Tissue and Cellular Localization of a Novel Polycystic Kidney Disease–Like Gene Product, Polycystin-L
Nuria Basora*,
Hideki Nomura,
Urs V. Berger,
Cherie Stayner,
Lei Guo,
Xiaohua Shen and
Jing Zhou
Renal Division and Membrane Biology Program, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts.
*Dr. Basora’s current affiliation: Département de physiologie et biophysique, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada.
Correspondence to Dr. Jing Zhou, Harvard Institutes of Medicine, Room 520, 77 Louis Pasteur Avenue, Boston, MA 02115. Phone: 617-525-5860; Fax: 617-525-5861; E-mail: zhou{at}rics.bwh.harvard.edu
ABSTRACT. Polycystin-L (PCL), the third member of the polycystinfamily of proteins, functions as a Ca2+-modulated nonselectivecation channel when expressed in Xenopus oocytes. Polycystin-1and -2 are mutated in autosomal-dominant polycystic kidney disease(ADPKD), but the role of PCL in disease has not been determined.In this study, an anti-peptide polyclonal antiserum was generatedagainst the carboxyl terminal domain of human PCL and used todetermine the patterns of expression and distribution of PCLby indirect immunofluorescence in both developing and adultmice. The results show that PCL is predominantly expressed inadult mouse tissues and has a more restricted pattern of expressionthan either polycystin-1 or -2. In the kidney, PCL expressionwas first detected at E16, and levels increased into adulthood.Localization of PCL was predominantly found in the apical regionof the principal cells of inner medullary collecting ducts.PCL was also found in discrete cell types of the retina, testis,liver, pancreas, heart, and spleen, but it was not detectedin the lung. These data in combination with evidence of PCLchannel activity are crucial for elucidating the physiologicrole of this novel cation channel and may shed light on thefunction of inner medullary collecting ducts and polycystins.The expression pattern of PCL suggests that it is unlikely tobe a candidate gene for ADPKD, but it remains a potential candidatefor other as yet unmapped human cystic disorders.
Polycystins comprise an expanding family of membrane proteinswith five members identified to date (1–6). Two membersof the polycystin family, polycystin-1 (PC1) and PC2, are mutatedin autosomal-dominant polycystic kidney disease (ADPKD). ADPKDis a common hereditary disorder that affects 1 in 1000 individualsand is associated with a 50% incidence of end-stage renal disease(7). A hallmark of this disease is the progressive replacementof renal parenchyma by fluid-filled cysts, although ADPKD alsoaffects other organs such as the liver, spleen, and pancreas.
Polycystin-L (PCL), the third cloned member of the family, ispredicted to be an integral membrane protein with 6 transmembranedomains. Its protein sequence shares 71% homology with PC2,and both show a striking structural similarity to Na+, K+, andCa2+ channels and to the Drosophila trp and trp-like capacitativecalcium entry channels (4,8,9). Both proteins contain recognizablemotifs, such as an EF-hand domain, with potential calcium-bindingand regulatory functions, as well as a coiled-coil domain thatmay mediate interaction with other proteins. The coiled-coildomain in PC2 has been shown to mediate homodimerization (10,11).
A fourth member of the polycystin family, polycystin-REJ, wasrecently identified. This member shows highest homology to PC1and to the sea urchin receptor for egg jelly (suREJ). Additionally,the distribution of PCREJ appears to be restricted to the testis,and it is hypothesized that it may be involved in human fertilization(5). Most recently, we and others (6,9) have identified a fifthmember of the polycystin family, polycystin-L2 (PCL2), encodedby PKD2L2. PCL2 shares significant homology with PCL and PC2,with amino acid identities of 58% and 59%, respectively. PKD2L2transcript is expressed in human brain, kidney, testis, andHepG2 cells, and there are at least three alternatively splicedvariants that were differentially expressed. PKD2L2 was mappedto human chromosome 5q31 and Pkd2L2 to mouse chromosome 18 inband C.
Expression studies have shown that the highest levels of PC1are found during development with low levels in adult life (12–14).PC2 is also expressed during development, but high levels ofexpression are maintained in the kidney in adult life (15–17),suggesting a more specific role for this protein in the maintenanceof differentiated renal tubular architecture. An essential developmentalrole for these proteins has been demonstrated by targeted disruptionof their genes in mice (15,18), because homozygous mice foreither mutation develop embryonic renal and pancreatic cystsand die perinatally.
