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Vol. 63, Issue 1, 243-252, January 2003
Department of Molecular Pharmacology, Medical School of the
Technical University of Aachen, Aachen, Germany
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.
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