifying currents reversed at 79.5 ± 1.0 mV ( n = 10). Finally, when the cells were reexposedto the hyperosmotic solution, the currents returned toward controllevel within 2-3 min. Overall, the ionomycin-induced K currents developed during hypotonicity in DCT cells from cftr / mice were quite similar to the swelling-activatedK currents measured in cftr  /  mice.

Fig. 7. Effect of extracellular Ca 2  on development ofhypotonicity-induced K currents in cultured DCT cells from cftr  /  mice. Membrane voltage was held at 50 mV andstepped to test potential of 100 to  120 mV in 20-mV increments.Whole cell currents were recorded in the presence of 5 mM EGTA and 5 mMMgATP in pipette: in control cells in the absence of extracellularCa 2  in bath solution ( A ), after 4-5 min ofextracellular perfusion of a 30% hypotonic solution withoutCa 2  ( B ), after replacement of extracellularsolution with a hypotonic solution Ca 2  ( C ), and after replacement of extracellular solution with ahypertonic solution Ca 2  ( D ) or ahypotonic solution containing 10 nM CTX apamin ( E ). F : currents measured 200 ms after onset of pulse at  100 mVin the same cell at rest. Values are means ± SE of 10 cells from8 monolayers.9 A, |5 q4 }. K9 [+ p$ P

Fig. 8. Effect of ionomycin on development of K currents incultured DCT cells from cftr / mice. Membrane voltage washeld at 50 mV and stepped to test potential of 100 to  120 mV in20-mV increments. Whole cell currents were recorded after 4-5 minof extracellular perfusion of a 30% hypotonic solution in the presenceof 20 mM EGTA and 5 mM MgATP in pipette solution and 1 mMCaCl 2 in extracellular bath. Whole cell currents weremeasured during hypotonic shock. A : control cells. B : 2 µM ionomycin. C : 10 nM CTX apamin. D : 1 mM TEA. E : average I-V relationships measured 200 ms after onset of pulse in the same cell atrest. Values are means ± SE of 10 cells from 10 monolayers.( L7 V, y/ b' M% @" s- l( ]
. f' U; D# Z, h. D
Role of extracellular adenosine. The results described above strongly suggest that swelling-activatedK and Cl currents in DCT cells are regulatedby an identical mechanism involving a Ca 2  influx. Inasmuchas we previously demonstrated that this Ca 2  influx couldbe due to stimulation of A 1 receptors by extracellular adenosine, experiments were performed to determine the role of adenosine in the K permeability of DCT cells from cftr  /  and cftr / mice. Adenosine (10 µM)activated an outwardly rectifying K conductance with E rev of 82.1 ± 4.1 ( n = 17 cells) and 78.1 ± 2.0 mV ( n = 18) for cftr  /  and cftr / DCT cells, respectively (Fig. 9, A and B). In thepresence of adenosine, the maximal slope conductances reached 18.0 ± 1.4 ( n = 17) and 18.7 ± 1.0 nS( n = 18) in cftr  /  and cftr / DCT cells, respectively. These adenosine-sensitive K currents were decreased in the presence of CTX apamin (10 nM) by84 ± 3% ( n = 9) in both types of DCT monolayers.To further study the influence of external Ca 2  onadenosine-sensitive K currents, experiments were performedin the absence of bath Ca 2 . The results are illustrated inFig. 10. As expected, removal ofexternal Ca 2  completely prevented adenosine from inducingK currents (Fig. 10, A and B ).Conversely, perfusion of a solution containing 1 mM freeCa 2  restored the response to adenosine (Fig. 10 C ).$ F* y  ]3 ?0 z1 V

