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Rab proteins regulate epithelial sodium channel activity in colonic epithelial HT-29 cells
BBRC Biochemical and Biophysical Research Communications 337 (2005) 1219–1223 www.elsevier.com/locate/ybbrc
Rab proteins regulate epithelial sodium channel activity in colonic epithelial HT-29 cells Sunil Saxena a,*, Madhurima Singh a, Kathrin Engisch b, Mitsunori Fukuda c, Simarna Kaur a a
Center for Cell and Molecular Biology, Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ 07030, USA b Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA c Fukuda Initiative Research Unit, RIKEN, Wako, Saitama 351-0198, Japan Received 29 September 2005 Available online 7 October 2005
Abstract ENaC, the sodium-selective amiloride-sensitive epithelial channel, mediates electrogenic sodium re-absorption in tight epithelia and is deeply associated with human hypertension. The ENaC expression at plasma membrane requires the regulated transport, processing, and macromolecular assembly in a defined and highly compartmentalized manner. Ras-related Rab GTPases regulate intracellular trafficking during endocytosis, regulated exocytosis, and secretion. To evaluate the role of these proteins in regulating amiloride-sensitive sodium channel activity, multiple Rab isoforms 3, 5, 6, and Rab27a were expressed in HT-29 cells. Rab3 and Rab27a inhibited ENaC currents, while the expression of other Rab isoforms failed to elicit any statistically significant effect on amiloride-sensitive currents. The immunoprecipitation experiments suggest protein–protein interaction of Rab3 and Rab27a with epithelial sodium channel. Biotinylation studies revealed that modulation of ENaC function is due to the reduced apical expression of channel proteins. Study also indicates that Rabs do not appear to affect the steady-state level of total cellular ENaC. Alternatively, introduction of isoform-specific small inhibitory RNA (SiRNA) reversed the Rab-dependent inhibition of amiloride-sensitive currents. These observations point to the involvement of multiple Rab proteins in ENaC transport through intracellular routes like exocytosis, recycling from ER to plasma membrane or degradation and thus serve as potential target for human hypertension. Ó 2005 Elsevier Inc. All rights reserved. Keywords: ENaC; Rab3; Rab27a; Rab6; Biotinylation; Trafficking; Regulation; Protein–protein interaction; HT-29 cells; Colonic epithelial cells
The epithelial sodium channel (ENaC) acquires primary importance in the control of sodium re-absorption in epithelial cells [1,2]. ENaC is an important component in the overall control of sodium balance, blood volume, and blood pressure. ENaC is widely expressed in multiple tissues including colon [3–6]. The mutations in ENaC are linked to genetic disorders of sodium channel activity (LiddleÕs Syndrome and Pseudohypoaldosteronism type 1) and are unique examples of its contrasting effects on blood pressure and hypertension [7,8]. The mineralocorti*
Corresponding author. Fax: +1 201 216 8240. E-mail address: [email protected] (S. Saxena).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.09.186
coid hormone aldosterone appears to affect ENaC function in multiple ways like mediating changes in gene transcription [9–12]. Other ENaC-activating pathways include aldosterone-induced serum and glucocorticoid-inducible kinase (SGK1) [13,14] and the small G protein, K-Ras 2A [11,12]. Understanding cellular mechanisms that control ENaC function may provide a comprehensive insight and a possible connection between abnormal renal sodium transport and essential hypertension. Rabs are small proteins (21–25 kDa), which are widely expressed and implicated in the cellular transport process like vesicle budding, tethering/docking of vesicles to their target compartment, membrane fusion, and interaction of
1220
S. Saxena et al. / Biochemical and Biophysical Research Communications 337 (2005) 1219–1223
vesicles with cytoskeletal elements [15–17]. The multiple functions associated with Rab proteins indicate that the transport process is highly regularized and all steps of vesicle transport converge and are coordinated by the same regulatory machinery. In the present investigation, we over-expressed human colonic epithelial cell line HT-29 cells which endogenously express ENaC and respond to aldosterone stimulation, with individual Rab isoforms 1a, 3, 4, 5, 6, and Rab27a and recorded amiloride-sensitive currents. This is the first report on the interaction of Rab proteins with sodium channel. Our data suggest that Rab3 and Rab27a substantially inhibit sodium currents. Additionally, the immunoprecipitation studies point to the protein–protein interaction between ENaC and these two Rab isoforms. We also estimated the apical expression of ENaC by biotinylation and Western blot analysis. Our data suggest that Rab proteins likely escort ENaC through its compartmental transport and exocytosis, recycling and lysosomal routes. Experimental procedures Materials. The ENaC antibodies were raised by Research genetics (Huntsville, AL). Alternatively, ENaC antibodies were also available from other sources. aENaC antibody was a kind gift from Dr. Peter Smith, Department of Physiology and Biophysics, UAB. The ENaC subunit-specific antibodies were generous gift by Dr. Bernard Rossier, University of Lausanne, Switzerland. Enhanced chemiluminescence kits for developing the Western blots were obtained from Amersham Pharmacia Biotech. The ENaC antibody was obtained from Eastman Kodak. Anti-Rab antibodies were purchased from BD Biosciences, Valencia, CA, and other reagents were obtained from Sigma. Chariot protein delivery system was purchased from Active Motif, Carlsbad, CA. RIPA buffer contained 20 mM Tris–Cl, pH 7.5, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 5 mM EDTA, pH 8.0, and 0.5% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride and 2 lg each of pepstatin, leupeptin, aprotinin, and soybean trypsin inhibitor with 0.2% Triton X-100. For statistical