The α4 subunit of rat α4β2 nicotinic receptors is phosphorylated in vivo

Viseshakul, Nareerat; Figl, Antonio; Lytle, Christian; Cohen, Bruce

doi:10.1016/s0169-328x(98)00128-4

Cited by 19 publications

(18 citation statements)

References 21 publications

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“…The molecular weights for the specific bands identified by antibodies against ␣3, ␣5, ␤2, and ␤4 subunits also closely match the molecular weight determined from the amino acid sequences as well as those reported in various species, including rats (Martin-Ruiz et al, 1999;Guan et al, 2000Guan et al, , 2001Gotti et al, 1997;Yeh et al 2001a,b). However, there are also observations that report molecular weights of ␣4 subunits that differ from those described in this study Viseshakul et al, 1998;ArroyaJimenez et al, 1999). Arroya-Jimenez et al (1999), utilizing a commercially available antibody against the ␣4 subunit, identified a 75 kD protein band in Western analysis from rat brain extract.…”

Section: Nicotinic Receptor Binding Sites In Rat Spinal Cordcontrasting

confidence: 61%

Nicotinic acetylcholine receptor distribution in relation to spinal neurotransmission pathways

Khan

Osaka

Stanislaus

et al. 2003

J of Comparative Neurology

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Neuronal nicotinic receptors (nAChR) are pentameric assemblies of subunits of a gene family where specified combinations of alpha and beta subunits form functional receptors. To extend our understanding of the role of spinal nAChR in the processing of sensory stimuli and regulation of autonomic and motor responses, we initiated investigations to localize nAChR subunit expression within discrete spinal regions and cell types. High-affinity epibatidine binding was present in the superficial dorsal and ventral horns, the mediolateral and central canal regions. RT-PCR identified transcripts for alpha3, alpha4, alpha5, beta2, and beta4 in both spinal cord parenchyma and dorsal root ganglia (DRG). Our affinity-purified antibodies against alpha3, alpha4, alpha5, beta2, and beta4 subunits identified specific protein bands of appropriate molecular mass (preadsorbed with the respective antigens) in specific tissues and cells that express nicotinic receptors, including the spinal cord and DRG neurons. Having established the absence of crossreactivity with related subunits, specific fluorescence labeling of nerve terminals and cell bodies was achieved and correlated with the distribution of defined marker proteins and nicotinic receptor binding sites determined autoradiographically. Our findings indicate that alpha3, alpha4, alpha5, beta2, and beta4 subunits are all expressed on primary afferents (IB4-positive terminals) in the spinal cord. The predominant presynaptic (synaptophysin colocalization) labeling is in the superficial layer of the dorsal horn. These receptor subunits, except for beta4, are also present in postsynaptic autonomic (anti-bNOS-positive) and somatic motor neurons (anti-VAChT-positive). The alpha3, alpha5, and beta2 subunits showed additional staining in glial (anti-GFAP-positive) cells. These studies reveal a dense and distinguishable distribution of nAChR subunits in the spinal cord and point toward future therapeutic targeting for specific spinal actions.

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Section: Nicotinic Receptor Binding Sites In Rat Spinal Cordcontrasting

confidence: 61%

Nicotinic acetylcholine receptor distribution in relation to spinal neurotransmission pathways

Khan

Osaka

Stanislaus

et al. 2003

J of Comparative Neurology

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“…It is important to mention that Wecker et al (2001)later referred this residue as S368 (numbered to include the signal peptide) and will be equivalent to S364 in our numbering. This residue is contained within the XRRXSX sequence that is phosphorylated by PKA (Wecker et al 2001) and is different from the residue mutated in this study, which is part of a putative consensus sequence phosphorylated by PKC (HHR SPR THT) (Viseshakul et al 1998). …”

Section: Introductionmentioning

confidence: 88%

“…Two of the serine residues, S336 and S516, in the α4 M3/ M4 intracellular domain, are part of PKC phosphorylation consensus sites. Viseshakul et al (1998) showed that the α4 subunit of α4β2 nAChRs was phosphorylated in vivo. Furthermore, Wecker et al (2001) demonstrated that the α4 M3/M4 intracellular domain was phosphorylated by PKA and PKC.…”

