Verlag des Forschungszentrums Jülich
JUEL-4018
In the present study, the subunit stoichiometry of the native CNG channel was investigated by
applying biochemical methods. For this purpose, the subunits of the CNG channel in ROS
membranes were cross-linked using thiol-specific as well as amino-specific cross-link reagents.
Both approaches resulted in distinct cross-link products which could also be observed when
thiol-specific and amino-specific cross-linking of subunits of the solubilised and purified CNG
channel were performed. Cross-linked and non-cross-linked channel subunits were subjected to
SDS-Polyacrylamidgelelectrophoresis carried out on gradient gels and transferred to PVDFmembranes.
In order to determine the subunit composition of each cross-link species the protein
bands were analysed by Western blotting using specific antibodies against the A- and Bsubunits.
Other A- and B-antibodies which recognize different epitopes of the channel subunits
show the same cross-link products. Therefore, epitope masking as a result of cross-linkink can be
widely excluded. In addition to the immunological verification, the apparent molecular weight of
the cross-link products was determined precisely. These results lead to the number of different
subunits that are present in a cross-link product.
The results of the present study show unambiguously that the native CNG channel from rod
photoreceptors is a tetrameric complex comprising three A-subunits and one B-subunit.
Furthermore, this study suggests that chemical cross-linking is a suitable method to investigate
the stoichiometry of CNG channels. This is of great interest since the subunit stoichiometry
determines many of the functional properties of these channels. In future it will be very
interesting to examine the subunit stoichiometry of CNG channels in other tissues like cones or
sperms by applying chemical cross-linking.
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Ficek, Nicole Yvonne
Über die Untereinheiten-Stöchiometrie des zyklisch Nukleotid-gesteuerten Ionenkanals aus Sehstäbchen
III, III, 96 S., 2003
The cyclic nucleotide-gated (CNG) channel from rod photoreceptors is a heterooligomer
composed of two different subunits. Due to electrophysiological studies with heterologously
expressed CNG channels, a subunit stoichiometry of two A- and two B-subunits has been
postulated for this channel complex (2A-2B).
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