analyses, two-sample comparisons were performed using StudentÕs t test. Cell line. HT-29 cells were cultured in McCoyÕs 5a medium with 1.5 mM L-glutamine and 10% fetal bovine serum in 5% CO2 at 37 °C. The cells were grown on Falcon 6- or 12-well inserts for all the experiments and maintained to determine the amiloride-sensitive currents described below. The cells were transfected with Rab constructs using lipofectamine (Gibco-BRL–Invitrogen) according to the manufacturerÕs instructions. The expression of Rabs was confirmed by Western blot analysis for each set of reactions outlined in this communication. Measurements of short circuit current (Isc). Amiloride-sensitive currents were recorded two ways. The Isc were also recorded with EVOM epithelial voltometer using STX2 electrode (WPI, Sarasota, FL). Alternatively, the confluent monolayers were mounted in a modified Ussing chamber (Trans-24 mini-perfusion chamber, Warner Instruments, Hamden, CT). Apical and basolateral chambers were continuously bathed with medium and Isc were measured with transepithelial voltage clamped at 0 mV with a DVC-1000 dual voltage clamp. Voltage pulses (10 mV) were applied every 3 min to monitor the transepithelial resistance. After the initial measurements, 10 lM amiloride were added to the apical side, and sodium currents expressed as the amiloridesensitive component of the Isc. Cell surface biotinylation and ENaC detection. Forty-eight hours after transfection, HT-29 cells were washed and harvested in phosphatebuffered saline and then incubated in 5 ml sulfo-NHS-SS-Biotin (Pierce
Biotechnology, Rockford, IL) (0.5 mg/ml) in at 4 °C for 30 min. After washing three times with ice-cold quenching buffer (192 mM glycine, 25 mM Tris, pH 8.3), cells were solubilized on ice in 500 ll RIPA buffer. The cell lysate was centrifuged for 20 min at 16,000g, and the supernatant was incubated in 50 ll of streptavidin resin (Pierce) (50% slurry in phosphate-buffered saline) overnight at 4 °C with gentle rocking. Samples were centrifuged for 2 min at 8000g, and the resin was washed five times with RIPA buffer. The protein was eluted from the resin by the addition of SDS–PAGE sample buffer containing 5% 2mercaptoethanol and incubation at 65 °C for 5 min. The samples were analyzed for ENaC expression by Western blotting using subunit-specific antibodies. The blots were raised using enhanced chemiluminescence (ECL) and the films were developed using autoradiography. Quantification was carried out with densitometry. Immunoprecipitation. Cells were solubilized in RIPA buffer. The supernatant recovered after 14,000 rpm centrifugation for 15 min was used for immunoprecipitation. The supernatants were incubated with a specific antibody for 2 h at 4 °C followed by incubation with Sepharose beads (Sigma) for 2 h at 4 °C. After washing with RIPA buffer and centrifugation at 10,000g for 15 s, beads were solubilized in SDS sample buffer and run on SDS–PAGE. The proteins were transferred to PVDF membrane. Bound antibody was detected by enhanced chemiluminescence and quantitated by phosphorimaging under conditions where there was a linear relationship between intensity and pixel number. Quantification was carried out with densitometry. SiRNA studies. One hundred nanomolar SiRNA (final conc.) was mixed with 20 ll of the transfection medium reagent (Santa Cruz Biotechnology, Santa Cruz, CA) per well and incubated. In another vial, 1.2 ll of the transfection reagent was mixed with 20 ll transfection medium and incubated. The contents of both vials were mixed incubated. After 20 min, the contents were mixed with 160 ml of the transfection medium and this solution was laid on top of the cells in each well. The cells growing on inserts in a 24-well plate (50% confluent for this experiment) were used for this experiment. The plate was incubated in 37 °C with 5% CO2 for 5–7 h, and then 200 ll of the culture medium with double serum was added on top of the inserts. The amiloride-sensitive currents were recorded 24 h post-SiRNA transfection as described before. Statistical analysis. A paired test or analysis of variance for multiple comparisons was used for statistical analysis. A p value less than 0.05 was considered significant.
Results and discussion Effect of Rabs on amiloride-sensitive currents To test the hypothesis that Rab GTPase functionally modulates ENaC function, amiloride-sensitive currents
Fig. 1. Rab isoforms regulate amiloride-sensitive currents in HT-29 cells. HT-29 cells grown on cell inserts were transfected with wild-type Rab constructs. The amiloride-sensitive currents were recorded 48 h later as described before. The data represent means of three individual experiments.
S. Saxena et al. / Biochemical and Biophysical Research Communications 337 (2005) 1219–1223
were assayed in HT-29 over-expressed with different Rab isoforms (Fig. 1). We observed high basal currents in these cells in the range of 2–3 lAmps that could be inhibited by the addition of 10 lM of amiloride to the bath solution. These amiloride-sensitive currents were inhibited in the cells over-expressing Rab3 or Rab27a, but not with a Rab5 or Rab6. These data indicate that functional regulation of ENaC is a Rab isoform-specific phenomenon. Effect of introduction of SiRNA on amiloride-sensitive currents We further tested the ability of Rab to influence amiloride-sensitive currents by introducing isoform-specific SiRNA in the cell. This effort was based on our observations that Rab isoforms are expressed in low to moderate levels in HT-29 cells (Fig. 2). In this study, we observed that Rab3 and Rab27a SiRNA reverse the inhibitory effect otherwise observed with the over-expression of these two Rab isoforms. Moreover, the introduction of Rab3 and Rab27a SiRNA potentiated the amiloride-sensitive currents higher than that recorded in control cells. The SiRNA effect on Rab expression was confirmed by Western blot analysis (Figs. 2B and C). These data further support our observations that Rab3 and Rab27a act as negative modulators of ENaC function.
