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Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling.

Reilly L, Howie J, Wypijewski K, Ashford ML, Hilgemann DW, Fuller W - FASEB J. (2015)

Bottom Line: Surprisingly, palmitoylation does not influence trafficking or localization of NCX1 to surface membranes, nor does it strongly affect the normal forward or reverse transport modes of NCX1.However, exchangers that cannot be palmitoylated do not inactivate normally (leading to substantial activity in conditions when wild-type exchangers are inactive) and do not promote cargo-dependent endocytosis that internalizes 50% of the cell surface following strong G-protein activation or large Ca transients.The palmitoylated cysteine in NCX1 is found in all vertebrate and some invertebrate NCX homologs.

View Article: PubMed Central - PubMed

Affiliation: *Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry, and Nursing, University of Dundee, Dundee, United Kingdom; and Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

No MeSH data available.


Related in: MedlinePlus

Impact of palmitoylation of NCX1 on massive endocytosis occurring in response to excessive G-protein activation and Ca influx. Stably transfected FT-293 cells expressing WT and C739A NCX1 were voltage clamped in the whole-cell mode with 8 mM MgATP in the pipette solution. A) Using a highly Ca-buffered cytoplasmic solution (10 mM EGTA with 1 mM Ca) with 0.25 mM GTPγS, cells expressing WT exchangers internalize 33 ± 3% of their membrane within 40 s of establishing the whole cell configuration, whereas cells expressing C739A exchangers internalize only 10 ± 2%. (P < 0.001). At 3 min, the difference between exchanger-expression cells is not significant, and membrane loss in cells without exchangers is negligible. B) Quantitation of endocytosis as the time required to decrease membrane area by 30%. C739A-expressing cells internalize at a rate that is >3 times less than cells expressing WT exchangers. C) Using a weakly Ca-buffered cytoplasmic solution (0.5 mM EGTA with 0.25 mM Ca), extracellular Ca (2 mM) is applied with 7 μM ionomycin to promote Ca influx. In WT-expressing cells, small initial exocytic responses (i.e., with increasing membrane area) are followed by loss of 50% of the cell surface within about 20 s. In C739 cells, the exocytic responses are larger and endocytic responses are delayed. The loss of membrane area in cells expressing WT exchangers amounts to 33 ± 4%, and loss of membrane in C739A-expressing cells amounts to only 13 ± 6% (P < 0.01). D) Quantitation of Ca-induced endocytic responses as the time required to remove 30% of the cell surface. Endocytosis times are increased by >50% in C739A cells, compared with WT cells (P < 0.05), and endocytosis times are doubled in cells that do not express NCX1 (P < 0.01). *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 7: Impact of palmitoylation of NCX1 on massive endocytosis occurring in response to excessive G-protein activation and Ca influx. Stably transfected FT-293 cells expressing WT and C739A NCX1 were voltage clamped in the whole-cell mode with 8 mM MgATP in the pipette solution. A) Using a highly Ca-buffered cytoplasmic solution (10 mM EGTA with 1 mM Ca) with 0.25 mM GTPγS, cells expressing WT exchangers internalize 33 ± 3% of their membrane within 40 s of establishing the whole cell configuration, whereas cells expressing C739A exchangers internalize only 10 ± 2%. (P < 0.001). At 3 min, the difference between exchanger-expression cells is not significant, and membrane loss in cells without exchangers is negligible. B) Quantitation of endocytosis as the time required to decrease membrane area by 30%. C739A-expressing cells internalize at a rate that is >3 times less than cells expressing WT exchangers. C) Using a weakly Ca-buffered cytoplasmic solution (0.5 mM EGTA with 0.25 mM Ca), extracellular Ca (2 mM) is applied with 7 μM ionomycin to promote Ca influx. In WT-expressing cells, small initial exocytic responses (i.e., with increasing membrane area) are followed by loss of 50% of the cell surface within about 20 s. In C739 cells, the exocytic responses are larger and endocytic responses are delayed. The loss of membrane area in cells expressing WT exchangers amounts to 33 ± 4%, and loss of membrane in C739A-expressing cells amounts to only 13 ± 6% (P < 0.01). D) Quantitation of Ca-induced endocytic responses as the time required to remove 30% of the cell surface. Endocytosis times are increased by >50% in C739A cells, compared with WT cells (P < 0.05), and endocytosis times are doubled in cells that do not express NCX1 (P < 0.01). *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: Compositions of solutions in electrophysiology experiments


Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling.

