cAMP-dependent activation of the renal-specificNa -K -2Cl cotransporter ismedia - 《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 13:38 |只看该作者 |倒序浏览 |打印

作者：PatriciaMeade, Robert S.Hoover, ConsueloPlata, NormaVázquez, Norma A.Bobadilla, GerardoGamba,  Steven C.Hebert作者单位：1 Molecular Physiology Unit, Instituto deInvestigaciones Biomédicas, Universidad NacionalAutónoma de México, and Instituto Nacional de CienciasMédicas y Nutrición Salvador Zubirán Tlalpan1400 Mexico City, Mexico; and Department of Cellular and Molecular Phys
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      【摘要】( |/ E( o  d# {/ f
   The murineapical bumetanide-sensitiveNa -K -2Cl cotransporter gene(mBSC1) exhibits two spliced isoform products that differ at theCOOH-terminal domain. A long COOH-terminal isoform (L-mBSC1) encodesthe Na -K -2Cl cotransporter, anda short isoform (S-mBSC1) exerts a dominant-negative effect on L-mBSC1cotransporter activity that is abrogated by cAMP. However, themechanism of this dominant-negative effect was not clear. In thisstudy, we used confocal microscopic analysis of an enhanced greenfluorescent protein (EGFP) fusion construct (L-mBSC1-EGFP) expressed tocharacterize the surface expression of the L-BSC1 isoform in Xenopus laevis oocytes. Functional expression was alsoassessed in L-mBSC1-injected oocytes by measuring the bumetanide-sensitive 86 Rb uptake. Oocytesinjected with L-mBSC1-EGFP cRNA developed a distinct plasmamembrane-associated fluorescence that colocalized with the fluorescentmembrane dye FM 4-64. The fluorescence intensity in L-mBSC1-EGFPoocytes did not change after cAMP was added to the extracellularmedium. In contrast, L-mBSC1-EGFP fluorescence intensity was reduced ina dose-dependent manner, with coexpression of S-mBSC1. The inhibitoryeffect of S-mBSC1 was abrogated by cAMP. Finally, the exocytosisinhibitor colchicine blocked the effect of cAMP on theL-mBSC1-EGFP/S-mBSC1-coinjected oocytes. All changes in L-mBSC1surface expression correlated with modification of bumetanide-sensitive 86 Rb uptake. Our data suggest that thedominant-negative effect of S-mBSC1 on L-mBSC1 transport function isdue to the effects of the cotransporter on trafficking.
      【关键词】 kidney thick ascending limb Xenopus laevis oocytes green fluorescent protein NKCC7 E: ?) e. J. U) d
   INTRODUCTION

SALT REABSORPTION IN THE THICK ascending limb of Henle (TAL) involves NaCl entryacross apical membranes by the apical bumetanide-sensitive Na -K -2Cl cotransporter (BSC1,NKCC2), apical recycling via ROMK K channels, andbasolateral Cl efflux by CLCNKB channels( 38 ). Loss-of-function mutations in any of the genesencoding these transport proteins in the human TAL cause Bartter'ssyndrome ( 34-36 ), an autosomal recessive disease featuring hypokalemic metabolic alkalosis with hypercalciuria and lowarterial blood pressure ( 23 ).( _+ ~$ {5 y* ]% n8 S

A fundamental mechanism for enhancing salt transport in the TAL is thegeneration of cAMP via activation of G s -coupledreceptors by hormones such as vasopressin, calcitonin, parathyroidhormone, glucagon, and catecholamine ( 13, 15, 16 ). Theeffects of these hormones are crucial to the normal functioning of theTAL in reabsorbing 10-15% of filtered NaCl, providing for normaldiluting and concentrating power and regulating divalent mineralexcretion. While vasopressin has been shown to directly activate theapical Na -K -2Cl cotransporterin mouse TAL ( 25 ), the molecular mechanism of thisactivation is not known.1 P9 }2 H; \: i! |% j* V. r

An emerging field of regulation of several membrane transporters,including the apical Na -K -2Cl cotransporter, appears to involve alternative splicing that generates isoforms with regulatory roles ( 9 ). In this regard, themurine renal specific Na -K -2Cl cotransporter gene SLC12A1, known as BSC1 or NKCC2, givesrise to six alternative spliced isoforms due to the combination of twoindependent splicing mechanisms ( 18, 27 ). One splicing event produces A, B, and F isoforms that vary in a portion of thepredicted second transmembrane segment and the contiguous intracellularloop connecting transmembrane domain 2 with 3 ( 18 ). The other splicing mechanism is analternative polyadenylation site that predicts two mBSC1 proteins thatare identical at the NH 2 -terminal and transmembranedomains, as well as in the first 74 amino acid residues of theCOOH-terminal domain ( 27 ). These isoforms differ in thesequence and length of the remaining COOH-terminal domain. The longerisoform, L-mBSC1 (previously known as mBSC1-9), contains 1,095 amino acids residues, with the last 383 residues being unique. Theshorter isoform, S-mBSC1 (previously known as mBSC1-4), consistsof 770 amino acids residues, with the initial 74 amino acids of theCOOH terminus being identical to that of L-mBSC1 but followed by ashort, unique segment of 55 residues. The shorter COOH-terminal domainof S-mBSC1 predicts distinct consensus PKA and PKC phosphorylationsites. Because the two splicing events are independent of each other,the combination of both mechanisms results in the production of sixdifferent isoforms: three with a long COOH-terminal domain (L-mBSC1A,B, or F) and three with a short COOH-terminal domain (S-mBSC1A, B, orF). Both L-mBSC1 and S-mBSC1 are expressed in the apical membrane ofthe TAL ( 27 ).! i9 n5 K# n8 W0 c  G4 L

