Cloning and immunocytochemical localization of a cyclic nucleotide-gated channel ?-subunit to all cone photoreceptors in the mouse retina

Abstract

Cyclic nucleotide-gated channels (CNGC) are ligand-gated ion channels that open and close in response to changes in the intracellular concentration of the second messengers, 3′,5′-cyclic adenosine monophosphate and 3′,5′-cyclic guanosine monophosphate. Most notably, they transduce the chemical signal produced by the absorption of light in photoreceptors into a membrane potential change, which is then transmitted to the ascending visual pathway. CNGCs have also been implicated in the signal transduction of other neurons downstream of the photoreceptors, in particular the ONbipolar cells, as well as in other areas of the central nervous system. We therefore undertook a search for additional cyclic nucleotide-gated channels expressed in the retina. Following a degenerate reverse transcription polymerase chain reaction approach to amplify low-copy number messages, a cDNA encoding a new splice variant of CNGC α-subunit was isolated from mouse retina and classified as mCNG3. An antiserum raised against the carboxy-terminal sequence identified the retinal cell type expressing mCNG3 as cone photoreceptors. Preembedding immunoelectron microscopy demonstrated its membrane localization in the outer segments, consistent with its role in phototransduction. Double-labeling experiments with cone-specific markers indicated that all cone photoreceptors in the murid retina use the same or a highly conserved cyclic nucleotide-gated channel. Therefore, defects in this channel would be predicted to severely impair photopic vision. Indexing termsrat; calcium/calmodulin; phototransduction Cyclic nucleotide-gated channels (CNGC) are nonselective cation channels, constructed out of αand β-subunits in a likely tetrameric configuration, with intracellular ligand-binding domain(s). When expressed in heterologous systems, the α-subunits can form functional ion channels. With the coexpression of the αand β-subunits, the channels acquire additional characteristics that reflect those seen in situ (Kaupp, 1995;Molday, 1996;Wei et al., 1998), for example, the ability to be modulated by calcium (Chen et al., 1993;Körschen et al., 1995). These channels are highly regulated (Zimmerman, 1995) and may be the downstream effectors of nitric oxide-signaling mechanisms (Ahmad et al., 1994;Savchenko et al., 1997).

Long-term and age-dependent restoration of visual function in a mouse model of CNGB3-associated achromatopsia following gene therapy

“…Rod and cone photoreceptor CNG channels are localized to the plasma membrane of the outer segment and play a pivotal role in phototransduction (6,7). Structurally, CNG channels belong to the superfamily of voltage-gated ion channels and comprise two structurally related subunit types, the A and B subunits: CNGA1/B1 in rods and CNGA3/B3 in cones.…”

Native cone photoreceptor cyclic nucleotide‐gated channel is a heterotetrameric complex comprising both CNGA3 and CNGB3: a study using the cone‐dominant retina of Nrl−/− mice

“…This is primarily because of the difficulty of investigating the cone system in a rod-dominant mammalian retina, as cones comprise only 3-5% of the total photoreceptor population. Although CNGA3 gene has been cloned in human, bovine, mouse, rat, chicken, and striped bass (Bonigk et al 1993;Biel et al 1994;Weyand et al 1994;Misaka et al 1997;Wissinger et al 1997;Hirano et al 2000;Paillart et al 2006) and CNGB3 gene has been cloned in human, canine, mouse, and striped bass (Gerstner et al 2000;Kohl et al 2000;Sidjanin et al 2002;Paillart et al 2006), the biochemical components of native cone CNG channel have never been determined. Studies using heterologous expression system (Peng et al 2003(Peng et al , 2004 and isolated cones from retinas of striped bass and ground squirrel (Rebrik et al 2000;Rebrik and Korenbrot 2004) have added significantly to our understanding of the functional properties of cone CNG channel and its modulation.…”

Localization of NMDA receptor subunits and mapping NMDA drive within the mammalian retina

“…Kalloniatis et al channels (Picco & Menini, 1993). If AGB entry occurs via cGMPgated channels, one possible reason for a lack of rod labelling is that rods and cones express different cGMP channel subunits (Hirano et al, 2000), and the effective pore size is known to be different in rods and cones (Picones & Korenbrot, 1992Picco & Menini, 1993;Stotz & Haynes, 1996). Mills and Massey (1994) reported that both type A and type B horizontal cells are localized throughout the rabbit retina, peaking at the visual streak.…”