|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01180
Research Article |
1 Departments of Physiology and Medicine, Gastroenterology Division, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
2 Department of Molecular Physiology and Biophysics and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
* Author for correspondence (e-mail: mdonowit{at}jhmi.edu)
Accepted 24 February 2004
The epithelial brush border (BB) Na+/H+ exchanger, NHE3, plays a major role in transcellular Na+ absorption in the renal proximal tubule. NHE3 activity is rapidly regulated by neurohumoral substances and growth factors via changes in its amount on the BB by a process partially involving vesicle trafficking. The PDZ domain-containing proteins, NHERF1/2, are scaffold proteins that link NHE3 to the actin cytoskeleton via their binding to both ezrin and NHE3. NHERF1/2 interact with both an internal C-terminal domain of NHE3 and the N-terminus of ezrin. We used fluorescence recovery after photobleaching (FRAP) to study the effect of NHERF1/2 on NHE3 mobility in the brush border of opossum kidney (OK) proximal tubule cells. A confocal microscope was used to allow the selective study of apical membrane versus intracellular NHE3. A chimera of NHE3-EGFP was transiently expressed in OK cells and its lateral diffusion in the apical membrane was measured with FRAP and confocal microscopy at 37°C. The contribution of intracellular NHE3-EGFP to recovery on the OK surface not directly over the juxtanuclear area (non-JN) was negligible as exposure to the water soluble crosslinker BS3 (10 mM) at 4°C resulted in no recovery of this component of surface NHE3-EGFP after photobleaching. The mobile fraction (Mf) of apical NHE3-EGFP was 47.5±2.2%; the effective diffusion coefficient (Deff) was (2.2±0.3) x10–10 cm2/second. Overexpression of NHERF2 in OK cells decreased the Mf to 29.1±3.1% without changing Deff. In the truncation mutant, NHE3585-EGFP (aa 1-585), which lacks the NHERF1/2 binding domain, Mf increased to 66.4±2.2%, with no change in Deff, whereas NHE3660-EGFP, which binds NHERF1/2, had Mf (48.3±3.0%) and Deff both similar to full-length NHE3. These results are consistent with the PDZ domain proteins NHERF1 and NHERF2 scaffolding NHE3 in macromolecular complexes in the apical membrane of OK cells under basal conditions, which limits the lateral mobility of NHE3. It is probable that this is one of the mechanisms by which NHERF1/2 affects rapid regulation of NHE3 by growth factors and neurohumoral mediators. By contrast, disrupting the actin cytoskeleton by latrunculin B treatment (0.05 µM, 30 minutes) reduced the NHE3 Mf (21.9±4.5%) without altering the Deff. Therefore the actin cytoskeleton, independently of NHERF1/2 binding, is necessary for apical membrane mobility of NHE3.
Key words: Na+/H+ exchange, Brush Border, NHE3, Na+ absorption, FRAP
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  R. W. Vandivier, T. R. Richens, S. A. Horstmann, A. M. deCathelineau, M. Ghosh, S. D. Reynolds, Y.-Q. Xiao, D. W. Riches, J. Plumb, E. Vachon, et al. Dysfunctional cystic fibrosis transmembrane conductance regulator inhibits phagocytosis of apoptotic cells with proinflammatory consequences Am J Physiol Lung Cell Mol Physiol, October 1, 2009; 297(4): L677 - L686. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. C. Zachos, X. Li, O. Kovbasnjuk, B. Hogema, R. Sarker, L. J. Lee, M. Li, H. de Jonge, and M. Donowitz NHERF3 (PDZK1) Contributes to Basal and Calcium Inhibition of NHE3 Activity in Caco-2BBe Cells J. Biol. Chem., August 28, 2009; 284(35): 23708 - 23718. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Di Sole, V. Babich, and O. W. Moe The Calcineurin Homologous Protein-1 Increases Na+/H+-Exchanger 3 Trafficking via Ezrin Phosphorylation J. Am. Soc. Nephrol., August 1, 2009; 20(8): 1776 - 1786. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. T. Alexander and S. Grinstein Tethering, recycling and activation of the epithelial sodium-proton exchanger, NHE3 J. Exp. Biol., June 1, 2009; 212(11): 1630 - 1637. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Donowitz, S. Mohan, C. X. Zhu, T.-E Chen, R. Lin, B. Cha, N. C. Zachos, R. Murtazina, R. Sarker, and X. Li NHE3 regulatory complexes J. Exp. Biol., June 1, 2009; 212(11): 1638 - 1646. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. A. Kruger, C. C. Yun, G. R. Monteith, and P. Poronnik Muscarinic-induced Recruitment of Plasma Membrane Ca2+-ATPase Involves PSD-95/Dlg/Zo-1-mediated Interactions J. Biol. Chem., January 16, 2009; 284(3): 1820 - 1830. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Yang, H.-C. Huang, H. Yin, R. J. Alpern, and P. A. Preisig RhoA required for acid-induced stress fiber formation and trafficking and activation of NHE3 Am J Physiol Renal Physiol, October 1, 2007; 293(4): F1054 - F1064. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Donowitz and X. Li Regulatory Binding Partners and Complexes of NHE3 Physiol Rev, July 1, 2007; 87(3): 825 - 872. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. Hryciw, J. Ekberg, C. Ferguson, A. Lee, D. Wang, R. G. Parton, C. A. Pollock, C. C. Yun, and P. Poronnik Regulation of Albumin Endocytosis by PSD95/Dlg/ZO-1 (PDZ) Scaffolds: INTERACTION OF Na+-H+ EXCHANGE REGULATORY FACTOR-2 WITH ClC-5 J. Biol. Chem., June 9, 2006; 281(23): 16068 - 16077. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. K. K. Leong, L. E. Yang, C. S. Landon, A. A. McDonough, and K.-P. Yip Phenol injury-induced hypertension stimulates proximal tubule Na+/H+ exchanger activity Am J Physiol Renal Physiol, June 1, 2006; 290(6): F1543 - F1550. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Cha, M. Tse, C. Yun, O. Kovbasnjuk, S. Mohan, A. Hubbard, M. Arpin, and M. Donowitz The NHE3 Juxtamembrane Cytoplasmic Domain Directly Binds Ezrin: Dual Role in NHE3 Trafficking and Mobility in the Brush Border Mol. Biol. Cell, June 1, 2006; 17(6): 2661 - 2673. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Gardet, M. Breton, P. Fontanges, G. Trugnan, and S. Chwetzoff Rotavirus Spike Protein VP4 Binds to and Remodels Actin Bundles of the Epithelial Brush Border into Actin Bodies. J. Virol., April 1, 2006; 80(8): 3947 - 3956. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. McGuire, J. Rong, S.-H. Li, and X.-J. Li Interaction of Huntingtin-associated Protein-1 with Kinesin Light Chain: IMPLICATIONS IN INTRACELLULAR TRAFFICKING IN NEURONS J. Biol. Chem., February 10, 2006; 281(6): 3552 - 3559. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. E. Yang, A. B. Maunsbach, P. K.K. Leong, and A. A. McDonough Redistribution of Myosin VI from Top to Base of Proximal Tubule Microvilli during Acute Hypertension J. Am. Soc. Nephrol., October 1, 2005; 16(10): 2890 - 2896. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. T. Alexander, W. Furuya, K. Szaszi, J. Orlowski, and S. Grinstein Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: Role in apical retention and targeting PNAS, August 23, 2005; 102(34): 12253 - 12258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Shiue, M. W. Musch, Y. Wang, E. B. Chang, and J. R. Turner Akt2 Phosphorylates Ezrin to Trigger NHE3 Translocation and Activation J. Biol. Chem., January 14, 2005; 280(2): 1688 - 1695. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Brone and J. Eggermont PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes Am J Physiol Cell Physiol, January 1, 2005; 288(1): C20 - C29. [Abstract] [Full Text] [PDF]
|
|