Using heterologous expression in Xenopus laevis oocytes, wehave previously shown that the L-mBSC1 isoform is abumetanide-sensitive Na -K -2Cl cotransporter ( 32, 33 ), whereas the S-mBSC1 isoform is a hypotonicity-activated, K -independent,bumetanide-sensitive Na -Cl cotransporterthat is inhibited by cAMP ( 31 ). Under isotonic conditions,however, S-mBSC1 is not trafficked to the plasma membrane whenexpressed alone but exerts a dominant-negative effect on theNa -K -2Cl cotransporterfunction. This latter effect can be abrogated with PKA activation bycAMP ( 33 ). Thus S-mBSC1 and L-mBSC1 interaction could becritical for activation of theNa -K -2Cl cotransporter byhormones such as vasopressin. This type of interaction betweenalternatively spliced isoforms has been observed in several renalcotransporters, but the mechanisms are not known ( 9 ). Competition between intracellular vesicles or heterodimers containing different isoforms has been suggested as possible mechanisms of interaction; however, no study has addressed this issue for membrane transporters.* O/ @+ d8 r9 T7 \1 _
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In the present study, we investigated the mechanism of interactionbetween the long and short isoforms of theNa -K -2Cl cotransporter. To thisend, we assessed the effects of the S-mBSC1 isoform on both the surfaceand functional expression of the L-mBSC1 isoform in X. laevis oocytes. Our results show that S-mBSC1 reduces the surfaceexpression, and hence the activity, of the L-mBSC1 Na -K -2Cl cotransporter isoformby a mechanism that involves PKA phosphorylation processes and theexocytosis machinery.

METHODS

X. laevis oocyte preparation and injection. Adult female X. laevis frogs were purchased from Nasco (FortAtkinson, MI). Frogs were maintained at the animal facility under constant control of room temperature and humidity at 16°C and 65%,respectively. Oocytes were harvested from tricaine(0.17%)-anesthetized frogs and incubated for 1 h with vigorousshaking in Ca 2  -free ND-96 [(in mM) 96 NaCl, 2 KCl, 1 MgCl 2, and 5 HEPES/Tris, pH 7.4] in the presence of 2 mg/ml of collagenase B. Oocytes were washed three times in regularND-96 [(in mM) 96 NaCl, 2 KCl, 1.8 CaCl 2, 1 MgCl 2, and 5 HEPES/Tris, pH 7.4], manually defolliculated, and incubated overnight at 16°C in incubation medium (ND-96supplemented with 2.5 mM sodium pyruvate and 5 mg/100 ml ofgentamicin). Oocytes ( 6 ) were injected with 50 nl of wateror a solution containing L-mBSC1 cRNA at 0.5 µg cRNA/µl (25 ngcRNA/oocyte). In coinjection experiments, oocytes were injected withthe same volume and amount of L-mBSC1 plus varying amounts of S-mBSC1cRNA. After injection, oocytes were incubated at 16°C during 4-5days, and the medium was changed every day. Oocytes were incubatedovernight in Cl -free ND-96 [(in mM) 96 sodiumisethionate, 2 potassium gluconate, 1.8 calcium gluconate, 1.0 magnesium gluconate, 5 HEPES, and 2.5 sodium pyruvate as well as 5 mg/100 ml gentamicin, pH 7.4 ( 11 )] before 86 Rb uptake experiments were performed.$ P5 t3 f5 ^6 L% s
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mBSC1 cDNA isoforms. The cDNA encoding the long and short COOH-terminal spliced isoforms ofthe apical Na -K -2Cl cotransporter, L-mBSC1 and S-mBSC1, respectively, was inserted in theplasmid pSPORT1 (Invitrogen, Carlsbad, CA) as described ( 27 ). For preparation of a cRNA template, each cDNAisoform was linearized at the 3'-end using Xba I (New EnglandBiolabs, Beverly, MA) restriction enzyme, and cRNA was in vitrotranscribed, using the T7 RNA polymerase mMESSAGE kit (Ambion, Austin,TX). Transcript integrity was confirmed on agarose gels, andconcentration was determined by absorbance at 260 nm (DU 640, Beckman,Fullerton, CA) and by densitometry of the corresponding bands inagarose gels. cRNA was stored frozen in aliquots at 80°C until use.
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Assessment of theNa -K -2Cl cotransporter function. The function of the Na -K -2Cl cotransporter was assessed by measuring tracer 86 Rb uptake (New England Nuclear) in groups of10-15 oocytes. Four days after water or cRNA injection, 86 Rb uptake was measured with the followingprotocol: a 30-min incubation period in mild hypotonic (~160mosmol/kgH 2 O) K - and Cl -freemedium [(in mM) 68 sodium gluconate, 4.6 calcium gluconate, 1.0 magnesium gluconate, and 5 HEPES/Tris, pH 7.4], with 1 ouabain; thiswas followed by a 60-min uptake period in a mild hypotonic (~160mosmol/kgH 2 O) uptake medium [(in mM) 62 NaCl, 10 KCl, 1.8 CaCl 2, 1 MgCl -2, 5 HEPES/Tris, pH 7.4], with 1 ouabain and 2.0 µCi/ml of 86 Rb  . To study theeffect of PKA activation onNa -K -2Cl cotransporterfunction, during the present study the 86 Rb uptake in groups of oocytes was analyzed in the absence or presence of10 3 M concentration of the cell membrane-permeabledibutyryl-cAMP (Roche, Mannheim, Germany) plus 10 6 Mconcentration of the phosphodiesterase inhibitor IBMX (Sigma, St.Louis, MO). Although the hypotonic conditions used in our experimentsinhibit the endogenous Na -K -2Cl cotransporter that is expressed in X. laevis oocytes( 10 ), uptakes were also measured in water-injected oocytes(data not shown), and the mean values for water groups were subtractedfrom corresponding L-mBSC1 groups to assess total 86 Rb uptake due to L-mBSC1. Ouabain (Sigma)was added to prevent 86 Rb entry viaNa -K -ATPase. Uptakes were performed at32°C, and at the end of the uptake period oocytes were washed fivetimes in ice-cold uptake solution without isotope to remove tracer inextracellular fluid. Oocytes were then dissolved in 10% SDS. Traceractivity was determined for each oocyte by -scintillation counting.