Fig. 9. Effect of adenosine on development of K currents incultured DCT cells from cftr  /  ( A ) and cftr / ( B ) mice. Membrane voltage was held at 50 mV and stepped to test potential of 100 to  120 mV in 20-mVincrements. Whole cell currents were recorded after 3 min ofextracellular perfusion of 10 µM adenosine in the presence of 5 mMEGTA and 5 mM MgATP in pipette solution and 1 mM CaCl 2.CTX apamin (10 nM) was perfused after development ofK currents. C : currents measured 200 ms afteronset of pulse at  100 mV in the same cell at rest. Values aremeans ± SE of 17 cells from 6 monolayers.
2 _; s% W9 k1 ?3 N7 f: B& A
Fig. 10. Effect of extracellular Ca 2  on development ofadenosine-induced K currents in cultured DCT cells from cftr / mice. Membrane voltage was held at 50 mV andstepped to test potential of 100 to  120 mV in 20-mV increments.Whole cell currents were recorded in the presence of 5 mM EGTA and 5 mMMgATP in pipette. A : control cells in the absence ofextracellular Ca 2  in bath solution. B : after 3 min of extracellular perfusion of a 10 µM adenosine solution withoutCa 2 . C : after replacement of extracellularsolution with 10 µM adenosine solution Ca 2 . D : after 3 min of extracellular perfusion of 10 nM CTX apamin. E : currents measured 200 ms after onset of pulseat  100 mV in the same cell at rest. Values are means ± SE of 4 cells from 4 monolayers.& a% e# r9 a. u7 D
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Finally, adenosine mimicked the effect of hypotonic shock, and itremains to be understood how this nucleotide is produced during thehypotonic cell swelling. Experiments were therefore carried out todetermine whether adenosine was generated by ATP degradation. For thispurpose, DCT cells from cftr  /  mice were subjected to ahypotonic shock in the presence of the selective ecto-ATPase inhibitorARL-67156. ARL-67156 (100 µM) completely blocked theswelling-activated K currents (Fig. 11 A ). Adenosine (10 µM)restored a swelling-activated K conductance, whichdisplayed an outwardly rectified I-V plot with E rev of 75.0 ± 1.1 mV ( n = 12; Fig. 11, B and D ) and was stronglyinhibited by CTX apamin (Fig. 11 C ).
1 H% ?4 ?( {; @/ E  q  h; g7 `
Fig. 11. Effects of ecto-ATPase antagonist (ARL-67156) onswelling-activated K currents in DCT cells from cftr  /  mice. Membrane voltage was held at 50 mV andstepped to test potential of 100 to  120 mV in 20-mV increments.Whole cell currents were recorded after 4-5 min of extracellularperfusion of a 30% hypotonic solution in the presence of 5 mM EGTA and5 mM MgATP in pipette and 100 µM ARL-67156 in extracellular bath( A ), 10 µM adenosine ( B ), and 10 nM CTX apamin ( C ). D : average I-V relationships measured 200 ms after onset of pulse in the same cell atrest. Values are means ± SE of 14 cells from 7 monolayers.

K Currents Activated by an OsmoticShock in Cultured DCT Cells Fromcftr / Mice Transfected WithCFTR cDNA- h4 k! J' O; _
* U5 w" F6 |: A2 ?) ?/ ~
The cftr / DCT cells in primary culture weretransfected with a plasmid pIres-CD8- cftr, allowingvisualization of transfected cells using anti-CD8 antibody-coatedbeads. Whole cell currents were recorded 48 h after thetransfection procedure. Currents recorded in labeled cells immediately(control) or 4-5 min after perfusion of the hypotonic solution areshown in Fig. 12 A. Asexpected, the developed currents reversed close to the equilibriumpotential of K ( 75 ± 9 mV, n = 8)and were blocked by CTX apamin and hypertonicity ( n = 3; Fig. 12, A and C ). Incontrast, in the same culture, the nonlabeled cells did not respond tohypotonic shock. These results clearly demonstrate that the transitorytransfection of cDNA encoding CFTR restored the ability of the cells toincrease their K conductance after a hypotonic shock.( _2 t0 ~' P% ?* L# R