Whether or not PCL plays a role in kidney development remainsto be established. The mouse orthologue, PkdL, is deleted togetherwith other genes, such as Pax2, in a 7-cM region in a mouseline presenting with kidney and retinal defects (Krd) (19).Homozygous Krd mice die early in utero, and heterozygotes developaplastic, hypoplastic, and cystic kidneys. Retinal defects inheterozygotes include a reduction in the cell density of theinner nuclear and ganglion cell layers as well as abnormal electroretinograms(19–20).
An emerging view is that PC1-like proteins are receptors thatregulate channel activity of PC2-like proteins through formationof a polycystin channel that can activate a diverse selectionof signaling pathways. PCL was the first member of this novelfamily to be identified to function as a channel and has beenshown to be permeable to Na+, K+, and Ca2+, which can be modulatedby Ca2+ (21). Subsequently, we and others (22–25) alsodemonstrated channel activities for PC2 that were shown to behighly similar to PCL. However, the physiologic role of polycystinchannels remains unknown. To extend the current knowledge ofpolycystins and as a first step in determining the physiologicrole of PCL, we have raised a polyclonal antiserum against apeptide in the intracellular carboxyl-terminal tail of humanPCL. We have determined the patterns of expression and distributionof this newly identified protein in fetal and adult mouse tissuesusing this antiserum. Our data demonstrate that PCL is expressedin a number of mouse tissues but is restricted to discrete celltypes within these organs and that these expression patternsare distinct from those of PC1 and PC2.
Generation of PCL Antibodies
Polyclonal antibodies were raised by immunizing New Zealandrabbits against the KLH-conjugated peptide TLRELGHAEHEITELTATFTKFD(Research Genetics Inc., Huntsville, AL), which correspondsto amino acids 624 to 646 of the predicted translation sequencefor human PCL (4). This region of PCL is located in the intracellularcarboxyl-terminal tail, which exhibits low homology to the otherknown polycystins. Antibody specificity was verified by Westernblot analysis with recombinant fusion proteins, including (3.1c,3.2c, and 3.3c) or excluding (3.4c) the region that containedthe peptide sequence used for immunization. The relevant fragmentsof PCL were amplified by PCR by using primers containing EcoRIand XhoI linkers. Recombinant fusion proteins were obtainedby subcloning the PCR-generated fragments into the EcoRI andXhoI sites of the pGEX4T-1 expression vector (Amersham-Pharmacia,Piscatway, NJ). Subclones were sequenced to confirm nucleotideand reading frame fidelity with respect to PCL. Recombinantproteins were expressed in DH5 and were purified by using glutathione-sepharose4B(Amersham-Pharmacia) according to the manufacturer’s protocol.
Western Blot Analysis
Purified recombinant fusion proteins were loaded onto a 4 to20% gradient gel (Biorad, Hercules, CA) and electrotransferredonto Hybond ECL nitrocellulose membranes (Amersham-Pharmacia).Membranes were incubated with anti-PCL (1/1000) or anti–glutathionesufatransferase (1/2000) before incubation with a peroxidase-conjugatedsecondary antibody (1/1000; Amersham) and were detected by enhancedchemiluminescense (ECL, Amersham-Pharmacia).
Indirect Immunofluorescence
Kidneys from 129SvJ mice were collected at stages E15, E16,E17.5, newborn (NB), 6 wk, and adult (12 to 18 mo). Other organswere collected at NB, 6 wk, and adult. All tissues were embeddedin optimal cutting temperature compound (VWR, Boston, MA). Aminimum of two individual mice were tested at each stage indicated.Mouse tissues were cross-sectioned at 5 µm in a cryostatat -20°C and were air-dried for 30 min. Optimization ofthe anti-PCL antiserum for indirect immunofluorescence witha variety of fixation conditions revealed that this antiserumonly worked on frozen sections fixed with methanol. No specificstaining was observed on the following types of fixed tissues:frozen sections fixed with 2% paraformaldehyde, acetone, ethanol,or on paraffin sections either with or without antigen-demaskingtechniques. Positive labeling was eliminated by preincubationof the antiserum with the immunizing peptide (50µM; Figure 2C),and no specific signal was observed with preimmune serum(data not shown).