Assessment of mBSC1 expression in oocyte plasma membranes. The surface expression of the L-mBSC1 isoform in the oocyte plasmamembrane was measured by fluorescence using an enhanced greenfluorescent protein (EGFP)-mBSC1 fusion construct. To obtain thepSPORT1/S-mBSC1-EGFP construct, the fragment containing the EGFPsequence was removed from pSPORT1/EGFP-L-mBSC1 and ligated intopSPORT1-S-mBSC1. L-mBSC1-EGFP cRNA was in vitro transcribed andmicroinjected into X. laevis oocytes in a volume of 50 nl at0.5 µg cRNA/µl (25 ng cRNA/oocyte). In coinjection experiments, oocytes were injected with the same volume and amount of L-mBSC1-EGFP plus varying amounts of S-mBSC1 cRNA. After injection, oocytes wereincubated at 16°C for 4-5 days. During this time, the incubation medium was changed every day. Individual oocytes were monitored forEGFP fluorescence (excitation = 488 n;. emittance = 505 nm), using a Zeiss confocal laser-scanning microscope LSM510 (×10objective lens, excitation with 488-nm line of a multiline argon ionlaser; Carl Zeiss). Fluorescent emissions were passed through a 505-nm band-pass filter. Water and wild-type L-mBSC1-injected oocytes wereused as controls. Background autofluorescence of water-injected oocyteswas minimized by adjusting brightness and contrast settings at aconstant pinhole size. These settings were then used to assess fluorescence of L-mBSC1-EGFP. To study the effect of PKA activation onthe surface expression of L-mBSC1-EGFP, the fluorescence intensity wasassessed in oocytes before and 30 min after exposure todibutyryl-cAMP IBMX. All confocal microscopic experiments wereperformed using conditions identical to those for the functionalexperiments (i.e., the same hypotonic solutions, incubation times, anddrug concentrations).

For membrane colocalization, oocytes were bathed at 4°C with 2 µMFM 46-4 [ N -(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)-hexatrienyl) pyridinium dibromide] (Molecular Probes, Eugene,OR), a fluorescent membrane marker. The low temperature was used tominimize endocytosis of FM 4-64 so as to ensure that fluorescence wasprimarily coming from dye localization in the plasma membrane. Fordetermination of fluorescence secondary to FM 4-64 membrane labeling,the excitation was at 543 nm and the emissions were passed through a650-nm band pass filter. To use this dye for colocalization experimentswith EGFP-tagged membrane proteins, the fluorescence emission of FM4-64 detected at ~670 nm becomes undetectable at wavelengths below580 nm (data from Molecular Probes). We found in preliminaryexperiments (data not shown) that FM 4-64 fluorescence becomesundetectable when measured at 505 nm, the emission wavelength for EGFPfluorescence. Fluorescence of both EGFP and FM 4-64 was quantified atequatorial focal sections of oocytes using SigmaScan Pro (JandelScientific, San Rafael, CA) image-analysis software.
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Western blotting. Oocyte homogenates were obtained 4 days after injection with water,S-mBSC1, or S-mBSC1-EGFP cRNA. Groups of 30-50 oocytes werehomogenized in 2 µl/oocyte of homogenization buffer [(in mM) 250 sucrose, 0.5 EDTA, and 5 Tris/HCl, pH 6.9, plus protease inhibitors],centrifuged twice at 100 g for 10 min at 4°C, and thesupernatant was recollected. Oocyte protein (6 oocytes/lane, 12 µl)was heated in sample buffer containing 6% SDS, 15% glycerol, 0.3%bromophenol blue, 150 mM Tris, pH 7.6, and 2% -mercaptoethanol, resolved by Laemmli SDS-PAGE (7.5%), and transferred in 10 × Tris/CAPS to a polyvinylidene difluoride membrane by semidryelectroblotting. Prestained molecular mass markers were used (Bio-Rad,Hercules, CA).5 J( u" ?$ V/ ?1 ]: |
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For immunodetection, we used a monoclonal antibody against GFP diluted1:1,000 (Clontech, Palo Alto, CA). The membrane was blocked for 1 h in 10% milk (10 mM Tris, pH 9.0, 150 mM NaCl, 0.1% Tween 20; TBS-T)and exposed to anti-GFP antibody diluted in 1% milk powder/TBS-Tovernight at 4°C. After a washing in TBS-T, the membrane was exposedto horseradish peroxidase-linked anti-mouse IgG secondary antibody(Amersham Life Science, Arlington Heights, IL) for 1 h at roomtemperature. After a washing in TBS-T, antigen-antibody complexes weredetected by autoradiography-enhanced chemiluminescence (ECL PlusWestern blot analysis system, Amersham Life Science).
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Statistical analysis. The significance of the differences between groups was tested byone-way ANOVA with multiple comparisons using Bonferroni correction.The significance within the same group was analyzed using the paired t -test. The results are presented as means ± SE.6 H  z( |: f4 D8 v5 D4 I