Fig. 12. Restoration of swelling-activated K currents bytransitory transfection of pIres - CD8- cftr.Transfected cells were visualized using anti-CD8 antibody-coated beads.Membrane potential was held at 50 mV and stepped to test potential of 100 to  120 mV in 20-mV increments. A : whole cell currentsin cells labeled with anti-CD8-coated beads. B : whole cellscurrents in unlabeled cells. C : average I-V relations measured 200 ms after onset of pulse in the same cell at rest( A ) during perfusion of hyposmotic solution, after perfusionof hyperosmotic solution, and after perfusion with 10 nM CTX apamin. Values are means ± SE of 8 cells from 6 transfectedmonolayers.8 W& y) D, v2 m3 L1 _4 B

DISCUSSION0 {+ {- v- R* v1 N
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In the majority of mammalian cells, an increase in cell volumeactivates Cl currents. In renal tissue, we previouslyshowed the presence of swelling-activated Cl currentsresponsible for the RVD after a hypotonic shock ( 26, 27 ).Such currents were also found in primary cultures of PCT, DCT, and CCTfrom wild-type mice ( 1 ). Moreover, the cftr knockout strongly decreased these currents in PCT, DCT, and corticalcollecting duct cells (CCD). In this study, we were interested in theconsequences of the cftr knockout on the K currents activated by cAMP and on swelling-activated K currents activated by a hypotonic shock.; W: P$ y. g6 \: X9 ]/ o
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In the present work, we showed the presence of cAMP-activatedK currents in DCT and CCD cells from wild-type mice only.K 90% of the trialsonly when the monolayers were treated with forskolin for 10 min beforethe seal formation. This could indicate that cAMP induced incorporation of new channels in the membrane of DCT or CCT cells. TheseK currents were completely inhibited by the chromanolanalog 293B. Because this antiarrhythmic is known to block mainly theKCNQ1 channels ( 15, 34 ), it is probable that thecAMP-sensitive K currents in mouse distal cells belong tothe KCNQ1 family of channels. Activation of KCNQ1 channels by the cAMPpathway has been reported in epithelial cells from many tissues,including inner ear, colon, small intestine, and airways (1a, 8, 33).In the kidney, although the presence of KCNQ1 transcripts has been demonstrated in the CCD of the outer medulla and the DCT and CCT of thecortex ( 8 ), the role of the protein remains unclear. Inour work, the putative presence of KCNQ1 in DCT and CCT cells raisedthe problem of its coexpression with a KCNE protein. In epithelia,KCNQ1 associates with the small regulatory -subunit KCNE1 or KCNE3to form a K channel that could be implicated in thecAMP-stimulated Cl secretion (1a). Using RT-PCR andSouthern blotting experiments, we previously identified transcriptsencoding the KCNQ1 and KCNE1 sequence in cultured PCT cells fromwild-type mice (unpublished observations). In contrast, DCT cells alsoexpressed KCNQ1, but not KCNE1, excluding a role for this subunit inthe cAMP-sensitive K channel in this part of the nephron.Whether KCNQ1 expression in DCT and CCT cells required an additionalsubunit such as KCNE3 remains to be elucidated.

