Phosphorylation of the α4 subunit of human α4β2 nicotinic receptors: role of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC)

Pacheco, Mary A.; Pastoor, Tina E.; Wecker, Lynn

doi:10.1016/s0169-328x(03)00138-4

Cited by 17 publications

(23 citation statements)

References 27 publications

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“…The effects of the rare CHRNA4 variants on nAChR binding site production, plasma membrane trafficking, and upregulation by nicotine are further supported by proteomic studies. The a4 subunit of the nAChR is phosphorylated on several sites within the M3-M4 loop (Wecker et al, 2001;Pacheco et al, 2003;Pollock et al, 2007Pollock et al, , 2009), and we augmented the proteomic study by enriching for phosphopeptides prior to LC-MS/MS to identify and profile potential shifts in the phosphorylation state of nAChR subunits and other putatively associated proteins attributable to the presence of the a4 rare variants. The degree of difference relative to the common variant may identify interesting targets for future mechanistic studies and is an indicator of the severity of effect of a single amino acid substitution on the function/assembly/trafficking of a4b2 nAChRs conferred by these rare a4 variants.…”

Section: Discussionmentioning

confidence: 99%

Rare Human Nicotinic Acetylcholine Receptor α4 Subunit (CHRNA4) Variants Affect Expression and Function of High-Affinity Nicotinic Acetylcholine Receptors

McClure-Begley

Papke

Stone

et al. 2014

J Pharmacol Exp Ther

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Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). a4b2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encode the a4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants (a4R336C, a4P451L, and a4R487Q) to the common variant to determine their effects on a4b2 nAChR pharmacology. We examined [ 3 H]epibatidine binding, interacting proteins, and phosphorylation of the a4 nAChR subunit with liquid chromatography and tandem mass spectrometry (LC-MS/ MS) in HEK 293 cells and voltage-clamp electrophysiology in Xenopus laevis oocytes. We observed significant effects of the a4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified a4b2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in X. laevis oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these a4 rare variants, as well as shifts in receptor function after incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of human a4b2 nAChRs, resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common a4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.

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Section: Discussionmentioning

confidence: 99%

Rare Human Nicotinic Acetylcholine Receptor α4 Subunit (CHRNA4) Variants Affect Expression and Function of High-Affinity Nicotinic Acetylcholine Receptors

McClure-Begley

Papke

Stone

et al. 2014

J Pharmacol Exp Ther

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“…This intracellular region has been shown to contribute to channel kinetics in muscle nAChR [69, 70] and contains determinants of channel conductance in 5-HT 3 A receptors [71-73]. It also contains phosphorylation sites, and it has been demonstrated that phosphorylation modulates expression, upregulation, desensitization, and interaction with cytoskeleton proteins of nAChRs [74][75][76][77][78][79][80].…”

Section: The Extracellular Domain: Location Of the Agonist Binding Sitesmentioning

confidence: 99%

Structural Basis of Activation of Cys-Loop Receptors: the Extracellular–Transmembrane Interface as a Coupling Region

2009

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Cys-loop receptors mediate rapid transmission throughout the nervous system by converting a chemical signal into an electric one. They are pentameric proteins with an extracellular domain that carries the transmitter binding sites and a transmembrane region that forms the ion pore. Their essential function is to couple the binding of the agonist at the extracellular domain to the opening of the ion pore. How the structural changes elicited by agonist binding are propagated through a distance of 50 A to the gate is therefore central for the understanding of the receptor function. A step forward toward the identification of the structures involved in gating has been given by the recently elucidated high-resolution structures of Cys-loop receptors and related proteins. The extracellular-transmembrane interface has attracted attention because it is a structural transition zone where beta-sheets from the extracellular domain merge with alpha-helices from the transmembrane domain. Within this zone, several regions form a network that relays structural changes from the binding site toward the pore, and therefore, this interface controls the beginning and duration of a synaptic response. In this review, the most recent findings on residues and pairwise interactions underlying channel gating are discussed, the main focus being on the extracellular-transmembrane interface.

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“…The net effect of targeted deletion of the ␣ 4 subunit on PKC activity in our system is difficult to predict because signaling through nAChRs can either increase or decrease PKC activity, depending on the preparation [e.g., increase in synaptosomes (Soliakov and Wonnacott, 2001) and in rat prefrontal cortex slices (Drew and Werling, 2003) and decrease in vivo (Sun et al, 2004)] and the sensitization state of the nAChRs (Sun et al, 2004). The situation is further complicated by the fact that the ␣ 4 nAChR subunit itself is phosphorylated by PKC (Pacheco et al, 2003), and the phosphorylation status influences the sensitization state of the molecule (Fenster et al, 1999).…”

Section: Downloaded Frommentioning

confidence: 99%

Mice Lacking the α4 Nicotinic Receptor Subunit Fail to Modulate Dopaminergic Neuronal Arbors and Possess Impaired Dopamine Transporter Function

Parish

Nunan²,

Finkelstein³

et al. 2005

Mol Pharmacol

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Neuronal nicotinic acetylcholine receptors (nAChRs) at presynaptic sites can modulate dopaminergic synaptic transmission by regulating dopamine (DA) release and uptake. Dopaminergic transmission in nigrostriatal and mesolimbic pathways is vital for the coordination of movement and is associated with learning and behavioral reinforcement. We reported recently that the D2 DA receptor plays a central role in regulating the arbor size of substantia nigra dopaminergic neurons. Given the known effects of nAChRs on dopaminergic neurotransmission, we assessed the ability of the ␣ 4 nAChR subunit to regulate arbor size of dopaminergic neurons by comparing responses of wild-type and ␣ 4 nAChR subunit knockout [␣ 4 (Ϫ/Ϫ)] mice to long-term exposure to cocaine, amphetamine, nicotine, and haloperidol, and after substantia nigra neurotoxic lesioning. We found that dopaminergic neurons in adult drug-naive ␣ 4 (Ϫ/Ϫ) mice had significantly larger terminal arbors, and despite normal shortterm behavioral responses to drugs acting on pre-and postsynaptic D2 DA receptors, they were unable to modulate their terminal arbor in response to pharmacological manipulation or after lesioning. In addition, although synaptosome DA uptake studies showed that the interaction of the D2 DA receptor and the dopamine transporter (DAT) was preserved in ␣ 4 (Ϫ/Ϫ) mice, DAT function was found to be impaired. These findings suggest that the ␣ 4 subunit of the nAChR is an independent regulator of terminal arbor size of nigrostriatal dopaminergic neurons and that reduced functionality of presynaptic DAT may contribute to this effect by impairing DA uptake.

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Phosphorylation of the α4 subunit of human α4β2 nicotinic receptors: role of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC)

Cited by 17 publications

References 27 publications

Rare Human Nicotinic Acetylcholine Receptor α4 Subunit (CHRNA4) Variants Affect Expression and Function of High-Affinity Nicotinic Acetylcholine Receptors

Rare Human Nicotinic Acetylcholine Receptor α4 Subunit (CHRNA4) Variants Affect Expression and Function of High-Affinity Nicotinic Acetylcholine Receptors

Structural Basis of Activation of Cys-Loop Receptors: the Extracellular–Transmembrane Interface as a Coupling Region

Mice Lacking the α4 Nicotinic Receptor Subunit Fail to Modulate Dopaminergic Neuronal Arbors and Possess Impaired Dopamine Transporter Function

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