RESULTS& }! |; Y: k' R8 e; J5 |
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Surface expression of L-mBSC1-EGFP protein in X. laevis oocytes. X. laevis oocytes injected with L-mBSC1-EGFP cRNA exhibiteda ring of fluorescence at their surfaces, as determined by confocal fluorescence microscopy (Fig. 1 b ). Control oocytes injectedwith water showed no significant fluorescence at the emission andexcitation wavelengths for EGFP (Fig. 1 a ). To determinewhether the L-mBSC1-EGFP-specific fluorescence was mainly at the oocyteplasma membrane, we loaded oocytes with FM 4-64 under conditions thatwould suppress endocytosis of the dye, i.e., 4°C ( 22 ).FM 4-64 is a lipophilic fluorophore that has been used to measuresurface expression of other membrane transporters ( 19, 20 ), including the rat thiazide-sensitive Na -Cl cotransporter in X. laevis oocytes ( 17 ), because it possesses the optimal propertiesfor a fluorescent membrane marker. Figure 1 c shows FM 4-64 fluorescence at 4°C obtained in the same L-mBSC1-EGFP-injected oocyteshown in Fig. 1 b. The red FM 4-64-specific fluorescence wasdetected in a surface ring pattern similar to that forL-mBSC1-EGFP-specific fluorescence (Fig. 1 b ). As shown inFig. 1 d, the superimposition of the two images (Fig. 1, b and c ) gives a yellow signal, indicating colocalization of FM 4-64 and L-mBSC1-EGFP protein. We observed 99%surface colocalization of L-mBSC1-EGFP and FM 4-64 fluorescence in alltested oocytes, suggesting that the L-mBSC1-EGFP fluorescence measuredat equatorial confocal sections in oocytes was indicative of expressionon plasma membrane.. L# ^7 S& s8 \* J

Fig. 1. Top : representative confocal images of X. laevis oocytes injected with water ( a ) or 25 ng of long COOH-terminal isoform of murine apical bumetanide-sensitiveNa -K -2Cl cotransporter gene(mBSC1)-enhanced green fluorescent protein (EGFP) fusion construct(L-mBSC1-EGFP) cRNA with ( b ) or without the FM 4-64 fluorescence dye ( c ). d : superimpositon of b and c. Middle : confocal images ofoocytes injected with 25 ng of short COOH-terminal isoform ofmBSC1-EGFP fusion construct (S-mBSC1-EGFP; a ) or withL-mBSC1-EGFP before ( g ) and after exposure todibutyryl-cAMP IBMX ( h ). f : Western blot usinganti-GFP monoclonal antibody of proteins obtained from 6 oocytesinjected with S-mBSC1-EGFP cRNA, S-mBSC1 cRNA, or water. Bottom : representative images of oocytes coinjected with 25 ng L-mBSC1-EGFP plus 25 ng of S-mBSC1 cRNA before ( i ) andafter exposure to dibutyryl-cAMP IBMX ( j ) and ( k )or colchicine ( l )as indicated.' h7 L' e: i0 G0 y4 v

To further exclude the possibility that we were detecting primarilyEGFP fluorescence in vesicles below the plasma membrane, we alsoexamined fluorescence from an EGPF-labeled protein that we hadpreviously shown to be exclusively expressed just below the plasmamembrane. S-mBSC1 protein does not reach the plasma membrane butremains in what appears to be a submembranal pool of vesicles in X. laevis oocytes incubated under isotonic conditions ( 31 ). S-mBSC1-EGFP-injected oocytes exhibited no EGFPsurface fluorescence (Fig. 1 e ). The absence of surfacefluorescence in the S-mBSC1-EGFP-injected oocytes was not due to thelack of protein expression because S-mBSC1-EGFP protein was detected byWestern blot analysis using anti-GFP monoclonal antibody (Fig. 1 f ). In addition, S-mBSC1-EGFP protein retained the abilityto interact with L-mBSC1 (see below).. Z6 R/ P$ @; d

To further demonstrate that L-mBSC1-EGFP fluorescence representscotransporter expression in oocyte plasma membranes, we assessed thecorrelation between L-mBSC1-EGFP surface expression and L-mBSC1 functional expression (Fig. 2 ). Activityof the Na -K -2Cl cotransporterassessed by measuring 86 Rb uptake increased asa function of the amount of cRNA injected in oocytes, reaching aplateau at ~20 ng/oocyte. The surface expression of L-mBSC1-EGFPprotein increased similarly as a function of the amount of cRNAinjected. All of these results when taken together indicate that thesurface EGFP fluorescence detected in our L-mBSC1-EGFP-injected oocyteswas predominantly coming from cotransporter expression in plasmamembranes.

Fig. 2. Correlation between the dose dependency of L-mBSC1-EGFPsurface expression, assessed by laser-scanning confocal microscopy( ), and the dose dependency of 86 Rb uptake by L-BSC1 ( ) in Xenopus laevis oocytes. Each point represents the mean ± SE of 20 oocytes. AU, arbitrary units.% C) k9 W; P, c; h) J