In previous studies ( 26 ), we clearly established that CFTRfunctions as a cAMP-activated Cl channel in the apicalmembrane of DCT cells. Although from these results the localization ofthe cAMP-sensitive K secretion cannot be specified, thischannel might participate in the driving force of apicalCl secretion ( 15, 17 ). Our results show thatforskolin did not activate K current in DCT and CCT cellsfrom cftr / mice. Although the role of CFTR in thecontrol of K channels is well established in varioustissues ( 2, 16, 20 ), the mechanism of the interactionbetween CFTR and K channel protein is far from beingcompletely understood. This mechanism probably depends on the nature ofthe K channel, because, in addition to KCNQ1, other typesof channels, such as the ROMK family, could interact directly with CFTR( 2, 11, 20 ). The data reported in the literature for therelation between KCNQ1 and CFTR are conflicting. Some studies lead tothe conclusion that KCNQ1 currents were activated by the cAMP pathway, independent of the presence of CFTR ( 2, 18, 34 ); other studies demonstrate that CFTR directly activates KCNQ1 conductances ( 7, 17 ) and that this conductance was not detected in the epithelial cell line CFPAC expressing F508-CFTR ( 16 ).This last finding corroborates our own observations and suggests that, in the distal tubule, KCNQ1 K currents are dependent onCFTR expression.; _( m6 O* O( A: J1 j& T4 w
" p4 f& y; o9 s* e1 U- [
As expected, a hypotonic shock activated K currents inPCT, DCT, and CCD cells from wild-type mice. In the monolayers from cftr / mice, development of these K currentswas completely impaired in DCT and CCT cells but was not modified inPCT cells. This suggests two different types of swelling-activatedK currents in PCT and DCT cells. The pharmacological studyperformed on these channels confirmed this hypothesis. In PCT cells,the swelling-activated K conductance shared someproperties with the K currents activated by cell swellingthat have been reported in gallbladder epithelium ( 31 ) andin Ehrlich ascites tumor cells ( 12, 22 ). It wasinsensitive to TEA but was strongly blocked by quinidine and clofilium.Moreover, this K conductance was not affected byintracellular Ca 2  concentration but was sensitive toexternal pH variations, with activation at alkaline pH and inhibitionat acidic pH. Finally, this pharmacological profile is consistent withTASK2 channels. Further experiments are required to definitivelycharacterize the nature of the swelling-activated K channels in PCT. However, it is now well established that TASK2 isstrongly expressed in the kidney ( 23 ) and, more precisely, in mouse proximal tubule, in situ or in primary culture( 29 ).( H3 i8 R7 l! z/ z6 x3 j
) j# D9 p& ]" Z7 E2 w) B
In DCT and CCT cells, the swelling-activated K conductances exhibited a different pharmacology: they were inhibited byTEA and CTX and were dependent on extracellular Ca 2 . Theinhibition by CTX indicates that Ca 2  activated BK andintermediate K (IK) channels. Furthermore, a minor role ofSK channels cannot be excluded, because this toxin inhibited theswelling-activated K currents by 27% and has been shownto be additive to the CTX effect.* A1 c, h/ Y" [! e/ Y2 D( ^