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Immunoprecipitation of ENaC with Rab The functional data revealed an interaction between ENaC and Rab proteins. In order to test this hypothesis, we performed co-immunoprecipitation studies in which we used isoform-specific antibodies to pull down Rabs in HT-29 cells. The blots were probed with subunit-specific antibody. ENaC proteins were co-immunoprecipitated with Rab3 and Rab27a (Fig. 3) but other Rab isoforms fail to report interaction. Similar data were observed when the ENaC complex was first precipitated with ENaC antibody followed by immunodetection with Rab isoform-specific antibody (data not shown). These data suggest that ENaC and Rabs physically associate in HT-29 cells. Cell surface biotinylation In view of the involvement of Rab proteins in intracellular transport, we next explored the hypothesis that the modulation of ENaC-mediated currents in the cells over-expressing Rab proteins was the consequence of reduced ENaC at cell surface. In order to prove this, we employed cell impermeant sulfo-NHS-SS biotin to label the cell surface proteins and pulled down the complex with Streptavidin-agarose. As expected, we observed reduced ENaC at the apical surface in the HT-29 cells expressing either Rab3 or Rab27a (Fig. 4). These observations were restrictive and made only with these two isoforms as expression of Rab5 or Rab6 could not effect changes in aENaC density. These data demonstrate that the expression of Rab3 and Rab27a interferes with the ENaC transport to the cell surface by either restraining the channel proteins inside the cell or obstructing ENaC exocytosis to the apical plasma membrane. Total cell ENaC determination We also reasoned that changes in ENaC surface proteins might be due to the alteration of overall or total ENaC
Fig. 2. Isoform-specific SiRNA reverses inhibition of amiloride-sensitive currents. The HT-29 cells were targeted with SiRNAs specific for Rab isoforms as described in the text. The amiloride-sensitive currents were recorded. Data represent three individual experiments. (A) Amiloridesensitive currents recorded in the absence () or presence (+) of isoformspecific SiRNA, (B) Western blot analysis of Rab proteins from protein lysates showing SiRNA inhibits Rab expression, and (C) densitometric analysis of Rab proteins from (B). For each Rab isoform () SiRNA is treated as 100%. The result is average of three individual experiments.
Fig. 3. Rab3 and Rab27a interact with ENaC in HT-29 cells. Isoformspecific anti-Rab antibodies were used to immunoprecipitate proteins ENaC from HT-29 cell lysates expressing Rab isoforms. The immune complex was adsorbed on protein A–agarose beads and then separated by SDS–polyacrylamide gel electrophoresis and transferred to PVDF membrane. The blots were probed with anti-aENaC antibody (top panel). IgG was used as control in all experiments. Alternatively, the Rab3 and Rab27a were immunoprecipitated using cENaC antibody (lower panel). The data show interaction of Rab3 and Rab27a with ENaC in vivo and point to its involvement and physiological significance in the regulation of ENaC in native cells.
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Fig. 4. Rab3 and Rab27a alter ENaC cell surface expression. HT-29 cells grown on cell inserts were over-expressed with Rab constructs. The cell surface proteins were biotinylated with sulfo-NHS-SS-biotin as described in the text. The biotinylated proteins were adsorbed on streptavidin– Sepharose beads, solubilized in SDS-buffer, separated on SDS–PAGE, and transferred to PVDF membrane. The blots were probed with aENaC antibody. The data reflect lower ENaC density in HT-29 cells expressing Rab3 and Rab27a. (B) Densitometric analysis of aENaC from blot (A).
pool in the cells over-expressing Rab3 and Rab27a isoforms. It was a valid assumption to see if there were adjustments in the steady-state level of total cell ENaC population. In order to provide an answer to this question, we extracted total cellular proteins from HT-29 cells and Western blot analysis was performed using subunit-specific ENaC antibodies (Fig. 5). We observed that total cellular ENaC remains unchanged in the cells expressing Rab3 or Rab27a. These results in conjunction with previous obser-
Fig. 5. Effect of Rab expression on steady-state level of ENaC. Rab expression does not alter steady-state level of ENaC. HT-29 cells were transiently transfected with Rab constructs and 48 h later the cells were homogenized in RIPA buffer and the cell lysate was subjected to SDS– PAGE and Western blot analysis as described in the text. The blot was probed with affinity-purified anti-aENaC antibody and developed with ECL.
