PKCε phosphorylates α4β2 nicotinic ACh receptors and promotes recovery from desensitization

Anna M Lee, D. F. Wu, J. Dadgar, D. Wang, T. McMahon, R. O. Messing

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Abstract

Background and Purpose Nicotinic (ACh) receptor recovery from desensitization is modulated by PKC, but the PKC isozymes and the phosphorylation sites involved have not been identified. We investigated whether PKCε phosphorylation of α4β2 nAChRs regulates receptor recovery from desensitization. Experimental Approach Receptor recovery from desensitization was investigated by electrophysiological characterization of human α4β2 nAChRs. Phosphorylation of the α4 nAChR subunit was assessed by immunoblotting of mouse synaptosomes. Hypothermia induced by sazetidine-A and nicotine was measured in Prkce-/- and wild-type mice. Key Results Inhibiting PKCε impaired the magnitude of α4β2 nAChR recovery from desensitization. We identified five putative PKCε phosphorylation sites in the large intracellular loop of the α4 subunit, and mutating four sites to alanines also impaired recovery from desensitization. α4 nAChR subunit phosphorylation was reduced in synaptosomes from Prkce-/- mice. Sazetidine-A-induced hypothermia, which is mediated by α4β2 nAChR desensitization, was more severe and prolonged in Prkce-/- than in wild-type mice. Conclusions and Implications PKCε phosphorylates the α4 nAChR subunit and regulates recovery from receptor desensitization. This study illustrates the importance of phosphorylation in regulating α4β2 receptor function, and suggests that reducing phosphorylation prolongs receptor desensitization and decreases the number of receptors available for activation.

Original languageEnglish (US)
Pages (from-to)4430-4441
Number of pages12
JournalBritish Journal of Pharmacology
Volume172
Issue number17
DOIs
StatePublished - Sep 1 2015

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Nicotinic Receptors
Cholinergic Receptors
Phosphorylation
Induced Hypothermia
Synaptosomes
Nicotine
Immunoblotting
Alanine
Isoenzymes

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PKCε phosphorylates α4β2 nicotinic ACh receptors and promotes recovery from desensitization. / Lee, Anna M; Wu, D. F.; Dadgar, J.; Wang, D.; McMahon, T.; Messing, R. O.

In: British Journal of Pharmacology, Vol. 172, No. 17, 01.09.2015, p. 4430-4441.

Research output: Contribution to journalArticle

Lee, Anna M ; Wu, D. F. ; Dadgar, J. ; Wang, D. ; McMahon, T. ; Messing, R. O. / PKCε phosphorylates α4β2 nicotinic ACh receptors and promotes recovery from desensitization. In: British Journal of Pharmacology. 2015 ; Vol. 172, No. 17. pp. 4430-4441.
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abstract = "Background and Purpose Nicotinic (ACh) receptor recovery from desensitization is modulated by PKC, but the PKC isozymes and the phosphorylation sites involved have not been identified. We investigated whether PKCε phosphorylation of α4β2 nAChRs regulates receptor recovery from desensitization. Experimental Approach Receptor recovery from desensitization was investigated by electrophysiological characterization of human α4β2 nAChRs. Phosphorylation of the α4 nAChR subunit was assessed by immunoblotting of mouse synaptosomes. Hypothermia induced by sazetidine-A and nicotine was measured in Prkce-/- and wild-type mice. Key Results Inhibiting PKCε impaired the magnitude of α4β2 nAChR recovery from desensitization. We identified five putative PKCε phosphorylation sites in the large intracellular loop of the α4 subunit, and mutating four sites to alanines also impaired recovery from desensitization. α4 nAChR subunit phosphorylation was reduced in synaptosomes from Prkce-/- mice. Sazetidine-A-induced hypothermia, which is mediated by α4β2 nAChR desensitization, was more severe and prolonged in Prkce-/- than in wild-type mice. Conclusions and Implications PKCε phosphorylates the α4 nAChR subunit and regulates recovery from receptor desensitization. This study illustrates the importance of phosphorylation in regulating α4β2 receptor function, and suggests that reducing phosphorylation prolongs receptor desensitization and decreases the number of receptors available for activation.",
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AU - Wu, D. F.

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AU - McMahon, T.

AU - Messing, R. O.

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N2 - Background and Purpose Nicotinic (ACh) receptor recovery from desensitization is modulated by PKC, but the PKC isozymes and the phosphorylation sites involved have not been identified. We investigated whether PKCε phosphorylation of α4β2 nAChRs regulates receptor recovery from desensitization. Experimental Approach Receptor recovery from desensitization was investigated by electrophysiological characterization of human α4β2 nAChRs. Phosphorylation of the α4 nAChR subunit was assessed by immunoblotting of mouse synaptosomes. Hypothermia induced by sazetidine-A and nicotine was measured in Prkce-/- and wild-type mice. Key Results Inhibiting PKCε impaired the magnitude of α4β2 nAChR recovery from desensitization. We identified five putative PKCε phosphorylation sites in the large intracellular loop of the α4 subunit, and mutating four sites to alanines also impaired recovery from desensitization. α4 nAChR subunit phosphorylation was reduced in synaptosomes from Prkce-/- mice. Sazetidine-A-induced hypothermia, which is mediated by α4β2 nAChR desensitization, was more severe and prolonged in Prkce-/- than in wild-type mice. Conclusions and Implications PKCε phosphorylates the α4 nAChR subunit and regulates recovery from receptor desensitization. This study illustrates the importance of phosphorylation in regulating α4β2 receptor function, and suggests that reducing phosphorylation prolongs receptor desensitization and decreases the number of receptors available for activation.

AB - Background and Purpose Nicotinic (ACh) receptor recovery from desensitization is modulated by PKC, but the PKC isozymes and the phosphorylation sites involved have not been identified. We investigated whether PKCε phosphorylation of α4β2 nAChRs regulates receptor recovery from desensitization. Experimental Approach Receptor recovery from desensitization was investigated by electrophysiological characterization of human α4β2 nAChRs. Phosphorylation of the α4 nAChR subunit was assessed by immunoblotting of mouse synaptosomes. Hypothermia induced by sazetidine-A and nicotine was measured in Prkce-/- and wild-type mice. Key Results Inhibiting PKCε impaired the magnitude of α4β2 nAChR recovery from desensitization. We identified five putative PKCε phosphorylation sites in the large intracellular loop of the α4 subunit, and mutating four sites to alanines also impaired recovery from desensitization. α4 nAChR subunit phosphorylation was reduced in synaptosomes from Prkce-/- mice. Sazetidine-A-induced hypothermia, which is mediated by α4β2 nAChR desensitization, was more severe and prolonged in Prkce-/- than in wild-type mice. Conclusions and Implications PKCε phosphorylates the α4 nAChR subunit and regulates recovery from receptor desensitization. This study illustrates the importance of phosphorylation in regulating α4β2 receptor function, and suggests that reducing phosphorylation prolongs receptor desensitization and decreases the number of receptors available for activation.

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