Absence of cAMP on functional and surface expression of L-mBSC1 inX. laevis oocytes. We have shown previously ( 33 ) that microinjection of X. laevis oocytes with L-mBSC1 cRNA results in a significantincrease in 22 Na uptake that is not affectedby PKA activation. BecauseNa -K -2Cl cotransport in nativeTAL cells is enhanced by cAMP and PKA ( 25 ), this previousexperiment suggested that some factor or protein was missing inL-mBSC1-injected oocytes that would allow the activation of thiscotransporter by cAMP. To determine the effect of dibutyryl-cAMP IBMX on the surface expression of the mouseNa -K -2Cl cotransporter in X. laevis oocytes, we analyzed 20 oocytes injected withL-mBSC1-EGFP cRNA before and 30 min after addition ofdibutyryl-cAMP IBMX. As shown in Fig. 3 A, no change in fluorescenceintensity was observed after PKA activation. Oocytes exhibited a meanof 37.5 ± 3.1 arbitraty units (AU) before and 35.8 ± 5.8 AUafter addition of dibutyryl-cAMP IBMX. A representative example of thefluorescence observed in a single L-mBSC1-EGFP cRNA-injected oocytebefore and after addition of dibutyryl-cAMP IBMX is shown in Fig. 1, g and h, respectively. Note that fluorescenceintensity is similar in both pictures. Similarly, dibutyryl-cAMP IBMXfailed to increase bumetanide-sensitive 86 Rb uptake in L-mBSC1-injected oocytes [15.8 ± 0.7 vs. 17.4 ± 1.4 nmol · oocyte 1 · h 1,respectively, P = not significant (NS)], consistentwith our previous results ( 32 ). Thus the plasma membraneand functional expression ofNa -K -2Cl cotransport induced byheterologous expression of the L-mBSC1 isoform were not affected by PKAactivation with dibutyryl-cAMP IBMX.0 X" Q8 q5 _8 u/ N$ t

Fig. 3. Effect of dibutyryl-cAMP and IBMX on the function andsurface expression of L-BSC1 or L-BSC1-EGFP in groups of X. laevis oocytes analyzed in the absence (open bars) or presence(hatched bars) of 1 mM cAMP 1 µM IBMX. A : laser-scanningconfocal microscopy of 80 oocytes from 5 different frogs injected withL-mBSC1-EGFP cRNA before and 30 min after dibutyryl-cAMP IBMX exposure.Representative images are shown in Fig. 1, g and h. B : bumetanide-sensitive 86 Rb uptake in 2 groups of oocytes injectedwith 25 ng of L-BSC1 cRNA. Bumetanide was added to the preincubationand uptake medium at 10 4 M. Each bar represents themean ± SE of ~100 oocytes from 5 different frogs.

Coexpression of the long and short mBSC1 isoform and regulation bycAMP. In native TAL cells, L-mBSC1 and S-mBSC1 isoforms are coexpressed inthe apical membrane ( 27 ). We have also previously shown that S-mBSC1 exerts a dominant-negative-like effect on thebumetanide-sensitive Na -K -2Cl cotransport activity of L-mBSC1 expressed in oocytes and that theinhibitory effect of S-mBSC1 was largely reversed by addition ofcAMP IBMX ( 33 ). Given that S-mBSC1 is exclusivelyexpressed in a submembranal pool when expressed alone( 31 ), we hypothesized that the dominant-negative effect ofS-mBSC1 on L-mBSC1 function may involve modulation in trafficking ofL-mBSC1 to or from the plasma membrane." U. T( _4 H7 f

To begin to assess the mechanism by which S-mBSC1 regulates L-mBSC1 ina cAMP-dependent manner, we examined the effects of the S-mBSC1 isoformon the surface expression of the L-mBSC1 isoform. Thus we assessed thefluorescence intensity in oocytes injected with L-mBSC1-EGFP cRNA aloneor with S-mBSC1 cRNA. We also measured 86 Rb uptake in the same batch of X. laevis oocytes injected withL-mBSC1 cRNA alone or together with S-mBSC1 cRNA. As shown in Fig. 4 A, the fluorescence intensityof oocytes injected with L-mBSC1-EGFP alone was significantly higher(73.9 ± 5.3 AU) than intensity observed in oocytes coinjectedwith L-mBSC1-EGFP and S-mBSC1 (42.9 ± 3.5 AU, P images from aL-mBSC1-EGFP oocyte and a L-mBSC1-EGFP S-mBSC1 oocyte is presented inFig. 1, g and i, respectively. Consistent with the reduced surface expression of L-mBSC1-EGFP induced by S-mBSC1 (Fig. 4 ), functional analysis confirmed a significant reduction inbumetanide-sensitive 86 Rb uptake inL-mBSC S-mBSC1-injected oocytes (7.76 ± 0.5 nmol · oocyte 1 · h 1 )compared with values obtained in L-mBSC1 oocytes (15.8 ± 0.7 nmol · oocyte 1 · h 1, P and in L-mBSC1 bumetanide-sensitive 86 Rb transport showed similar relationships tothe amount of coinjected S-mBSC1. As shown in Fig. 5 A, when oocytes injected with25 ng of L-mBSC1-EGFP cRNA were additionally injected with increasing concentrations of S-mBSC1 cRNA, from 6 to 50 ng/oocyte, a significant dose-dependent decrease in fluorescence intensity was observed. Weobserved a dose-dependent inhibition of 86 Rb uptake within the same range of S-mBSC1 coinjections (Fig. 5 B ). Thus the lower the L-mBSC1-to-S-mBSC1 ratio, the lowerthe surface expression and activity of theNa -K -2Cl cotransporter.- H. s$ Y8 [$ m1 b/ ]' _
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Fig. 4. Reduction of theNa -K -2Cl cotransporter surfaceand functional expression induced by S-mBSC1 cRNA in X. laevis oocytes injected with 25 ng of L-mBSC1-EGFP or L-BSC1 cRNAalone (open bars) or together with 25 ng of S-mBSC1 cRNA (filled bars). A : laser-scanning confocal microscopy fluorescenceintensity. Representative images are depicted in Fig. 1, g and i.** P B : bumetanide-sensitive 86 Rb uptake innmol · oocyte 1 · h 1.Bumetanide was added to the preincubation and uptake mediums at10 4 M concentration. Each bar depicts the mean ± SEof 100 oocytes from 5 different frogs. * P* l8 f/ I0 w5 a! l5 G