vations indicate that co-injection of Rab redistributes ENaC complex from surface membrane to the internal stores. We utilized human colorectal epithelial cell line HT-29 cell system to explore the role of Rabs in the regulation of indigenously expressed amiloride-sensitive epithelial sodium channel (ENaC). In order to achieve this goal, we transiently expressed Rabs, though several Rab isoforms are natively expressed in these cells at low levels. The Rab expression was detected with Western blot analysis using isoform-specific antibodies. However, since these proteins are detected at low levels and we were interested in a formidable cell system, which has indigenous ENaC expression, we utilized these cells to monitor the transient over-expression of Rab proteins on ENaC function. The major conclusions of the studies include: (I) ENaC is functionally modulated by Rab3 and Rab27a, (ii) ENaC proteins physically interact with Rab3 and Rab27a, (iii) these Rab isoforms impair the trafficking of ENaC proteins to the plasma membrane, (iv) Rab expression does not alter the steady-state amount of total cellular ENaC protein, and (v) Rab3 and Rab27a reduce ENaC cell surface expression. The maturation and trafficking of transmembrane proteins from the ER to the Golgi and subsequently to the plasma membrane is a result of specialized machinery and targeted translocation of transport vesicles [18,19]. Subsequent vesicular fusion results in enhanced channel density at the surface. However, the underlying mechanism by which ENaC transport vesicles are targeted to apical membranes, docked and finally fused, remains an open question. The ability of multiple Rabs to govern the regularized traffic defines the molecular mechanisms by which they control the translocation of specialized vesicles inside the cell. By using the HT-29 expression system, we have demonstrated that Rab isoforms can modulate amiloridesensitive currents associated with ENaC. Both Rab3 and Rab27a act as negative regulators of ENaC activity. Other Rab isoforms fail to alter ENaC function. Our study does not address active (GTP bound) or inactive (GDP bound) status of Rab, yet, the importance of these regulatory aspects remains the core of our future studies. However, our current study, which is the first of its kind, shows that Rabs can regulate ENaC function. Moreover, we also established that both Rab isoforms physically interact with ENaC as demonstrated by the immunoprecipitation experiments. Our biotinylation studies (Fig. 4) clearly demonstrate that ENaC density at the cell membrane is weakened in HT-29 cells expressing Rab3 and Rab27a, suggesting that these two isoforms are involved in ENaC trafficking. The association between ENaC and Rab isoforms appears to be specific as Rab5, and Rab6 conferred (i) no substantial modulation of ENaC function or (ii) interaction with channel. Our data further emphasize that Rab protein expression does not interfere with the steady-state level of total ENaC but possibly reallocates ENaC to the intracellular
S. Saxena et al. / Biochemical and Biophysical Research Communications 337 (2005) 1219–1223
compartment viz. Rab27a which is implicated in lysosomal trafficking [20] that might lead to the eventual degradation of channel proteins. We are currently involved in defining the molecular mechanism by which Rabs affect ENaC in HT-29 cells including the compartmentalization of channel proteins. It needs to be mentioned that occasionally we encountered reduction in total ENaC pool in Rab27a expressing cells. In conclusion, ENaC physically and functionally interacts with at least two Rab isoforms: Rab3 and Rab27a, each of which acts as a negative regulator of ENaC function. In view of the multiple Rabs, their effectors [21,22] that have been described, it will be important to define the molecular mechanism of Rab(s) interactions with the ENaC complex. Although our studies did not address a vital issue related to ENaC mutants associated with LiddleÕs syndrome or PHA-1 [23,24], it would be necessary to explore the possibility of Rab proteins regulating the transport of mutant ENaC. We sincerely believe that a therapeutic approach with Rabs may emerge that may provide an alternative to control ENaC activity. Acknowledgments
[8] [9]
[10]
[11]
[12]
[13]
[14] [15] [16]
Authors are grateful to Constantine George for help and encouragement during the course of study. Technical support of Amanda Rogers is greatly appreciated. The work was supported in part by the grant from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) awarded to SS (DK57727).
[17]
[18]
[19]
References [20] [1] E. Hummler, Epithelial sodium channel, salt intake, and hypertension, Curr. Hypertens. Rep. 5 (2003) 11–18. [2] Y.R. Su, A.G. Menon, Epithelial sodium channels and hypertension, Drug Metab. Dispos. 29 (2001) 553–556. [3] E.R. Greig, R.P. Boot-Handford, V. Mani, G.I. Sandle, Decreased expression of apical Na+ channels and basolateral Na+, K+-ATPase in ulcerative colitis, J. Pathol. 204 (2004) 84–92. [4] T. Coric, N. Hernandez, D.A. de la Rosa, D. Shao, T. Wang, C.M. Canessa, Expression of ENaC and serum- and glucocorticoid-induced kinase1 in the rat intestinal epithelium, Am. J. Physiol. Gastrointest. Liver Physiol. 286 (2004) G663–G670. [5] L. Dijkink, A. Hartog, C.H. van Os, R.J. Bindels, The epithelial sodium channel (ENaC) is intracellularly located as a tetramer, Pflugers Arch. 444 (2002) 549–555. [6] G.M. Fraser, L.M. Blendis, P. Smirnoff, E. Sikular, Y. Niv, B. Schwartz, Portal hypertension induces sodium channel expression in colonocytes from the distal colon of the rat, Am. J. Physiol. Gastrointest. Liver Physiol. 279 (2000) G886–G892. [7] S. Pradervand, A. Vandewalle, M. Bens, I. Gautschi, J. Loffing, E. Hummler, L. Schild, B.C. Rossier, Dysfunction of the epithelial