Figure 2. PCL is expressed in the developing and adult mouse kidney. (A) Schematic representation of the different regions of the kidney, PCL expression is restricted to the boxed region. (B) PCL expression at E16. Staining of the papillary urothelium is nonspecific. (C) anti-PCL antiserum preincubated with the immunizing peptide. (D) PCL expression in adult kidney. (E) Reverse transcriptase–PCR (RT-PCR) amplification of PKD in adult mouse papilla. PKD𝓁-specific primers in exon 2 and exon 5 of the mouse cDNA sequence amplify a product of 611-by (+RT). Each reverse transcription reaction was performed in duplicate with one of the two samples lacking the reverse transcriptase anzyme (RT-). This sample was included in each PCR reaction. The H2O lane was a PCR control that contained water instead of cDNA. Glyceraldehyde-3-phosphate dehydrogenase–PCR (GAPDH-PCR) reactions were performed in parallel with identical amounts of cDNA. Magnification: x20 in B through D.
Sections were fixed in methanol (prechilled) at -20°C for10 min. The sections were then incubated for 1 h at room temperaturewith primary polyclonal antibodies and diluted as follows: anti-PCL,1/100; aquaporin-1 (AQP1), 1/100; and AQP3, 1/100 (generousgifts of Dr. Dennis Brown, Charlestown, MA). For colabelingexperiments, antibodies were incubated sequentially in the followingorder: rabbit anti-PCL, goat anti-rabbit-FITC (Vector, Burlingame,CA), AQP1 or AQP3, goat anti-rabbit-CY3 (Sigma Chemical, St.Louis,MO). Control sections were individually incubated with eachantibody to establish the patterns of expression. Pictures weretaken under fluorescence illumination and a spot digital camera.Sections incubated with anti-PCL and preimmune sera were photographedwith the same exposure settings, and contrasts were then identicallyenhanced with Adobe Photoshop software (Adobe, Mountain View,CA).
Reverse Transcriptase–PCR of PkdL
The papilla region was dissected from adult mouse kidneys (n= 2). Total RNA was isolated by using Trizol (Life Technologies,Rockville, MD), and 5 µg was subjected to DNase I digestionfor 15 min. First-strand cDNA synthesis was performed by usingthe First-Strand Synthesis System (Life Technologies) for reversetranscriptase–PCR (RT-PCR) with oligo-dT primers. Primersin exon 4 (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]forward: 5'-TCACCATCTTCCAGGAGCG-3') and exon 8 (GAPDH reverse:5'-CTGCTTCACCACCTTCTTGA-3') of the rat GAPDH gene were usedin a PCR reaction to confirm the quality of the cDNA and tostandardize the amount of cDNA obtained from different reversetranscription reactions. The GAPDH PCR product was observedafter 18 to 24 cycles with an annealing temperature of 52°Cfor 30 s and an extension at 72°C of 30 s. The PkdL-specificfragment (confirmed by gel-isolation of the fragment and directsequencing) was obtained by using a forward primer in exon 2of the mouse cDNA (F605: 5'-CACAGCCGAGAACAGGGAGCTT-3') and areverse primer in exon 5 (F605: 5'-CAGTCCCTCCTGGAGTCCTTGG-3')with the following parameters: 94°C for 10 s, 67°C for30 s, 72°C for 40 s. Products were observed between cycles38 to 40. PCR reactions were repeated at least three times each.
Characterization of PCL Antibodies
Polyclonal antibodies against human PCL were generated to apeptide sequence located in the intracellular carboxyl-terminaltail (Figure 1A), which exhibits low homology to other knownpolycystins. Recombinant GST-PCL fusion proteins, with or withoutthe immunizing peptide sequence, were generated to test thespecificity of the antibody (Figure 1B). Western blot analysisof purified GST-PCL fusion proteins with anti-GST antibodiesdetected all four fusion proteins (Figure 1C, right panel).However, anti-PCL antibodies recognized only those GST-PCL fusionproteins that contained the epitope (Figure 1C, left panel;3.1c, 3.2c, and 3.3c) and not the fusion protein lacking theepitope (Figure 1C, left panel; 3.4c), thus confirming the specificityof the antibody. The smaller, less intense bands are likelydegradation products, which are commonly seen in fusion proteinpreparations. We could not detect PCL by Western blot in wholekidney tissue protein homogenates, probably due to the low abundanceof this protein, because human PCL transiently overexpressedin HEK293 cells was detected only after immunoprecipitation(data not shown).
Figure 1. Characterization of anti-human polycystin-L (PCL) antiserum. (A) Proposed membrane topology for PCL. (B) Schematic representation of the regions of the intracellular C tail of human PCL fused with GST. TM6 indicates transmembrane domain 6; predicted EF-hand; spans the peptide sequence used for immunization. (C) Western blot analysis of the purified glutathione sufatransferase fusion proteins shown in panel B; left panel, blotted with an anti-PCL antiserum; right panel, anti-GST.
