Binding of the cGMP-gated channel to the Na/Ca-K exchanger in rod photoreceptors

Abstract

The intracellular Ca2+ concentration in rod outer segments of vertebrate photoreceptors is controlled by Ca2+ influx through cGMP-gated channels and by Ca2+ efflux driven by Na/Ca-K exchangers. Previously, we suggested that channel and exchanger are associated (Bauer, P. J., and Drechsler, M. (1992) J. Physiol. (Lond.) 451, 109-131). This suggestion has been thoroughly examined using a variety of biochemical approaches. First, we took advantage of the fact that cGMP-gated channels bind calmodulin (CAM). Using CAM affinity chromatographic purification of the channel in 10 mM CHAPS, a significant fraction of exchanger was co-eluted with the channel indicating a binding affinity between channel and exchanger. Binding of channel and exchanger was examined more directly by crosslinking of proteins in the rod outer segment membranes. Activation of the channel with cyclic 8- bromo-GMP lead to exposure of a cysteine, which allowed cross-linking of the channel to the exchanger with the thiol-specific reagent DL-1,4-bismaleimido- 2,3-butanediol. Cleavage of the cross-links and electrophoretic analysis indicated that a cross-link between the α-subunit of the channel and the exchanger formed. Furthermore, a cross-link between two adjacent α-subunits of the channel was found, suggesting that the α-subunits of the native channel are dimerized. Further support for an interaction between α-subunit and exchanger was obtained by in vitro experiments. Specific binding of the exchanger to the α-subunit but not to the β-subunit of the channel was observed in Western blots of purified channel incubated with purified exchanger. This study suggests that two exchanger molecules bind to one cGMP- gated channel and, more specifically, that binding of exchanger molecules occurs at the α-subunits, which in the native channel are dimerized. The implications of these findings regarding the possibility of local Ca2+ signaling in vertebrate photoreceptors will be discussed.