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sodium channel expressed in the kidney of a mouse model for Liddle syndrome, J. Am. Soc. Nephrol. 14 (2003) 2219–2228. D.G. Warnock, The epithelial sodium channel in hypertension, Curr. Hypertens. Rep. 1 (1999) 158–163. W. Lim, D. Kim, J.B. Park, S.H. Kim, Y. Lee, Sodium chloride regulation of the alpha epithelial amiloride-sensitive sodium channel (alphaENaC) gene requires syntheses of new protein(s), J. Steroid Biochem. Mol. Biol. 88 (2004) 305–310. V.E. Mick, O.A. Itani, R.W. Loftus, R.F. Husted, T.J. Schmidt, C.P. Thomas, The alpha-subunit of the epithelial sodium channel is an aldosterone-induced transcript in mammalian collecting ducts, and this transcriptional response is mediated via distinct cis-elements in the 5 0 -flanking region of the gene, Mol. Endocrinol. 15 (2001) 575– 588. D.C. Eaton, B. Malik, N.C. Saxena, O.K. Al-Khalili, G. Yue, Mechanisms of aldosteroneÕs action on epithelial Na+ transport, J. Membr. Biol. 184 (2001) 313–319. F. Verrey, D. Pearce, R. Pfeiffer, B. Spindler, L. Mastroberardino, V. Summa, M. Zecevic, Pleiotropic action of aldosterone in epithelia mediated by transcription and post-transcription mechanisms, Kidney Int. 57 (2000) 1277–1282. F. Verrey, J. Loffing, M. Zecevic, D. Heitzmann, O. Staub, SGK1: aldosterone-induced relay of Na+ transport regulation in distal kidney nephron cells, Cell. Physiol. Biochem. 13 (2003) 21–28. D. Pearce, SGK1 regulation of epithelial sodium transport, Cell. Physiol. Biochem. 13 (2003) 13–20. J. Somsel Rodman, A. Wandinger-Ness, Rab GTPases coordinate endocytosis, J. Cell Sci. 113 (Pt. 2) (2000) 183–192. J. Armstrong, How do Rab proteins function in membrane traffic? Int. J. Biochem. Cell Biol. 32 (2000) 303–307. M.P. Stein, J. Dong, A. Wandinger-Ness, Rab proteins and endocytic trafficking: potential targets for therapeutic intervention, Adv. Drug Deliv. Rev. 55 (2003) 1421–1437. D. Hanwell, T. Ishikawa, R. Saleki, D. Rotin, Trafficking and cell surface stability of the epithelial Na+ channel expressed in epithelial Madin–Darby canine kidney cells, J. Biol. Chem. 277 (2002) 9772–9779. M.B. Butterworth, S.I. Helman, W.J. Els, cAMP-sensitive endocytic trafficking in A6 epithelia, Am. J. Physiol. Cell Physiol. 280 (2001) C752–C762. G. Bossi, S. Booth, R. Clark, E.G. Davis, R. Liesner, K. Richards, M. Starcevic, J. Stinchcombe, C. Trambas, E.C. DellÕangelica, G.M. Griffiths, Normal lytic granule secretion by cytotoxic T lymphocytes deficient in BLOC-1, -2 and -3 and myosins Va, VIIa and XV, Traffic 6 (2005) 243–251. S. Torii, T. Takeuchi, S. Nagamatsu, T. Izumi, Rab27 effector granuphilin promotes the plasma membrane targeting of insulin granules via interaction with syntaxin 1a, J. Biol. Chem. 279 (2004) 22532–22538. E. Korobko, S. Kiselev, S. Olsnes, H. Stenmark, I. Korobko, The Rab5 effector Rabaptin-5 and its isoform Rabaptin-5delta differ in their ability to interact with the small GTPase Rab4, FEBS J. 272 (2005) 37–46. S. Pradervand, P.M. Barker, Q. Wang, S.A. Ernst, F. Beermann, B.R. Grubb, M. Burnier, A. Schmidt, R.J. Bindels, J.T. Gatzy, B.C. Rossier, E. Hummler, Salt restriction induces pseudohypoaldosteronism type1 in mice expressing low levels of the beta-subunit of the amiloride-sensitive epithelial sodium channel, Proc. Natl. Acad. Sci. USA 96 (1999) 1732–1737. D.G. Warnock, Liddle syndrome: genetics and mechanisms of Na+ channel defects, Am. J. Med. Sci. 322 (2001) 302–307.
Rab proteins regulate epithelial sodium channel activity in colonic epithelial HT-29 cells Sunil Saxena a,*, Madhurima Singh a, Kathrin Engisch b, Mitsunori Fukuda c, Simarna Kaur a a
Center for Cell and Molecular Biology, Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ 07030, USA b Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA c Fukuda Initiative Research Unit, RIKEN, Wako, Saitama 351-0198, Japan Received 29 September 2005 Available online 7 October 2005
Abstract ENaC, the sodium-selective amiloride-sensitive epithelial channel, mediates electrogenic sodium re-absorption in tight epithelia and is deeply associated with human hypertension. The ENaC expression at plasma membrane requires the regulated transport, processing, and macromolecular assembly in a defined and highly compartmentalized manner. Ras-related Rab GTPases regulate intracellular trafficking during endocytosis, regulated exocytosis, and secretion. To evaluate the role of these proteins in regulating amiloride-sensitive sodium channel activity, multiple Rab isoforms 3, 5, 6, and Rab27a were expressed in HT-29 cells. Rab3 and Rab27a inhibited ENaC currents, while the expression of other Rab isoforms failed to elicit any statistically significant effect on amiloride-sensitive currents. The immunoprecipitation experiments suggest protein–protein interaction of Rab3 and Rab27a with epithelial sodium channel. Biotinylation studies revealed that modulation of ENaC function is due to the reduced apical expression of channel proteins. Study also indicates that Rabs do not appear to affect the steady-state level of total cellular ENaC. Alternatively, introduction of isoform-specific small inhibitory RNA (SiRNA) reversed the Rab-dependent inhibition of amiloride-sensitive currents. These observations point to the involvement of multiple Rab proteins in ENaC transport through intracellular routes like exocytosis, recycling from ER to plasma membrane or degradation and thus serve as potential target for human hypertension. Ó 2005 Elsevier Inc. All rights reserved. Keywords: ENaC; Rab3; Rab27a; Rab6; Biotinylation; Trafficking; Regulation; Protein–protein interaction; HT-29 cells; Colonic epithelial cells
The epithelial sodium channel (ENaC) acquires primary importance in the control of sodium re-absorption in epithelial cells [1,2]. ENaC is an important component in the overall control of sodium balance, blood volume, and blood pressure. ENaC is widely expressed in multiple tissues including colon [3–6]. The mutations in ENaC are linked to genetic disorders of sodium channel activity (LiddleÕs Syndrome and Pseudohypoaldosteronism type 1) and are unique examples of its contrasting effects on blood pressure and hypertension [7,8]. The mineralocorti*
Corresponding author. Fax: +1 201 216 8240. E-mail address: [email protected] (S. Saxena).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.09.186
coid hormone aldosterone appears to affect ENaC function in multiple ways like mediating changes in gene transcription [9–12]. Other ENaC-activating pathways include aldosterone-induced serum and glucocorticoid-inducible kinase (SGK1) [13,14] and the small G protein, K-Ras 2A [11,12]. Understanding cellular mechanisms that control ENaC function may provide a comprehensive insight and a possible connection between abnormal renal sodium transport and essential hypertension. Rabs are small proteins (21–25 kDa), which are widely expressed and implicated in the cellular transport process like vesicle budding, tethering/docking of vesicles to their target compartment, membrane fusion, and interaction of