Fig. 5. Effect of increasing concentrations of S-mBSC1 cRNA on surfaceexpression of L-mBSC1-EGFP and functional expression of L-mBSC1 in X. laevis oocytes. A : fluorescence intensity inoocytes injected with 25 ng of L-mBSC1-EGFP cRNA alone or together withincreasing amounts of S-BSC1 cRNA as indicated. ** P B :bumetanide-sensitive 86 Rb uptake was assessedin groups of oocytes that were injected with 25 ng of L-mBSC1 cRNAalone or together with increasing amounts of S-BSC1 cRNA as stated.Bumetanide was added to the preincubation and uptake mediums at10 4 M concentration. Each bar depicts the mean ± SEof 50 oocytes from 3 different frogs. * P. R. O( H; C% p1 @
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Given the reduction in Na -K -2Cl cotransporter surface expression and function induced by S-mBSC1isoform in X. laevis oocytes, we measured the effect ofdibutyryl-cAMP IBMX on the fluorescence intensity and 86 Rb uptake in coinjected oocytes. As shown inFig. 6 A,the reduction in the surface expression of L-mBSC1-EGFP in S-mBSC1coinjected oocytes was partially reversed by the addition ofdibutyryl-cAMP IBMX (30.3 ± 4.8 without vs. 49 ± 7.1 AUwith dibutyryl-cAMP IBMX, P L-mBSC1-EGFP and S-mBSC1 isoformsbefore and 30 min after exposure to dibutyryl-cAMP IBMX is shown inFig. 1, i and j, respectively. A clear increasein fluorescence intensity is observed in Fig. 1 j comparedwith Fig. 1 i. As depicted in Fig. 6 B, similarresults were obtained when functional expression was assessed(7.76 ± 0.5 in the absence vs. 10.9 ± 0.7 nmol · oocyte 1 · h 1 in the presence of dibutyryl-cAMP IBMX, P Fig. 6 C shows that the S-mBSC1-EGFP construct isalso able to reduce the function of L-mBSC1 and that this reduction canalso be abrogated by dibutyryl-cAMP IBMX. Thus in X. laevis oocytes, the surface expression, and hence the function, of theNa -K -2Cl cotransporter can bemodulated by dibutyryl-cAMP only when L-mBSC1 and S-mBSC1 isoforms arecoexpressed but not when the L-mBSC1 isoform is expressed alone (Fig. 3 ). Furthermore, similar 86 Rb responses todibutyryl-cAMP IBMX are observed whether the EGFP reporter is onS-mBSC1 or L-mBSC1. Moreover, experiments were performed in whichL-mBSC1 and S-mBSC1-EGFP were coinjected and EGFP fluorescenceintensity was assessed in the absence and presence of cAMP. NoS-mBSC1-EGFP fluorescence was detected 30 min after cAMP[H 2 O injected 0.18 ± 0.1 AU ( n = 10); L-mBSC1 S-mBSC1 coinjected, 0.28 ± 0.06 AU ( cAMP; n = 18) and 0.23 ± 0.04 AU ( cAMP; n = 19); P = NS compared withH 2 O injected], suggesting that S-mBSC1 may be rapidlyremoved from the membrane after coinsertion with L-mBSC1 after cAMP.- e, v: Z/ z2 b* A

Fig. 6. Effect of dibutyryl-cAMP and IBMX on S-mBSC1-inducedreduction of surface expression and in S-mBSC1 or S-mBSC1-EGFP-inducedreduction in functional expression of theNa -K -2Cl cotransporter. A : fluorescence intensity in oocytes injected with 25 ng ofL-mBSC1-EGFP cRNA alone (open bar) or together with 25 ng of S-mBSC1 inthe absence (filled bar) or presence (hatched bar) of 1 mM cAMP and 1 µM IBMX. The filled and hatched bars depict the fluorescenceintensity in the same group of oocytes before and 30 min after exposureto dibutyryl-cAMP IBMX. Representative images are shown in Fig. 1, i and j. B : bumetanide-sensitive 86 Rb uptake in oocytes injected with 25 ng ofL-mBSC1 cRNA alone (open bar) or together with 25 ng of S-mBSC1 cRNA inthe absence (filled bar) and presence (hatched bar) ofdibutyryl-cAMP IBMX. C : bumetanide-sensitive 86 Rb uptake in oocytes injected with 25 ng ofL-mBSC1 cRNA alone (open bar) and together with 25 ng of S-mBSC1-EGFPcRNA in the absence (filled bar) and presence (hatched bar) ofdibutyryl-cAMP IBMX. In both B and C, bumetanide,cAMP, and IBMX were added to the preincubation and uptake media at10 4, 10 3, and 10 6 Mconcentration, respectively. Each bar represents the mean ± SE of40 oocytes from 3 different frogs.$ M, Z* i, ^( N
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The results in Fig. 6 suggest that the mechanism by which the S-mBSC1isoform reduces the function of theNa -K -2Cl cotransporter includesa reduction in the surface expression of L-mBSC1, which is partiallyabrogated after PKA activation with dibutyryl-cAMP IBMX. The modulationof L-mBSC1 surface expression could result from alterations intrafficking to (exocytosis) or from (endocytosis) the plasma membrane.To study this possible mechanism, we assessed the effects ofcolchicine, an inhibitor of microtubules ( 26 ), on thesurface and functional expression of theNa -K -2Cl cotransporter inoocytes coinjected with L-mBSC1-EGFP or L-mBSC1 cRNA, together withS-mBSC1 cRNA. As shown in Fig. 7 A, the fluorescence intensityin L-mBSC1-EGFP S-mBSC1-coinjected oocytes was lower than oocytes aloneand the dibutyryl-cAMP IBMX increment was prevented by prior exposureto colchicine. Colchicine alone had no effect on the fluorescenceintensity either in the L-mBSC1-EGFP-injected group (data not shown) orin the L-mBSC1-EGFP S-mBSC1-coinjected group. Representative images ofan oocyte exposed to colchicine alone or to colchicine anddibutyryl-cAMP IBMX are shown in Fig. 1, k and l,respectively. No change in fluorescence intensity is observed. Similarresults were observed when 86 Rb uptake wasassessed. The 86 Rb uptake observed inL-mBSC1 S-mBSC1-injected oocytes (6.2 ± 0.6 nmol · oocyte 1 · h 1 )was not affected in the presence of colchicine (5.9 ± 0.8 nmol · oocyte 1 · h 1 )or by dibutyryl-cAMP IBMX in the presence of colchicine (6.3 ± 0.8 nmol · oocyte 1 · h 1 ),suggesting that blocking the exocytosis mechanisms with colchicine prevents the L-mBSC1 response to dibutyryl-cAMP IBMX (Fig. 7 B ).

