Abstract
We investigated the transmembrane topology of the P2X2 receptor subunit expressed in HEK 293 cells. Initial studies using two P2X subunits expressed in tandem indicated that the amino- and carboxy-termini are on the same side of the membrane. Immunofluorescence studies showed the cytoplasmic orientation of the amino- and carboxy-termini. Finally, N-glycosylation scanning mutagenesis revealed that reporter sites inserted into the central loop, but not those in the amino- or carboxy-terminal regions, were glycosylated, thus suggesting an extracellular placement for that domain. Our results support a two-transmembrane arrangement for P2X receptors with intracellular amino- and carboxy-termini.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphate / analogs & derivatives*
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Adenosine Triphosphate / pharmacology
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Animals
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Cell Line
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DNA, Complementary
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Fluorescent Antibody Technique, Indirect
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Humans
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Ion Channel Gating / drug effects*
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Membrane Proteins / chemistry*
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Membrane Proteins / genetics
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Membrane Proteins / physiology
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Protein Conformation
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Rats
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Receptors, Purinergic P2 / chemistry*
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Receptors, Purinergic P2 / genetics
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Receptors, Purinergic P2 / physiology
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Receptors, Purinergic P2X2
Substances
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DNA, Complementary
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Membrane Proteins
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P2RX2 protein, human
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Receptors, Purinergic P2
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Receptors, Purinergic P2X2
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Adenosine Triphosphate
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alpha,beta-methyleneadenosine 5'-triphosphate