1220
S. Saxena et al. / Biochemical and Biophysical Research Communications 337 (2005) 1219–1223
vesicles with cytoskeletal elements [15–17]. The multiple functions associated with Rab proteins indicate that the transport process is highly regularized and all steps of vesicle transport converge and are coordinated by the same regulatory machinery. In the present investigation, we over-expressed human colonic epithelial cell line HT-29 cells which endogenously express ENaC and respond to aldosterone stimulation, with individual Rab isoforms 1a, 3, 4, 5, 6, and Rab27a and recorded amiloride-sensitive currents. This is the first report on the interaction of Rab proteins with sodium channel. Our data suggest that Rab3 and Rab27a substantially inhibit sodium currents. Additionally, the immunoprecipitation studies point to the protein–protein interaction between ENaC and these two Rab isoforms. We also estimated the apical expression of ENaC by biotinylation and Western blot analysis. Our data suggest that Rab proteins likely escort ENaC through its compartmental transport and exocytosis, recycling and lysosomal routes. Experimental procedures Materials. The ENaC antibodies were raised by Research genetics (Huntsville, AL). Alternatively, ENaC antibodies were also available from other sources. aENaC antibody was a kind gift from Dr. Peter Smith, Department of Physiology and Biophysics, UAB. The ENaC subunit-specific antibodies were generous gift by Dr. Bernard Rossier, University of Lausanne, Switzerland. Enhanced chemiluminescence kits for developing the Western blots were obtained from Amersham Pharmacia Biotech. The ENaC antibody was obtained from Eastman Kodak. Anti-Rab antibodies were purchased from BD Biosciences, Valencia, CA, and other reagents were obtained from Sigma. Chariot protein delivery system was purchased from Active Motif, Carlsbad, CA. RIPA buffer contained 20 mM Tris–Cl, pH 7.5, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 5 mM EDTA, pH 8.0, and 0.5% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride and 2 lg each of pepstatin, leupeptin, aprotinin, and soybean trypsin inhibitor with 0.2% Triton X-100. For statistical analyses, two-sample comparisons were performed using StudentÕs t test. Cell line. HT-29 cells were cultured in McCoyÕs 5a medium with 1.5 mM L-glutamine and 10% fetal bovine serum in 5% CO2 at 37 °C. The cells were grown on Falcon 6- or 12-well inserts for all the experiments and maintained to determine the amiloride-sensitive currents described below. The cells were transfected with Rab constructs using lipofectamine (Gibco-BRL–Invitrogen) according to the manufacturerÕs instructions. The expression of Rabs was confirmed by Western blot analysis for each set of reactions outlined in this communication. Measurements of short circuit current (Isc). Amiloride-sensitive currents were recorded two ways. The Isc were also recorded with EVOM epithelial voltometer using STX2 electrode (WPI, Sarasota, FL). Alternatively, the confluent monolayers were mounted in a modified Ussing chamber (Trans-24 mini-perfusion chamber, Warner Instruments, Hamden, CT). Apical and basolateral chambers were continuously bathed with medium and Isc were measured with transepithelial voltage clamped at 0 mV with a DVC-1000 dual voltage clamp. Voltage pulses (10 mV) were applied every 3 min to monitor the transepithelial resistance. After the initial measurements, 10 lM amiloride were added to the apical side, and sodium currents expressed as the amiloridesensitive component of the Isc. Cell surface biotinylation and ENaC detection. Forty-eight hours after transfection, HT-29 cells were washed and harvested in phosphatebuffered saline and then incubated in 5 ml sulfo-NHS-SS-Biotin (Pierce
Biotechnology, Rockford, IL) (0.5 mg/ml) in at 4 °C for 30 min. After washing three times with ice-cold quenching buffer (192 mM glycine, 25 mM Tris, pH 8.3), cells were solubilized on ice in 500 ll RIPA buffer. The cell lysate was centrifuged for 20 min at 16,000g, and the supernatant was incubated in 50 ll of streptavidin resin (Pierce) (50% slurry in phosphate-buffered saline) overnight at 4 °C with gentle rocking. Samples were centrifuged for 2 min at 8000g, and the resin was washed five times with RIPA buffer. The protein was eluted from the resin by the addition of SDS–PAGE sample buffer containing 5% 2mercaptoethanol and incubation at 65 °C for 5 min. The samples were analyzed for ENaC expression by Western blotting using subunit-specific antibodies. The blots were raised using enhanced chemiluminescence (ECL) and the films were developed using autoradiography. Quantification was carried out with densitometry. Immunoprecipitation. Cells were solubilized in RIPA buffer. The supernatant recovered after 14,000 rpm centrifugation for 15 min was used for immunoprecipitation. The supernatants were incubated with a specific antibody for 2 h at 4 °C followed by incubation with Sepharose beads (Sigma) for 2 h at 4 °C. After washing with RIPA buffer and centrifugation at 10,000g for 15 s, beads were solubilized in SDS sample buffer and run on SDS–PAGE. The proteins were transferred to PVDF membrane. Bound antibody was detected by enhanced chemiluminescence and quantitated by phosphorimaging under conditions where there was a linear relationship between intensity and pixel number. Quantification was carried out with densitometry. SiRNA studies. One hundred nanomolar SiRNA (final conc.) was mixed with 20 ll of the transfection medium reagent (Santa Cruz Biotechnology, Santa Cruz, CA) per well and incubated. In another vial, 1.2 ll of the transfection reagent was mixed with 20 ll transfection medium and incubated. The contents of both vials were mixed incubated. After 20 min, the contents were mixed with 160 ml of the transfection medium and this solution was laid on top of the cells in each well. The cells growing on inserts in a 24-well plate (50% confluent for this experiment) were used for this experiment. The plate was incubated in 37 °C with 5% CO2 for 5–7 h, and then 200 ll of the culture medium with double serum was added on top of the inserts. The amiloride-sensitive currents were recorded 24 h post-SiRNA transfection as described before. Statistical analysis. A paired test or analysis of variance for multiple comparisons was used for statistical analysis. A p value less than 0.05 was considered significant.