Fig. 7. Colchicine prevents the effect of the dibutyryl-cAMP andIBMX on surface expression and transport in L-mBSC1 and S-mBSC1coinjected oocytes. A : fluorescence intensity in oocytesinjected with 25 ng of L-mBSC1-EGFP cRNA alone (open bar) or togetherwith 25 ng of S-mBSC1 (filled bars) with or without colchicine anddibutyryl-cAMP IBMX as indicated. The filled bars depict thefluorescence intensity in the same group of oocytes before and 30 minafter exposure to colchicine and 30 min after exposure to colchicineand dibutyryl-cAMP IBMX. Representative images are shown in Fig. 1, k and l. ** P B : bumetanide-sensitive 86 Rb uptake in oocytes injected with 25 ng ofL-mBSC1 cRNA alone (open bar) and together with 25 ng of S-mBSC1 cRNA(filled bars) with or without colchicine and/or dibutyryl-cAMP IBMX asindicated. Colchicine was added to the preincubation medium at 20 µM,15 min before the addition of cAMP IBMX. Each bar represents themean ± SE of 25 oocytes from 3 different frogs.* P0 ]% \  G3 ~' b
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DISCUSSION
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The present work describes the effect of the COOH-terminaltruncated, alternatively spliced isoform of the murine SLC12A1 gene S-mBSC1 on the surface and functionalexpression of the Na -K -2Cl cotransporter (L-mBSC1). Using the X. laevis oocyte'sheterologous expression system, we show by confocal laser imageanalysis that the S-mBSC1 isoform exerts a dominant-negative effect onthe surface expression of the longer isoform L-mBSC1, which results ina reduction of the cotransporter activity (Figs. 1, 4, and 5 ). Thedominant-negative effect on surface expression, and hence functionalactivity, was partially abrogated by PKA activation withdibutyryl-cAMP IBMX (Fig. 6 ). Finally, we demonstrate that the effectof dibutyryl-cAMP IBMX was prevented by the exocytosis inhibitorcolchicine (Fig. 7 )./ n$ F+ W( Z; H" A2 }9 `
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It is well established that generation of cAMP by hormones, such asvasopressin, activates transepithelial salt transport in the murine TAL( 1, 12, 15 ). However, the mechanism of this activation wasunknown. We previously showed that PKA activation with cAMP did notenhance the uptake of 22 Na by L-mBSC1expressed in X. laevis oocytes, suggesting that other factors/proteins not present in oocytes are required to reconstitute the observed cAMP activation of apicalNa -K -2Cl cotransport in murineTAL ( 33 ). We had previously suggested that the criticaladditional protein required for cAMP-dependent modulation of thefunctional cotransporter isoform, L-mBSC1, was the short COOH-terminalmBSC1 splice variant. This was based on the following observations.First, both isoforms are expressed in the mouse TAL. The L-mBSC1isoform was expressed almost equally in both medullary (MTAL) andcortical TAL (CTAL) segments, but expression of the S-mBSC1 isoform washighest in the MTAL and diminished significantly along the CTAL towardthe cortical surface ( 27 ). This gradient of S-mBSC1expression parallels the medullary-to-cortical magnitude ofhormone-induced cAMP accumulation in the TAL and may account for theobservation that vasopressin enhances salt reabsorption predominantlyin MTAL rather than CTAL ( 14 ). In addition, we had alsopreviously reported that the short COOH-terminal isoform, S-mBSC1,exerts a dominant-negative-like effect on ion transport by the L-mBSC1Na -K -2Cl cotransporter( 33 ). This was not due to competition for translation inS-mBSC1 L-mBSC1-coinjected oocytes because unrelated cRNAs such asrenin or the Shaker K channel did notsignificantly affect transport by L-mBSC1. Thus S-mBSC1 would reducethe functional expression of L-mBSC1 in native TAL cells. Importantly,we showed that cAMP reversed the negative effect of S-mBSC1 on L-mBSC1function ( 33 ). The latter could provide a mechanism forcAMP-mediated activation ofNa -K -2Cl cotransport in nativeTAL cells.

