Abstract
Purpose: :
Cyclic nucleotide-gated (CNG) channels are localized exclusively to the plasma membrane of rod outer segments (ROS), where they generate the electrical response to light. However, little is known about the molecular machinery required for CNG channel targeting and maintenance in rod photoreceptors. In this study, we determined the role of the adaptor protein ankyrin-G which localizes exclusively to ROS in targeting and maintenance of the CNG channels.
Methods: :
We used immunofluorescence to localize ankyrin-G in mouse and Xenopus rods. To study interaction between CNG channels and ankyrin-G, we performed immunoprecipitation from isolated bovine ROS. We determined the role of ankyrin-G in localizing CNG channels to ROS in vivo by protein knockdown which was accomplished by electroporation of shRNA into mouse retina. We used a HEK 293 cell based assay to determine the ability of ankyrin-G to bind to rod CNG channel subunits and to the CNG channel beta subunit (CNG-ß1) mutants. Transgenic Xenopus tadpoles were used to localize wild type CNG-ß1 and CNG-ß1 mutants in rod photoreceptors
Results: :
Ankyrin-G co-immunoprecipitates with CNG channels and binds directly to the C-terminal domain of the CNG ß-subunit. Depleting ankyrin-G in mouse rods drastically reduced the ROS content of the CNG channel and markedly shortened the outer segment length. We next demonstrated that the deletion of 28 carboxyl terminal amino acids from CNG-ß1, which causes retinitis pigmentosa in humans, abolishes ankyrin-G binding. Further analysis indicated that ankyrin-G binding is abolished by mutating just two of these 28 amino acids at positions IL1237. Using transgenic expression in Xenopus tadpoles, we found that ankyrin-G binding defective CNG-ß1 human mutant proteins localize to photoreceptor cell bodies in marked contrast to the wild type ß subunit which localized to ROS. Targeting of CNG-ß1 to ROS was unaffected by replacing the native ankyrin-G binding region with one from ß-dystroglycan indicating that ankyrin-G binding was sufficient for ROS targeting.
Keywords: photoreceptors • cytoskeleton • ion channels