Reilly L, Howie J, Wypijewski K, Ashford ML, Hilgemann DW, Fuller W - FASEB J. (2015)

Impact of palmitoylation of NCX1 on massive endocytosis occurring in response to excessive G-protein activation and Ca influx. Stably transfected FT-293 cells expressing WT and C739A NCX1 were voltage clamped in the whole-cell mode with 8 mM MgATP in the pipette solution. A) Using a highly Ca-buffered cytoplasmic solution (10 mM EGTA with 1 mM Ca) with 0.25 mM GTPγS, cells expressing WT exchangers internalize 33 ± 3% of their membrane within 40 s of establishing the whole cell configuration, whereas cells expressing C739A exchangers internalize only 10 ± 2%. (P < 0.001). At 3 min, the difference between exchanger-expression cells is not significant, and membrane loss in cells without exchangers is negligible. B) Quantitation of endocytosis as the time required to decrease membrane area by 30%. C739A-expressing cells internalize at a rate that is >3 times less than cells expressing WT exchangers. C) Using a weakly Ca-buffered cytoplasmic solution (0.5 mM EGTA with 0.25 mM Ca), extracellular Ca (2 mM) is applied with 7 μM ionomycin to promote Ca influx. In WT-expressing cells, small initial exocytic responses (i.e., with increasing membrane area) are followed by loss of 50% of the cell surface within about 20 s. In C739 cells, the exocytic responses are larger and endocytic responses are delayed. The loss of membrane area in cells expressing WT exchangers amounts to 33 ± 4%, and loss of membrane in C739A-expressing cells amounts to only 13 ± 6% (P < 0.01). D) Quantitation of Ca-induced endocytic responses as the time required to remove 30% of the cell surface. Endocytosis times are increased by >50% in C739A cells, compared with WT cells (P < 0.05), and endocytosis times are doubled in cells that do not express NCX1 (P < 0.01). *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 7: Impact of palmitoylation of NCX1 on massive endocytosis occurring in response to excessive G-protein activation and Ca influx. Stably transfected FT-293 cells expressing WT and C739A NCX1 were voltage clamped in the whole-cell mode with 8 mM MgATP in the pipette solution. A) Using a highly Ca-buffered cytoplasmic solution (10 mM EGTA with 1 mM Ca) with 0.25 mM GTPγS, cells expressing WT exchangers internalize 33 ± 3% of their membrane within 40 s of establishing the whole cell configuration, whereas cells expressing C739A exchangers internalize only 10 ± 2%. (P < 0.001). At 3 min, the difference between exchanger-expression cells is not significant, and membrane loss in cells without exchangers is negligible. B) Quantitation of endocytosis as the time required to decrease membrane area by 30%. C739A-expressing cells internalize at a rate that is >3 times less than cells expressing WT exchangers. C) Using a weakly Ca-buffered cytoplasmic solution (0.5 mM EGTA with 0.25 mM Ca), extracellular Ca (2 mM) is applied with 7 μM ionomycin to promote Ca influx. In WT-expressing cells, small initial exocytic responses (i.e., with increasing membrane area) are followed by loss of 50% of the cell surface within about 20 s. In C739 cells, the exocytic responses are larger and endocytic responses are delayed. The loss of membrane area in cells expressing WT exchangers amounts to 33 ± 4%, and loss of membrane in C739A-expressing cells amounts to only 13 ± 6% (P < 0.01). D) Quantitation of Ca-induced endocytic responses as the time required to remove 30% of the cell surface. Endocytosis times are increased by >50% in C739A cells, compared with WT cells (P < 0.05), and endocytosis times are doubled in cells that do not express NCX1 (P < 0.01). *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: Compositions of solutions in electrophysiology experiments

Bottom Line: Surprisingly, palmitoylation does not influence trafficking or localization of NCX1 to surface membranes, nor does it strongly affect the normal forward or reverse transport modes of NCX1.However, exchangers that cannot be palmitoylated do not inactivate normally (leading to substantial activity in conditions when wild-type exchangers are inactive) and do not promote cargo-dependent endocytosis that internalizes 50% of the cell surface following strong G-protein activation or large Ca transients.The palmitoylated cysteine in NCX1 is found in all vertebrate and some invertebrate NCX homologs.

View Article: PubMed Central - PubMed

Affiliation: *Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry, and Nursing, University of Dundee, Dundee, United Kingdom; and Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

No MeSH data available.


Related in: MedlinePlus