There is precedence for a dominant-negative type of effect ofalternatively spliced isoforms of several genes, including several renal cotransporters ( 9 ). For example, theNa -phosphate cotransporter gene can express truncatedisoforms with dominant-negative effects on the cotransporter function( 37 ). One interesting example of dominant-negativeregulation occurs in the human KvLQT1 K channel that isassociated with the congenital long QT syndrome. One of the twoalternatively spliced KvLQT1 variants forms the functionalK channel. The other, a COOH-terminal truncated isoform,does not possess channel function but exerts a strong dominant-negative effect on channel function ( 4 ). It has been shown thattransgenic mice overexpressing the truncated isoform develop severalinteresting cardiac arrhythmias ( 5 ) and that, in humanswith the recessive form of the long QT syndrome (Jervell andLange-Nielsen syndrome), mutations in the dominant-negative isoformcorrelate with the phenotype of the cardiac arrhythmia( 24 ). The mechanisms by which truncated isoforms producetheir negative effects are still poorly understood.
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The competition between intracellular vesicles containing differentisoforms or formation of heterodimers between isoforms has beensuggested as possible mechanisms. In the glucocorticoid receptor, theformation of nonfunctional heterodimers seems to be at least part ofthe mechanism by which the dominant-negative isoform reduces thefunction of the receptor ( 29 ). However, to our knowledgeno study has addressed this issue in membrane transporters.# h2 p. ^2 U) a  D& Y3 T1 S/ D
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The interaction between the long and short isoforms of mBSC1 could bedue to competition between S-mBSC1 and L-mBSC1 containing vesicles.L-mBSC1 has been detected in subapical vesicles ( 28 ). Thustrafficking of L-mBSC1 vesicles to the apical membrane could play arole in cotransporter regulation. We observed no S-mBSC1-EGFP fluorescence in L-mBSC1 S-mBSC1-EGFP-coinjected oocytes after cAMPtreatment, suggesting that S-mBSC1-EGFP protein may be rapidly removedfrom the membrane after coinsertion with L-mBSC1. In addition, we haveshown that S-mBSC1 antibody staining was predominantly in a subapicaldistribution in mouse kidney ( 27 ). Therefore, it ispossible that the alternatively spliced S-mBSC1 isoform affects theinteraction of the transporter proteins with the cytoskeleton ( 7 ) and/or the vesicular trafficking machinery. If so,then coassociation of S-mBSC1 and L-mBSC1 isoforms may result in"trapping" of the L-mBSC1 complex within subapical vesicles.
9 S9 Z2 F% \& n4 {: ?- U$ ?8 \
To begin to understand the interaction between the short and longisoforms of the murine Na -K -2Cl cotransporter, we assessed the effect of the short mBSC1 isoform on thesurface expression of an EGFP-tagged long mBSC1 isoform using the X. laevis heterologous expression system. Several lines ofevidence suggest that the EGFP fluorescence observed in our presentstudy quantified cotransporter localization in the oocyte plasmamembrane. First, EGFP has been used to assess surface expression ofmany membrane proteins expressed in oocytes ( 2, 3, 8, 21 )as well as cultured cells ( 30 ). In oocytes, intracellular EGFP fluorescence is not detected with confocal microscopy because thelaser does not penetrate deeply enough to visualize the intracellular EGFP pool at equatorial sections in these large cells. Even if therewere some intracellular light penetration, the distribution of the EGFPsignal in these large cells is so diffuse that it would likely fallbelow the level of detection ( 3 ). Second, we show in Fig. 1 that L-mBSC1-EGFP fluorescence is colocalized with the membranemarker FM 4-64 under conditions that would reduce FM 4-64 endocytosisand potential labeling of a subplasma membrane pool. Third, we showthat the S-mBSC1-EGFP isoform, which under isotonic conditions remainsin the submembranal compartment of the oocyte ( 31 ), wasnot detected by confocal analysis. Finally, we show in Fig. 2 adirect correlation between EGFP fluorescence and the magnitude ofbumetanide-sensitive 86 Rb uptake. When takentogether, these results demonstrate that the L-mBSC1-EGFP fluorescencedetected in the present study was predominantly at the oocyte plasmamembrane. While our results in oocytes suggest one mechanism forregulation of BSC1 function by cAMP, verification of this mechanism inTAL cells would be informative; however, no one has been able to stablyexpress BSC1 in a mammalian cell line.

Our results indicate that the S-mBSC1 isoform regulates cotransporterfunction by inhibiting L-mBSC1 trafficking to the plasma membrane. Thefollowing observations support this conclusion. The alternativelyspliced S-mBSC1 isoform reduces, in a dose-dependent manner, both 86 Rb uptake by, and plasma membrane expressionof, the L-mBSC1 Na -K -2Cl cotransporter. We also show that in the presence of the S-mBSC1 isoform, but not in its absence, the surface expression of L-mBSC1-EGFP is increased by PKA activation with dibutyryl-cAMP IBMX. This increasein plasma membrane expression is also associated with an increase in 86 Rb uptake by the cotransporter. Finally, thedibutyryl-cAMP IBMX-induced increase in surface and functionalexpression was blocked by colchicine, an inhibitor of the exocytosismachinery. Thus the presence of the S-mBSC1 isoform precludes theL-mBSC1 complex from migrating to the plasma membrane, and thisinhibitory effect is abrogated by cAMP. To our knowledge, this is thefirst study to address the mechanisms of interaction betweenalternatively spliced isoforms of membrane cotransporters. Furtherstudies will be required to determine whether S-mBSC1 and L-mBSC1 formheterodimers and the role of PKA phosphorylation processes on thisinteraction and the trafficking mechanism.
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ACKNOWLEDGEMENTS: i: P+ ^. L: z; ]8 g, }/ j
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We are grateful to Dr. Jorge Sosa-Melgarejo for help in using thelaser-scanning confocal microscope and to members of the MolecularPhysiology Unit for suggestions and assistance.5 o. \/ }- F) Q& \9 y+ l8 W
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