Abstract
Purpose: :
G-protein coupled signal transduction pathways play important roles in visual function. For example, metabotropic glutamate receptors coupled to a Goα cascade underlie the ON signaling pathway in retinal bipolar cells. In this study, we used co-immunoprecipitation followed with MOLDI-TOF MS techniques to identify proteins that communicate with the Goα subunit in the mammalian retina.
Methods: :
Baboon retinal samples were mixed with a monoclonal anti-Goα antibody and immunoprecipitated with protein-G conjugated agarose beads. The precipitated protein samples were separated on SDS-PAGE and stained overnight in SYPRO Ruby Gel Staining Solution. Bands of interest were cut from the gel. After trypsin digestion, MALDI-TOF MS was performed by the Research Resource Center Protein Lab at UIC. For immunohistochemisty, rat and baboon retinas were fixed with 4% paraformaldehyde, cryosectioned, and stained with a polyclonal antibody to kv12.1 potassium channel.
Results: :
Several unique protein bands, distinct from those representing immunoglobin molecules, were clearly visible on SDS-PAGE of Goα co-precipitates. MALDI-TOF MS analysis revealed the identities for several of these Goα interactors. Among the proteins identified were a G-protein β subunit, a calcium channel regulatory subunit (α2Δ4), and a potassium channel (kv12.1). Kv12.1, which is also known as KCNH8, is an Elk family potassium channel that contains cyclic nucleotide binding domains. Immunohistochemical localization of kv12.1 in mammalian retina demonstrates prominent staining for kv12.1 in the outer plexiform layer in both the rat and baboon retina. In addition, photoreceptor cell bodies were positive for the kv12.1 potassium channel.
Conclusions: :
We identified the kv12.1 potassium channel as a novel interactor of the G-protein Goα subunit. The interaction between kv12.1 and Goα might participate in the metabotropic glutamate signaling cascade in ON bipolar cells. In addition, the presence of kv12.1 on photoreceptors suggests that these potassium channels likely shape the output signal from these first order retinal neurons.
Keywords: ion channels • excitatory neurotransmitters • signal transduction