Expression of PCL in Mouse Kidney
We first examined PCL expression in the kidney. Optimizationof the anti-PCL antiserum for indirect immunofluorescence witha variety of fixation conditions revealed that this antiserumonly worked on frozen sections fixed with methanol (see Materialsand Methods). PCL expression appeared at E16 in the inner medullaregion (Figure 2B) in a small number of tubules. No expressionwas detected in the nephrogenic zone, including the uretericbud, comma and S-shaped bodies, and undifferentiated mesenchyme,at all embryonic stages studied (E15-NB). The fluorescence seenin the papillary epithelium is probably nonspecific, as determinedby comparison with the anti-PCL serum neutralized with the immunizingpeptide (Figure 2, B and C). In the adult, localization remainedmuch the same, although the number of tubules and staining intensitywere increased (Figure 2D). We next isolated the papilla regionsfrom adult kidney samples to amplify the mRNA coding for PCLby RT-PCR. The primers were designed to span several exons toensure amplification of mRNA as opposed to genomic DNA. A bandof the correct expected size, 611 bp, was amplified in the papillasamples (Figure 2E), and sequencing confirmed that this bandcorresponded to the expected sequence of PkdL.
To determine the precise tubular segment that expressed PCL,we carried out double labeling of PCL and AQP1 and AQP3. Aquaporinscomprise a family of water channels, the members of which areexpressed in a nephron segment–specific pattern in thekidney (26,27). AQP1 was located in the apical membrane of epithelialcells of the proximal tubules and descending thin limbs (Figure 3A,blue box). AQP3 was found exclusively in the basolateralmembranes of the principal cells of collecting ducts from thecortex to the inner medulla (Figure 3A, red box). Double labelingof AQP1 and PCL illustrated that PCL expression was found adjacentto the descending thin limbs labeled by AQP1 in the inner medulla,i.e., these two proteins were restricted to distinct segmentsof renal tubules (Figure 3, B and C). Colabeling of PCL andAQP3, however, revealed that these two proteins were localizedin the same tubules in the inner medulla (Figure 3, D and E),where all tubules positive for PCL labeling were also positivefor AQP3 labeling. PCL expression was restricted to inner medullarycollecting ducts (Figure 3A, green box), and no signal was detectedin the cortical or outer medulla collecting ducts. Careful inspectionof the colabeled tubules further revealed that PCL was predominantlylocated in the apical region of those cells expressing AQP3(Figure 3E, arrows). Intercalated cells, i.e., those cells notexpressing AQP3, were also negative for PCL expression (Figure 3E,i). PCL was not detected in proximal tubules, descendinglimbs, ascending thick limbs, or in the distal convoluted tubules.Glomeruli, interstitial cells, and small vessels were also negativefor PCL expression.
Figure 3. PCL colocalizes with aquaporin-3 (AQP3) to the inner medullary collecting ducts in the mouse kidney. (A) Schematic representation showing where AQP1 (blue box), AQP3 (red box), and PCL (green box) are expressed in the nephron. (B) Double-labeling indirect immunofluorescence on adult mouse kidney transverse sections using anti-PCL (green) and anti-AQP1 (red). (C) higher magnification of panel B. (D) Anti-PCL (green) and anti-AQP3 (red). (E) Higher magnification of panel D. lu, lumen; arrows, apical PCL staining; I, intercalated cell that was negative for both PCL and AQP3 expression. Magnifications: x20 in B and D; x63 in C and E.
As mentioned above, PCL localization in embryonic mouse kidneyat E16 (Figure 4A) was similar to that observed in the adultmouse kidney (Figure 4D), where it appeared to be predominantlyexpressed in the apical region of tubular epithelial cells.Overlay images, which combine specific PCL indirect immunofluorescencedetection with Nomarsky imaging (Figure 4, B and E), clearlyshowed that PCL expression was indeed concentrated in the apicalregion (Figure 4, C and F). Some punctuate intracellular stainingwas also observed (Figure 4, A and D; arrowheads), althoughthe resolution of these images was not sufficient to identifywhether PCL was in the apical membrane or in structures verynear the plasma membrane or both.
