NHERF3 (PDZK1) contributes to basal and calcium inhibition of NHE3 activity in Caco-2BBe cells

Nicholas Zachos, Xuhang Li, Olga N Kovbasnjuk, Boris Hogema, Rafiquel Sarker, Luke J. Lee, Min Li, Hugo de Jonge, Mark Donowitz

Research output: Contribution to journalArticle

Abstract

Elevated intracellular Ca2+ ([Ca2+]i) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca2+]i regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the ionophore, 4-bromo-A23187 (0.5 μM): 1) inhibited NHE3 Vmax activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca2+]i by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca2+]i inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca2+]i; 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca2+.

Original languageEnglish (US)
Pages (from-to)23708-23718
Number of pages11
JournalJournal of Biological Chemistry
Volume284
Issue number35
DOIs
StatePublished - Aug 28 2009

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Carbachol
Cell membranes
Small Interfering RNA
Calcium
Confocal microscopy
Ionophores
Brushes
Fibroblasts
Fluorescence Resonance Energy Transfer
Amino Acids
Cell Membrane
Multiprotein Complexes
Proteins
Calcimycin
Microvilli
Immunoprecipitation
Confocal Microscopy
Fluorescent Antibody Technique
Epithelial Cells
sodium-hydrogen exchanger regulatory factor

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

NHERF3 (PDZK1) contributes to basal and calcium inhibition of NHE3 activity in Caco-2BBe cells. / Zachos, Nicholas; Li, Xuhang; Kovbasnjuk, Olga N; Hogema, Boris; Sarker, Rafiquel; Lee, Luke J.; Li, Min; de Jonge, Hugo; Donowitz, Mark.

In: Journal of Biological Chemistry, Vol. 284, No. 35, 28.08.2009, p. 23708-23718.

Research output: Contribution to journalArticle

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abstract = "Elevated intracellular Ca2+ ([Ca2+]i) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca2+]i regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the ionophore, 4-bromo-A23187 (0.5 μM): 1) inhibited NHE3 Vmax activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca2+]i by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50{\%}) in Caco2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca2+]i inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca2+]i; 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca2+.",
author = "Nicholas Zachos and Xuhang Li and Kovbasnjuk, {Olga N} and Boris Hogema and Rafiquel Sarker and Lee, {Luke J.} and Min Li and {de Jonge}, Hugo and Mark Donowitz",
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T1 - NHERF3 (PDZK1) contributes to basal and calcium inhibition of NHE3 activity in Caco-2BBe cells

AU - Zachos, Nicholas

AU - Li, Xuhang

AU - Kovbasnjuk, Olga N

AU - Hogema, Boris

AU - Sarker, Rafiquel

AU - Lee, Luke J.

AU - Li, Min

AU - de Jonge, Hugo

AU - Donowitz, Mark

PY - 2009/8/28

Y1 - 2009/8/28

N2 - Elevated intracellular Ca2+ ([Ca2+]i) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca2+]i regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the ionophore, 4-bromo-A23187 (0.5 μM): 1) inhibited NHE3 Vmax activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca2+]i by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca2+]i inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca2+]i; 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca2+.

AB - Elevated intracellular Ca2+ ([Ca2+]i) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca2+]i regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the ionophore, 4-bromo-A23187 (0.5 μM): 1) inhibited NHE3 Vmax activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca2+]i by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca2+]i inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca2+]i; 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca2+.

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