Expression of P2X6, a Purinergic Receptor Subunit, Is Affected by Dietary Zinc Deficiency in Rat Hippocampus

Chu, Ye; Mouat, Michael F.; Coffield, Julie A.; Orlando, Ron; Grider, Arthur

doi:10.1385/bter:91:1:77

Cited by 15 publications

(13 citation statements)

References 45 publications

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“…Because the P2X 6 subunit can increase the calcium permeability of the P2X 2 receptor (Egan and Khakh, 2004) and change the pharmacology of both P2X 2 and P2X 4 , it might be "designed" to operate as a modulatory subunit rather than a receptor in its own right. This suggestion is substantiated by many reports of overlapping distributions of P2X 6 with P2X 2 and P2X 4 in both the central nervous system and the periphery (Collo et al, 1996;Soto et al, 1996;Rubio and Soto, 2001;Glass et al, 2002;Turner et al, 2003) and by the demonstration that P2X 6 is up-regulated under pathological conditions such as cancer and zinc deficiency (Urano et al, 1997;Nawa et al, 1999;Chu et al, 2003;Park et al, 2005). More recently, Liang et al (2005) provided evidence of endogenous P2X 4/6 heteromers in a human bronchial epithelial cell line.…”

Section: Discussionmentioning

confidence: 82%

An Uncharged Region within the N Terminus of the P2X₆ Receptor Inhibits Its Assembly and Exit from the Endoplasmic Reticulum

Ormond

Barrera²,

Qureshi³

et al. 2006

Mol Pharmacol

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ATP-gated P2X receptors are trimeric complexes formed by the homomeric or heteromeric assembly of seven different subunits. We have shown previously that, unlike all of the other P2X subunits, the P2X 6 subunit cannot form homomeric receptors and when expressed alone is retained in the endoplasmic reticulum (ER) in monomeric form (J Biol Chem 280:107591-10765, 2005). However, other studies have shown that P2X 6 can form functional heteromeric receptors with P2X 2 and P2X 4 subunits. In this study, we used a combination of immunocytochemistry, surface biotinylation, and atomic force microscopy to investigate the assembly and trafficking of the P2X 6 subunit, both alone and as part of a heteromer. We show that as a heteromer, it exits the ER and is either stably expressed at the cell surface or constitutively internalized, depending on its partner. Through the use of targeted mutation, we demonstrate that an uncharged region at the N terminus of P2X 6 exerts an inhibitory effect on its assembly and export from the ER. When this region is removed, or when charge is added to it, P2X 6 forms homotrimeric assemblies, undergoes complex glycosylation and is delivered to the plasma membrane, albeit less efficiently than the P2X 2 receptor. The N-terminal mutants were, however, nonfunctional. Substituting the uncharged 14-amino acid N-terminal region for the equivalent region of P2X 2 increased ER retention but was not sufficient to prevent the formation of functional homomeric receptors. We propose that the N terminus of the P2X 6 subunit contributes to a mechanism that prevents the inappropriate export and plasma membrane expression of nonfunctional P2X receptors.

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Section: Discussionmentioning

confidence: 82%

An Uncharged Region within the N Terminus of the P2X₆ Receptor Inhibits Its Assembly and Exit from the Endoplasmic Reticulum

Ormond

Barrera²,

Qureshi³

et al. 2006

Mol Pharmacol

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“…Furthermore, the role of P2X 6 as a modulatory subunit might be regulated in response to changes in circumstances. For example, expression of P2X 6 is known to change under pathological conditions such as cancer and zinc deficiency [20,51,58,71]. We have begun to examine the control of P2X heteromer assembly, and these results are discussed below.…”