Figure 4. PCL is located in the apical region of tubular epithelia. (A) PCL expression at E16 in the apical region (arrows). Punctuate intracellular staining can also be seen (arrowheads). (B) Nomarski optics image of the same field as in panel A. (C) Overlay of images in panels A and B. (D) PCL expression of the adult kidney. (E) Nomarski optics and (F) an overlay of the two images in panels D and E. lu, lumen; *, principal cell that has been cut across the apical plane (as if viewed from the top); i, intercalated cell. Magnification: x40 in A through F.
PCL Expression in Extrarenal Tissue
Extrarenal expression was not observed at the embryonic or newbornstages but was detected in adult tissues only and included avariety of cell types. cDNA for PKDL was originally isolatedfrom the retina, and mice with one PkdL allele also developretinal defects (4,19,20). We, therefore, examined PCL expressionin this tissue. We found that PCL was indeed expressed in theadult mouse retina and was located in the neurons of the ganglioncell and inner nuclear cell layers (Figure 5A).
Figure 5. PCL expression in various adult mouse tissues. Indirect immunofluorescence using anti-PCL antiserum on adult mouse (A) retina (* indicates the inner nucleated and ganglion cell layers). (B) liver (arrowheads indicate the bile canaliculi), (C) pancreas (arrow and arrowheads indicate the positively stained smaller and larger pancreatic ducts [weaker staining], respectively), (D) heart (e indicates epicardium), (E) testis (lu indicates lumina of an individual seminiferous tubule), and (F) spleen. Magnification: x20 in all.
We also examined other tissues that are affected in ADPKD. Inthe liver, PCL was detected in the smaller ducts of the biliarytree (canaliculi) (Figure 5B), although staining in sinusoidalcells cannot be excluded. The larger bile ducts, as well ashepatocytes, were not labeled. In the pancreas, PCL was expressedin the epithelial cells of both the small and large pancreaticducts (Figure 5C). The islets of Langerhans and pancreatic aciniwere not stained. Positive staining was also observed in theheart (Figure 5D), particularly in the epicardium and in theendothelial cells of the larger blood vessels that were markedby using an anti-PECAM antibody (data not shown). No expressionwas detected in the myocytes or in any of the smaller bloodvessels. Other cell types found to express PCL included spermatocytesin the testis (Figure 5E) and reticular cells in the red pulpof the spleen (Figure 5F).
In this study, we generated a polyclonal PCL-specific antiserumand used it to define the tissue and cellular localization ofPCL. Immunostaining of the developing mouse kidney showed thatexpression of PCL was first detectable at E16 in the inner medulla,which coincides with the maturation of inner medullary collectingducts. Higher levels of PCL were found in the adult kidney,where its expression in the inner medulla was further supportedby detection of Pkd mRNA from this region. Unlike PC1 and PC2,PCL was detected in only one type of nephron segment, as demonstratedby co-localization experiments with AQP3.
This study suggests that PCL’s direct involvement in ADPKDis unlikely. The restricted localization of PCL to the innermedullary collecting ducts is not compatible with the wide distributionof cystic lesions in ADPKD, which involves all nephron segments.However, a secondary role for PCL in cystogenesis should notbe excluded, because the functional relationships between PCLand PC1 and/or PC2 have not yet been determined. PC1 and PCLappear to colocalize in some of the same structures of the adultmouse kidney (12–14), including the apical region of thecollecting ducts. In contrast, PC2 is weakly expressed in collectingducts and is found in the basolateral compartment in mice (15–17).Protein relocalization due to missorting, is a common featureof ADPKD during cystogenesis (28,29), and it is possible thatthe mistargeting of either PCL and/or PC2 could favor theirinteraction. For example, a recent study has demonstrated thata known human mutation in PKD2 (R742X), which removes most ofthe carboxyl-terminal tail, results in its protein product beingtargeted to the plasma membrane instead of to the intracellularmembranes, where the wild-type protein resides (23,30). Therefore,it is possible that PCL could associate with PC1 and/or PC2in a physiologic or pathologic situation and thus contributeindirectly to cyst formation in ADPKD.
A common feature of PC1, PC2, and PCL in localization studieswas the intracellular granular/punctuate staining in the innermedullary collecting ducts. Whether these proteins are locatedin the same intracellular structures remains to be determined.Colocalization studies of polycystins may have significant functionalrelevance because it has been shown that polycystins can interactwith each other in vitro (10,11), and in fact, PC2 functionat the cell membrane appears to depend on association with PC1(25).