Results and discussion Effect of Rabs on amiloride-sensitive currents To test the hypothesis that Rab GTPase functionally modulates ENaC function, amiloride-sensitive currents
Fig. 1. Rab isoforms regulate amiloride-sensitive currents in HT-29 cells. HT-29 cells grown on cell inserts were transfected with wild-type Rab constructs. The amiloride-sensitive currents were recorded 48 h later as described before. The data represent means of three individual experiments.
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were assayed in HT-29 over-expressed with different Rab isoforms (Fig. 1). We observed high basal currents in these cells in the range of 2–3 lAmps that could be inhibited by the addition of 10 lM of amiloride to the bath solution. These amiloride-sensitive currents were inhibited in the cells over-expressing Rab3 or Rab27a, but not with a Rab5 or Rab6. These data indicate that functional regulation of ENaC is a Rab isoform-specific phenomenon. Effect of introduction of SiRNA on amiloride-sensitive currents We further tested the ability of Rab to influence amiloride-sensitive currents by introducing isoform-specific SiRNA in the cell. This effort was based on our observations that Rab isoforms are expressed in low to moderate levels in HT-29 cells (Fig. 2). In this study, we observed that Rab3 and Rab27a SiRNA reverse the inhibitory effect otherwise observed with the over-expression of these two Rab isoforms. Moreover, the introduction of Rab3 and Rab27a SiRNA potentiated the amiloride-sensitive currents higher than that recorded in control cells. The SiRNA effect on Rab expression was confirmed by Western blot analysis (Figs. 2B and C). These data further support our observations that Rab3 and Rab27a act as negative modulators of ENaC function.
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Immunoprecipitation of ENaC with Rab The functional data revealed an interaction between ENaC and Rab proteins. In order to test this hypothesis, we performed co-immunoprecipitation studies in which we used isoform-specific antibodies to pull down Rabs in HT-29 cells. The blots were probed with subunit-specific antibody. ENaC proteins were co-immunoprecipitated with Rab3 and Rab27a (Fig. 3) but other Rab isoforms fail to report interaction. Similar data were observed when the ENaC complex was first precipitated with ENaC antibody followed by immunodetection with Rab isoform-specific antibody (data not shown). These data suggest that ENaC and Rabs physically associate in HT-29 cells. Cell surface biotinylation In view of the involvement of Rab proteins in intracellular transport, we next explored the hypothesis that the modulation of ENaC-mediated currents in the cells over-expressing Rab proteins was the consequence of reduced ENaC at cell surface. In order to prove this, we employed cell impermeant sulfo-NHS-SS biotin to label the cell surface proteins and pulled down the complex with Streptavidin-agarose. As expected, we observed reduced ENaC at the apical surface in the HT-29 cells expressing either Rab3 or Rab27a (Fig. 4). These observations were restrictive and made only with these two isoforms as expression of Rab5 or Rab6 could not effect changes in aENaC density. These data demonstrate that the expression of Rab3 and Rab27a interferes with the ENaC transport to the cell surface by either restraining the channel proteins inside the cell or obstructing ENaC exocytosis to the apical plasma membrane. Total cell ENaC determination We also reasoned that changes in ENaC surface proteins might be due to the alteration of overall or total ENaC
Fig. 2. Isoform-specific SiRNA reverses inhibition of amiloride-sensitive currents. The HT-29 cells were targeted with SiRNAs specific for Rab isoforms as described in the text. The amiloride-sensitive currents were recorded. Data represent three individual experiments. (A) Amiloridesensitive currents recorded in the absence () or presence (+) of isoformspecific SiRNA, (B) Western blot analysis of Rab proteins from protein lysates showing SiRNA inhibits Rab expression, and (C) densitometric analysis of Rab proteins from (B). For each Rab isoform () SiRNA is treated as 100%. The result is average of three individual experiments.
Fig. 3. Rab3 and Rab27a interact with ENaC in HT-29 cells. Isoformspecific anti-Rab antibodies were used to immunoprecipitate proteins ENaC from HT-29 cell lysates expressing Rab isoforms. The immune complex was adsorbed on protein A–agarose beads and then separated by SDS–polyacrylamide gel electrophoresis and transferred to PVDF membrane. The blots were probed with anti-aENaC antibody (top panel). IgG was used as control in all experiments. Alternatively, the Rab3 and Rab27a were immunoprecipitated using cENaC antibody (lower panel). The data show interaction of Rab3 and Rab27a with ENaC in vivo and point to its involvement and physiological significance in the regulation of ENaC in native cells.