Section: Homo-oligomeric Receptorsmentioning

confidence: 99%

Determination of the architecture of ionotropic receptors using AFM imaging

Barrera

Henderson

Edwardson

2007

Pflugers Arch - Eur J Physiol

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Fast neurotransmission in the nervous system is mediated by ionotropic receptors, all of which contain several subunits surrounding an integral ion channel. There are three major families of ionotropic receptors: the 'Cysloop' receptors (including the nicotinic receptor for acetylcholine, the 5-HT 3 receptor, the GABA A receptor and the glycine receptor), the glutamate receptors (including the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate and N-methyl-D-aspartic acid receptors) and the P2X receptors for adenosine triphosphate. These receptors are often built from multiple types of subunit, raising the question of the stoichiometry and subunit arrangement within the receptors. This question is of therapeutic significance because in some cases drug-binding sites are located at subunit-subunit interfaces. In this paper, we describe a general method, based on atomic force microscopy imaging, to solve the architecture of multi-subunit proteins, such as the ionotropic receptors. Specific epitope tags are engineered onto each receptor subunit. The subunits are then expressed exogenously in cultured cells, and the receptors are isolated from detergent extracts of membrane fractions by affinity chromatography. The receptors are imaged both alone and in complex with anti-epitope antibodies. The size of the imaged particles provides an estimate of the subunit stoichiometry, whereas the geometry of the receptor-antibody complexes produces more detailed information about the receptor architecture. We use an automated, unbiased system to identify receptors and receptor-antibody complexes and to determine the geometry of the complexes. We are also able to determine the orientation of the receptors on the mica substrate, which will allow us to solve the subunit arrangement within receptors, such as the GABA A receptor, which contain three types of subunits.

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“…The excised protein spots were prepared for in-gel tryptic digestion [14]. Each gel plug was incubated with 150 µl of 50 mM ammonium bicarbonate containing 12.2 M acetonitrile.…”

Section: Mass Spectrometrymentioning

confidence: 99%

Analysis of the liver soluble proteome from bull terriers affected with inherited lethal acrodermatitis

Grider

Mouat

Mauldin

et al. 2007

Molecular Genetics and Metabolism

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Lethal acrodermatitis (LAD) is a genetic disease affecting bull terrier dogs. The phenotype is similar to that for acrodermatitis enteropathica in humans, but is currently without treatment. The purpose of the research presented here is to determine the biochemical defects associated with LAD using proteomic methodologies. Two affected (male and female) and one unaffected (male) bull terrier pups were euthanized at 14 weeks of age, their livers dissected and prepared for twodimensional gel electrophoresis (2DE) and densitometry. Approximately 200 protein spots were observed. The density of the spots within each gel was normalized to the total spot volume of the gel; only those soluble liver protein spots that were consistently different in both of the livers of the affected pups compared to the unaffected pup were excised manually and submitted for MALDI mass spectrometry. Thirteen proteins were identified as differentially expressed in the affected, compared to the unaffected, pups. The proteins were involved in numerous cellular physiological functions, including chaperones, calcium binding, and energy metabolism, as well as being associated with the inflammatory response. Of note were haptoglobin, glutamine synthetase, prohibitin and keratin 10 which exhibited at least a 4-fold level of differential expression. These data represent the first proteomic analysis of this mutation. The differentially expressed proteins that were identified may be key in understanding the etiology of LAD, and may lead to diagnostic tools for its identification within the bull terrier population.

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Expression of P2X6, a Purinergic Receptor Subunit, Is Affected by Dietary Zinc Deficiency in Rat Hippocampus

Cited by 15 publications

References 45 publications

An Uncharged Region within the N Terminus of the P2X₆ Receptor Inhibits Its Assembly and Exit from the Endoplasmic Reticulum

An Uncharged Region within the N Terminus of the P2X₆ Receptor Inhibits Its Assembly and Exit from the Endoplasmic Reticulum

Determination of the architecture of ionotropic receptors using AFM imaging

Analysis of the liver soluble proteome from bull terriers affected with inherited lethal acrodermatitis

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Expression of P2X6, a Purinergic Receptor Subunit, Is Affected by Dietary Zinc Deficiency in Rat Hippocampus

Cited by 15 publications

References 45 publications

An Uncharged Region within the N Terminus of the P2X6 Receptor Inhibits Its Assembly and Exit from the Endoplasmic Reticulum

An Uncharged Region within the N Terminus of the P2X6 Receptor Inhibits Its Assembly and Exit from the Endoplasmic Reticulum

Determination of the architecture of ionotropic receptors using AFM imaging

Analysis of the liver soluble proteome from bull terriers affected with inherited lethal acrodermatitis

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Product

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An Uncharged Region within the N Terminus of the P2X₆ Receptor Inhibits Its Assembly and Exit from the Endoplasmic Reticulum

An Uncharged Region within the N Terminus of the P2X₆ Receptor Inhibits Its Assembly and Exit from the Endoplasmic Reticulum