These K conductances were activated by hypotonicity in thepresence of a high concentration of EGTA in the pipette solution. Thesimplest interpretation of this finding could be that an increase inintracellular Ca 2  is not necessarily required to activateK currents and that Ca 2  influx is sufficientto increase the K conductance during hypotonicity( 25, 27 ). This conclusion is supported by the recent dataof Grunnet et al. ( 9 ), who reported that stimulation of BKand SK channels during swelling was not mediated by a change inintracellular Ca 2 .
/ Y, }$ P! x: `" T: ~# f* [
Although they belong to a distinct channel family, theK channels activated by hypotonicity in PCT and DCT cellsof wild-type mice participate in RVD, together with swelling-activatedCl conductances ( 27 ). In both monolayers,the swelling-activated K currents were insensitive to thechromanol derivative 293B, indicating that KCNQ1 is not implicated inthis conductance. This conclusion is at variance with that reached byLock and Valverde ( 14 ), who proposed that the KCNQ1-KCNE1complex was implicated in the maintenance of K secretionlinked to RVD in murine airway cells. As we previously demonstrated( 1 ), null mutation of CFTR strongly impaired the RVD inPCT and DCT cells. Obviously, in PCT cells this inhibition was due to adefect in swelling-activated Cl conductances, because theaccompanying activated K channels remained functional in cftr / monolayers. In contrast, in DCT cells,swelling-activated Cl and K conductanceswere affected by the knockout of CFTR. At this step in our work, thequestion arose as to what extent the Cl andK channels in DCT cells were modulated by a commonmechanism that was altered in the absence of CFTR. As demonstrated forswelling-activated Cl conductance, ionomycin in thepresence of a high concentration of EGTA inside the cells restored theswelling-activated K currents in the DCT and CCT cellsfrom cftr / mice, confirming the role of Ca 2  entry in these cells. Moreover, adenosine mimicked the effect ofionomycin in the presence of external Ca 2  only. Therefore,in the case of the Cl channels, this Ca 2  influx might be due to stimulation of A 1 receptors byadenosine generated by the degradation of ATP by membrane ectoenzymes.According to Braunstein et al. ( 3 ), lack of CFTR leads toimpairment of ATP release during hypotonic shock ( 5, 10, 24 ). The final result is loss of the RVD process, because thefirst element (ATP release) of the cascade leading to activation ofCl and K channels is missing.& V/ s# ?1 z. S, Q) C% U5 }5 C
( J2 D( j' g6 S6 V
The results of these experiments are summarized in Fig. 13. In PCT and DCT cells, RVD afterhypotonic challenge is dependent on CFTR. However, in PCT cells, onlythe swelling-activated Cl conductance is affected by thisregulation. In DCT cells, swelling-activated Cl and K currents are regulated by CFTR during hypotonicshock. Although swelling-activated Cl currents share thesame properties in PCT and DCT cells, this is not the case forswelling-activated K currents: in PCT cells theK current could be due to TASK2 channels, whereas in DCTcells the K current could flow throughCa 2  -dependent BK and SK channels. With regard to thecAMP-sensitive conductances, the data strengthen the hypothesis thatCFTR mediates forskolin-activated Cl and K currents in DCT cells. The K conductance could be formedby KCNQ1-like channels. In contrast, forskolin-activatedCl and K currents are not detectable in PCTcells. Therefore, it appears that, in renal epithelium, CFTR not onlyfunctions as a cAMP-activated Cl channel but alsoparticipates in regulation of cell volume. These two functions areprobably independent, because PCT exhibits only the latter, whereas DCTexhibits both functions. This suggests that the form of CFTR expressedin PCT is different from that in DCT. We have no evidence of suchdifferences. However, it may be recalled that an alternative spliceform of CFTR (TNR-CFTR) was found in inner medullary collecting duct,indicating a possible tissue-specific processing of CFTR( 13 )., m/ R& a2 f' |1 {5 m6 @- X0 o
0 P  }# \# v: q9 G1 X0 ]" B
Fig. 13. Working model of CFTR and purinergic signaling in normal proximaland distal cells. Loss of CFTR and purinergic signaling leads todefects in cell volume regulation. A 1, A 1 adenosine receptor; DPCPX, 8-cyclopentyl-1,3-diproxylxanthine.

The question arises of whether the inhibition of swelling-activatedK and Cl currents in cftr / has physiological consequences. The cftr / mice exhibitdrastic growth and intestinal dysfunctions and generally die 6 wk afterbirth. To optimize their lifetime, the animals are fed a special liquiddiet. Despite these precautions, the animals remain very weak, andtheir renal function has not been investigated. Therefore, kidneyfunction could be impaired, but the very poor physiological state ofthe animals could mask this specific alteration. There is no apparentdifference in growth between cell cultures from cftr / and control cells from cftr  /  mice, at least when they arekept in constant control conditions, such as an incubator. However,preliminary experiments (data not shown) indicate that a hypotonicshock stopped division of the cftr /, but not the cftr  / , cells.

The situation is quite different in humans, because the dysfunction ofCFTR was mainly due to F508 mutation. However, according toBraunstein et al. ( 3 ), it is possible that the F508mutation resulted in an alteration of RVD. Thus we can suppose that the human kidney from CF patients will also exhibit altered RVD. The consequences of such a modification remain to be analyzed in terms of arenal role of CFTR.* \1 U5 G4 g4 q1 F7 u! ^
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