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Fig. 4. Rab3 and Rab27a alter ENaC cell surface expression. HT-29 cells grown on cell inserts were over-expressed with Rab constructs. The cell surface proteins were biotinylated with sulfo-NHS-SS-biotin as described in the text. The biotinylated proteins were adsorbed on streptavidin– Sepharose beads, solubilized in SDS-buffer, separated on SDS–PAGE, and transferred to PVDF membrane. The blots were probed with aENaC antibody. The data reflect lower ENaC density in HT-29 cells expressing Rab3 and Rab27a. (B) Densitometric analysis of aENaC from blot (A).
pool in the cells over-expressing Rab3 and Rab27a isoforms. It was a valid assumption to see if there were adjustments in the steady-state level of total cell ENaC population. In order to provide an answer to this question, we extracted total cellular proteins from HT-29 cells and Western blot analysis was performed using subunit-specific ENaC antibodies (Fig. 5). We observed that total cellular ENaC remains unchanged in the cells expressing Rab3 or Rab27a. These results in conjunction with previous obser-
Fig. 5. Effect of Rab expression on steady-state level of ENaC. Rab expression does not alter steady-state level of ENaC. HT-29 cells were transiently transfected with Rab constructs and 48 h later the cells were homogenized in RIPA buffer and the cell lysate was subjected to SDS– PAGE and Western blot analysis as described in the text. The blot was probed with affinity-purified anti-aENaC antibody and developed with ECL.
vations indicate that co-injection of Rab redistributes ENaC complex from surface membrane to the internal stores. We utilized human colorectal epithelial cell line HT-29 cell system to explore the role of Rabs in the regulation of indigenously expressed amiloride-sensitive epithelial sodium channel (ENaC). In order to achieve this goal, we transiently expressed Rabs, though several Rab isoforms are natively expressed in these cells at low levels. The Rab expression was detected with Western blot analysis using isoform-specific antibodies. However, since these proteins are detected at low levels and we were interested in a formidable cell system, which has indigenous ENaC expression, we utilized these cells to monitor the transient over-expression of Rab proteins on ENaC function. The major conclusions of the studies include: (I) ENaC is functionally modulated by Rab3 and Rab27a, (ii) ENaC proteins physically interact with Rab3 and Rab27a, (iii) these Rab isoforms impair the trafficking of ENaC proteins to the plasma membrane, (iv) Rab expression does not alter the steady-state amount of total cellular ENaC protein, and (v) Rab3 and Rab27a reduce ENaC cell surface expression. The maturation and trafficking of transmembrane proteins from the ER to the Golgi and subsequently to the plasma membrane is a result of specialized machinery and targeted translocation of transport vesicles [18,19]. Subsequent vesicular fusion results in enhanced channel density at the surface. However, the underlying mechanism by which ENaC transport vesicles are targeted to apical membranes, docked and finally fused, remains an open question. The ability of multiple Rabs to govern the regularized traffic defines the molecular mechanisms by which they control the translocation of specialized vesicles inside the cell. By using the HT-29 expression system, we have demonstrated that Rab isoforms can modulate amiloridesensitive currents associated with ENaC. Both Rab3 and Rab27a act as negative regulators of ENaC activity. Other Rab isoforms fail to alter ENaC function. Our study does not address active (GTP bound) or inactive (GDP bound) status of Rab, yet, the importance of these regulatory aspects remains the core of our future studies. However, our current study, which is the first of its kind, shows that Rabs can regulate ENaC function. Moreover, we also established that both Rab isoforms physically interact with ENaC as demonstrated by the immunoprecipitation experiments. Our biotinylation studies (Fig. 4) clearly demonstrate that ENaC density at the cell membrane is weakened in HT-29 cells expressing Rab3 and Rab27a, suggesting that these two isoforms are involved in ENaC trafficking. The association between ENaC and Rab isoforms appears to be specific as Rab5, and Rab6 conferred (i) no substantial modulation of ENaC function or (ii) interaction with channel. Our data further emphasize that Rab protein expression does not interfere with the steady-state level of total ENaC but possibly reallocates ENaC to the intracellular
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compartment viz. Rab27a which is implicated in lysosomal trafficking [20] that might lead to the eventual degradation of channel proteins. We are currently involved in defining the molecular mechanism by which Rabs affect ENaC in HT-29 cells including the compartmentalization of channel proteins. It needs to be mentioned that occasionally we encountered reduction in total ENaC pool in Rab27a expressing cells. In conclusion, ENaC physically and functionally interacts with at least two Rab isoforms: Rab3 and Rab27a, each of which acts as a negative regulator of ENaC function. In view of the multiple Rabs, their effectors [21,22] that have been described, it will be important to define the molecular mechanism of Rab(s) interactions with the ENaC complex. Although our studies did not address a vital issue related to ENaC mutants associated with LiddleÕs syndrome or PHA-1 [23,24], it would be necessary to explore the possibility of Rab proteins regulating the transport of mutant ENaC. We sincerely believe that a therapeutic approach with Rabs may emerge that may provide an alternative to control ENaC activity. Acknowledgments
[8] [9]
[10]
[11]
[12]
[13]
[14] [15] [16]
Authors are grateful to Constantine George for help and encouragement during the course of study. Technical support of Amanda Rogers is greatly appreciated. The work was supported in part by the grant from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) awarded to SS (DK57727).
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