Andreas Schwarzer and Paul J. Bauer

Institute f. Biological Information Processing, Research Center, POB 1913, D-52425 Juelich, Germany

Contact Person: Paul J. Bauer (p.j.bauer@fz-juelich.de)

Ca ions play an important role in vertebrate photoreceptors, both in signal transduction and light adaptation. The intracellular Ca concentration is controlled by Ca influx through cGMP-gated cation channels and Ca extrusion via Na/Ca,K exchange. Here, we report a cross-linking study which demonstrates that the Na/Ca,K exchanger associates in the plasma membrane to form a homo-dimer. We found that this protein could be cross-linked with many cysteine-specific reagents. Purification of the exchanger after cross-linking with a cleavable reagent, and 2D-SDS-PAGE analysis of the cleaved cross-links showed that the Na/Ca,K exchanger formed a homo-dimer. The observed cross-links were specific because no unspecific cross-links could be detected upon re-probing of the Western blots with monoclonal antibodies against cGMP-gated channels or against rhodopsin. - Furthermore, we investigated the influence of cysteine cross-linking on the rate of Na/Ca,K exchange. Using Ca-flux experiments, we found that the Na-induced Ca-flux depended on the spacer of the dimaleimide; compared to the non-cross-linked exchanger, the Ca-flux increased with increasing spacer length, and the Ca-flux decreased if the cysteines were directly cross-linked by a disulfide bridge (’zero spacer cross-link’). Thus by interpolation, the distance of the adjacent cysteines could be estimated to be about 3.5 Angstroms.

Keywords: photoreceptor, Na/Ca,K exchanger, oligomerization, Ca-flux.