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| Home > Publications database > Über die Untereinheiten-Stöchiometrie des zyklisch Nukleotid-gesteuerten Ionenkanals aus Sehstäbchen |
| Home > Publications database > Über die Untereinheiten-Stöchiometrie des zyklisch Nukleotid-gesteuerten Ionenkanals aus Sehstäbchen |
| Dissertation / PhD Thesis/Book | PreJuSER-26442 |
2002
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/2564
Report No.: Juel-4018
Abstract: 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). 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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