Section: Introductionmentioning

confidence: 99%

Contribution of Position α4S336 on Functional Expression and Up-regulation of α4β2 Neuronal Nicotinic Receptors

et al. 2008

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Phosphorylation of the nicotinic acetyl-choline receptor (nAChR) is believed to play a critical role in its nicotine-induced desensitization and up-regulation. We examined the contribution of a consensus PKC site in the α4 M3/M4 intracellular loop (α4S336) on the desensitization and up-regulation of α4β2 nAChRs expressed in oocytes. Position α4S336 was replaced with either alanine to abolish potential phosphorylation at this site or with aspartic acid to mimic phosphorylation at this same site. Mutations α4S336A and α4S336D displayed a threefold increase in the ACh-induced response and an increase in ACh EC50. Epibatidine binding revealed a three and sevenfold increase in surface expression for the α4S336A and α4S336D mutations, respectively, relative to wild-type, therefore, both mutations enhanced expression of the α4β2 nAChR. Interestingly, the EC50’s and peak currents for nicotine activation remained unaffected in both mutants. Both mutations abolished the nicotine-induced up-regulation that is normally observed in the wild-type. The present data suggest that adding or removing a negative charge at this phosphorylation site cannot be explained by a simple straightforward on-and-off mechanism; rather a more complex mechanism(s) may govern the functional expression of the α4β2 nAChR. Along the same line, our data support the idea that phosphorylation at multiple consensus sites in the α4 subunit could play a remarkable role on the regulation of the functional expression of the α4β2 nAChR.

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“…The major consensus phosphorylation sequences on nAChR subunits lie within the large intracellular loop between TM domains 3 and 4 (Swope et al, 1992). Several of these sites have been shown to be directly phosphorylated in vitro by PKA or PKC (Vijayaraghavan et al, 1990;Nakayama et al, 1993;Moss et al, 1996;Wecker et al, 2001), and may underlie the observed phosphorylation of certain nAChRs in vivo (Viseshakul et al, 1998). In particular, serine 368 on the ␣4 subunit, may underlie the enhanced recovery from desensitization of oocyteexpressed ␣4␤2 nAChRs mediated by Ca 2ϩ /PKC, because mutation of this residue limits recovery (Fenster et al, 1999a) and, although it has a higher affinity for PKA, serine 368 is a substrate for PKC (Wecker et al, 2001).…”

Section: Regulation Of Desensitizationmentioning

confidence: 99%

Desensitization of neuronal nicotinic receptors

2002

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ABSTRACT:The loss of functional response upon continuous or repeated exposure to agonist, desensitization, is an intriguing phenomenon if not as yet a welldefined physiological mechanism. However, detailed evaluation of the properties of desensitization, especially for the superfamily of ligand-gated ion channels, reveals how the nervous system could make important use of this process that goes far beyond simply curtailing excessive receptor stimulation and the prevention of excitotoxicity. Here we will review the mechanistic basis of desensitization and discuss how the subunit-dependent properties and regulation of nicotinic acetylcholine receptor (nAChR) desensitization contribute to the functional diversity of these channels. These studies provide the essential framework for understanding how the physiological regulation of desensitization could be a major determinant of synaptic efficacy by controlling, in both the short and long term, the number of functional receptors. This type of mechanism can be extended to explain how the continuous occupation of desensitized receptors during chronic nicotine exposure contributes to drug addiction, and highlights the potential significance of prolonged nAChR desensitization that would also occur as a result of extended acetylcholine lifetime during treatment of Alzheimer's disease with cholinesterase inhibitors. Thus, a clearer picture of the importance of nAChR desensitization in both normal information processing and in various diseased states is beginning to emerge.

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The α4 subunit of rat α4β2 nicotinic receptors is phosphorylated in vivo

Cited by 19 publications

References 21 publications

Nicotinic acetylcholine receptor distribution in relation to spinal neurotransmission pathways

Nicotinic acetylcholine receptor distribution in relation to spinal neurotransmission pathways

Contribution of Position α4S336 on Functional Expression and Up-regulation of α4β2 Neuronal Nicotinic Receptors

Desensitization of neuronal nicotinic receptors

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