Monomeric and dimeric byproducts are the principal functional elements of higher order P2X1 concatamers.
Heteromultimeric assembly of ion channel subunits generates high diversity in ion channel subtypes with distinct pharmacological and functional properties. To determine the subunit stoichiometry and order of ion channels, constructs with several concatenated subunits have been widely used in electrophysiological studies. Here we used primarily biochemical techniques to analyze the synthesis, assembly, and surface expression of P2X1 concatamers. We found that full-length concatamers consisting of two to six contiguous copies of the P2X1 subunit, although readily synthesized in Xenopus laevis oocytes, were entirely retained as aggregates in the endoplasmic reticulum. In contrast, minute levels of lower order byproducts, such as monomers and dimers, that were inherently formed with all the concatamers combined to form defined protein complexes equal in mass to the homotrimeric P2X1 receptor assembled from P2X1 monomers. Besides these complexes consisting of three monomers or one monomer plus one concatenated dimer, only small amounts of concatenated P2X1 trimers reached the plasma membrane. Complexes comprising more than three subunits were not observed in the plasma membrane. The byproduct complexes can account fully for the ATP-gated currents arising from expression of concatenated P2X1 subunits. These results strongly corroborate a trimeric architecture for P2X receptors yet indicate that formation of lower order by-products can be a pitfall of the concatamer approach.
Nicke
Annette
A
Department of Molecular Pharmacology, Medical School of the Technical University of Aachen, Aachen, Germany.
Rettinger
Jürgen
J
Schmalzing
Günther
G
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D006031:Glycosylation; D008715:Methionine / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000378:metabolism*; D058469:Receptors, Purinergic P2X; D014982:Xenopus laevis
D001704:Biopolymers; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D000255:Adenosine Triphosphate; D008715:Methionine