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Volume 17, Number 17,
Issue of September 1, 1997
pp. 6529-6533
Copyright ©1997 Society for Neuroscience
Baculovirus Expression Provides Direct Evidence for Heteromeric
Assembly of P2X2 and P2X3 Receptors
Received April 18, 1996; revised June 9, 1997; accepted June 11, 1997.
Kathryn M. Radford,
Caterina Virginio,
Annmarie Surprenant,
R. Alan North, and
Eric Kawashima
Geneva Biomedical Research Institute, Glaxo Wellcome Research and
Development, 1228 Geneva, Switzerland
P2X2 and P2X3 are subunits of P2X
receptors, cation channels opened by binding extracellular ATP. cDNAs
encoding P2X2 and P2X3 receptor subunits, each
with one of two C-terminal epitope tags, were cloned into baculovirus.
Virally infected insect cells (Spodoptera frugiperda)
expressed moderate to high levels of the corresponding proteins, as
detected by Western blotting, by the specific binding of
[35S]ATP and by whole-cell recordings of membrane
current evoked by ATP or   methylene-ATP. In cells infected at the
same time with two viruses encoding P2X2 and
P2X3 receptors, the two proteins could be
cross-immunoprecipitated with antibodies specific for either of the
epitope tags. Whole-cell recordings from these cells showed that ATP
and methylene-ATP evoked currents with agonist sensitivity and
desensitization quite distinct from those observed when
P2X2 or P2X3 receptors were expressed alone.
The results offer a method to express large amounts of P2X receptor
protein, and they provide direct evidence that P2X2 and
P2X3 subunits assemble to form heteromeric channels having
distinct properties from those formed as homomers.
Key words:
ATP;
P2X receptors;
baculovirus;
cation channels;
coimmunoprecipitation;
subunit assembly
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