The possibility that PCL may play an indirect role in ADPKDwas also suggested by preliminary results that showed an increasein PCL expression in the cystic kidneys of transgenic Pkd1del34mice (18,31); N. Basora and J. Zhou, unpublished results). Thesemice have been genetically modified to delete exon 34 of thePkd1 gene, mimicking many known human truncation mutations.Mice that are homozygous for this mutation develop severe kidneydisease, and in a significant number of the older heterozygotemice, kidney cysts are readily visible (31). The reasons forthis apparent increase in PCL expression are unknown but mayoccur in response to altered ion absorption in the diseasedkidneys.
PCL functions as a Ca2+-regulated nonselective cation channelthat is permeable to Ca2+, Na+, and K+ ions when expressed inXenopus oocytes (21). Expression of PCL in the apical regionof tubular epithelia in the inner medullary collecting ductssuggests that PCL could play a role in Na+ reabsorption, becauseextracellular Ca2+ and K+ levels are very low in the tubularfluid of this segment. Na+ reabsorption in inner medullary collectingducts was reported to be mediated by amiloride-sensitive nonselectivecation channels that are inhibited by atrial natriuretic peptidevia its second messenger cGMP (32) and perhaps also by epithelialNa+ channels (ENaC) that are not cGMP-sensitive (33,34). Thesechannels have single-channel conductances of 28 pS and 4–6pS, respectively. The PCL channel is pH-sensitive but does notappear to be regulated by protein kinase A- and C-dependentphosphorylation events, nor is it cAMP sensitive (21). The presenceof PCL channels with a large single-channel conductance of 137pS in inner medullary collecting ducts may provide the nephronwith an alternative way for fine-tuning extracellular fluidionic composition and volume. Future studies of the regulationof PCL channel are required to more thoroughly understand themechanism of volume regulation in this nephron segment.
It is interesting to note that PCL was found in a variety ofcell types, including epithelial, neuronal, and endothelialcells. The subcellular distribution of PCL expression appearedto be different in these cell types. In polarized epithelialcells, such as in the kidney and the epicardium, staining wasclearly predominant in or near the plasma membrane. In retinalneurons, however, the staining pattern of PCL was distinctlyintracellular. The reasons for these differences in distributionare currently unknown, but they could suggest diverse functionsof PCL in specific cell types or diverse targeting of PCL asa result of alternative splicing (35). Similar tissue-specificdifferences in subcellular distribution have been reported forPC2 (16,17,36).
PCL may be a candidate gene for other unmapped human geneticcystic disorders, particularly autosomal dominant medullarycystic kidney disease (MCD) (37). This condition resembles familialjuvenile nephronophthisis (FJN) and is characterized by renalcysts at the corticomedullary junction or medulla and by saltwasting (38). The main distinction between MCD and FJN is theage of onset and their mode of inheritance. MCD occurs muchlater in life and is dominant; FJN is recessively inherited.The gene loci for FJN and MCD have been mapped to human chromosomes2q13 (29) and 1q21 (38,39), respectively, but several studies(38,39) have shown genetic heterogeneity for the MCD locus,which suggests the existence of unidentified loci.
In summary, by generating a specific antiserum, we were ableto demonstrate expression and localization of a newly identifiedpolycystin, PCL, in mouse tissues. These data in combinationwith evidence of PCL channel activity are crucial for elucidatingthe physiologic role of this novel cation channel and may shedlight into the function of other polycystins.
Acknowledgments
We thank Drs. Dennis Brown and Alfred Van Hoek of the Programin Membrane Biology, Massachusetts General Hospital, for theantibodies against AQP1 and AQP3 and for helpful discussions.We also thank Drs. Anna Pavlova and Gui Lan Yao for helpfuldiscussions and Mr. Eric Williams for technical assistance.NB is a recipient of the Fonds de Recherche et Sante au Quebec(FRSQ) fellowship. CS is a recipient of a postdoctoral fellowshipfrom the New Zealand Foundation for Research, Science and Technology(BWH901). This work was supported by grants from the NationalInstitutes of Health (NIDDK) to JZ.
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Received for publication September 24, 2001.
Accepted for publication October 30, 2001.
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Top
Abstract
Introduction
and were purified by using glutathione-sepharose4B (Amersham-Pharmacia) according to the manufacturer’s protocol. 
predicted EF-hand; 
spans the peptide sequence used for immunization. (C) Western blot analysis of the purified glutathione sufatransferase fusion proteins shown in panel B; left panel, blotted with an anti-PCL antiserum; right panel, anti-GST.
