A cGMP-gated channel subunit in Limulus photoreceptors

Abstract

The phototransduction cascade in invertebrate photoreceptors has not been fully elucidated. It has been proposed that in Limulus ventral photoreceptor cGMP is the intracellular second messenger that directly controls the gating of the light-dependent channels (Johnson et al., 1986: Bacigalupo et al., 1991). Recently, a putative cGMP-gated channel cDNA, Lcng1, has been cloned from Limulus and shown to be expressed in the brain and the ventral eye (Chen et al., 1999). In this study, we sought to more specifically localize the LCNG1 transcript and protein. In situ hybridization was used to determine whether the gene is expressed in glia or photoreceptor cells in the ventral eye. The results clearly demonstrated that Lcngl mRNA is transcribed in the ventral photoreceptors. On Western blots probed with a polyclonal antibody raised against the C-terminus of LCNGI, a 100-kDa band and an 80-kDa band was labeled in the membrane protein preparations from brain and ventral eye, respectively. The labeling of these bands was blocked by preabsorption of the antibody with the antigen, indicating the labeling specificity. Immunocytochemistry and confocal microscopy were applied to investigate the subcellular localization of this antigen. Immunolabeling was highly localized in the transducing lobes of ventral eye photoreceptors and lateral eye photoreceptors. In both cases, the labeling was associated with membrane regions specialized for phototransduction, but the exact pattern appeared to be somewhat different in the two eyes. Preabsorption of the antiserum with antigen abolished the labeling, confirming specificity. The results lend support to the hypothesis that a cGMP-gated channel is directly involved in the phototransduction process.

Phototransduction Motifs and Variations

“…Other studies have suggested as many as three different kinds of light-sensitive channels possibly operating in parallel, controlled by distinct G protein-mediated pathways: one signaling via PLC and InsP 3 , a second via GC, and a third via adenylate cyclase (Dorlochter and Stieve, 1997; Nasi et al, 2000). Consistent with the electrophysiological evidence mentioned above, a cGMP-gated cation channel homologous to the vertebrate CNG channels has been cloned and immunolocalized to the microvillar membrane (Chen et al, 2001). A Limulus trp homolog was also found in the mRNA from the ventral photoreceptor (Bandyopadhyay and Payne, 2004).…”

“…Even in rods and/or cones, G α11 and PLCβ4 are present (Ferreira and Pak, 1994; Peng et al, 1997) but are apparently not involved in the phototransduction pathway (Jiang et al, 1996). Conversely, a cGMP-gated channel has been found in the rhabdomeric ventral photoreceptor of Limulus (Chen et al, 2001), which may be responsible for at least one component of the light response. CNG channels and a soluble GC are also expressed in Drosophila photoreceptors, although in this case they are implicated in axonal path-finding during development rather than phototransduction (Baumann et al, 1994; Gibbs and Truman, 1998).…”

The excitation cascade of Limulus ventral photoreceptors: guanylate cyclase as the link between InsP3-mediated Ca2+release and the opening of cGMP-gated channels

“…Most recently, a putative cyclic nucleotide-gated channel gene has been cloned from Limulus [22]. The mRNA for the channel is expressed in photoreceptors and the protein product was specifically localized in the R-lobe [21]. …”

“…The Limulus cascade ends with the opening of cGMP-gated channels which, in this system, can be directly studied in cell-attached and excised patches [19,20]. Photoreceptor cells contain mRNA for a putative Limulus cyclic nucleotide-gated channel protein, and antibodies to the expressed protein specifically label the light-sensitive rhabdomeric lobe [21,22]. Furthermore either intracellular injection of cGMP [23,24] or elevation of cGMP by inhibition of phosphodiesterase [25,26] excites the cell.…”

Variants of TRP ion channel mRNA present in horseshoe crab ventral eye and brain

“…It has also been suggested that multiple second messenger pathways and channels may mediate the response in Limulus photoreceptors (Nagy 1993). The search for alternative mechanisms has led to physiological and molecular evidence for a cyclic nucleotide gated channel in the light-sensitive membrane of Limulus ventral photoreceptors (Johnson et al 1986;Bacigalupo et al 1991;Chen et al 1999Chen et al , 2001. There is pharmacological evidence for the linkage of the opening of this channel to the PI pathway via a guanylate cyclase (Garger et al 2004), but no biochemical or molecular pathway has yet been identified.…”