Molecular Cloning of a Putative Cyclic Nucleotide‐Gated Ion Channel cDNA from Limulus polyphemus

Abstract

Cyclic nucleotide-gated channels have been proposed to mediate the electrical response to light in the ventral photoreceptor cells of the horseshoe crab, Limulus polyphemus. However, a cyclic nucleotide-gated channel has not been identified from Limulus. We have cloned a putative full-length cyclic nucleotide-gated channel cDNA by screening cDNA libraries constructed from Limulus brain using a probe developed from Limulus ventral eye nerves. The putative full-length cDNA was derived from two overlapping partial cDNA clones. The open reading frame encodes 905 amino acids; the sequence shows 44% identity to that of the ␣ subunit of the bovine rod cyclic GMP-gated channel over the region containing the transmembrane domains and the cyclic nucleotide binding domain. This Limulus channel has a novel C-terminal region of ϳ200 amino acids, containing three putative Src homology domain 3 binding motifs and a putative coiled-coil domain. The possibility that this cloned channel is the same as that detected previously in excised patches from the photoreceptive membrane of Limulus ventral photoreceptors is discussed in terms of its sequence and its expression in the ventral eye nerves. Key Words: Cyclic nucleotide-gated channels-Limulus polyphemus. J. Neurochem. 72, 461-471 (1999).Cyclic GMP (cGMP) is the intracellular second messenger for phototransduction in vertebrate photoreceptors, where it directly activates cyclic nucleotide-gated (CNG) channels in the plasma membrane of the outer segment (Fesenko et al., 1985;Kaupp et al., 1989). It has also been proposed as an intracellular second messenger that activates ion channels in horseshoe crab (Limulus polyphemus) photoreceptors. Injection of cGMP into Limulus ventral photoreceptor cells partially depolarizes the photoreceptors, and channels in excised patches from the photoreceptive membranes can be activated directly by cGMP (Johnson et al., 1986;Bacigalupo et al., 1991).As a first step toward resolving the role of CNG channels in Limulus phototransduction, we have cloned a putative CNG channel cDNA, Lcng1, from Limulus. This putative Limulus CNG channel subunit (LCNG1) shares significant homology with the ␣ subunit of the bovine rod cGMP-gated channel. The sequence also reveals a novel long C terminus. RT-PCR experiments indicate that the Lcng1 gene is expressed in both Limulus brain and ventral eye nerve. MATERIALS AND METHODS Materials and tissue preparationHorseshoe crabs were either purchased from the Martin Fish Co. (Ocean City, MD, U.S.A.) or the Marine Biological Laboratory (Woods Hole, MA, U.S.A.) or caught in the Indian River of Florida. Dissected, live tissues were either used immediately or frozen in liquid nitrogen and stored at Ϫ80°C.Ventral eye nerves were cut at least several millimeters away from the brain to avoid brain tissue contamination. In desheathing nerves, both the sheath around the nerve and the modified patch of cuticle at the eye terminal were removed. Brains were dissected free of surrounding nerves. The coax extensor muscle of th...

Opsin co-expression in Limulus photoreceptors: differential regulation by light and a circadian clock

“…Forward (OpsF1) and reverse (OpsR2) primers (Table1) based on this initial sequence were used to screen various cDNA libraries and other EST collections from Limulus. An anticipated 250-bp product with a sequence identical to the new opsin was obtained from a LE cDNA library (Smith et al, 1993), VE and adult brain cDNA libraries (Chen et al, 1999) and VE and adult CNS EST collections. Using the VE EST collection as template, the 250-bp sequence was extended with a RACE (rapid amplification of cDNA ends) strategy, using the 5Ј and 3Ј adaptor primers Lu4st11 and Lu4 TRSA (Matz, 2003) (Table1).…”

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

“…2000). Our pharmacological observations and our characterization of a novel Limulus TRP channel provide a testable alternative to the proposed role of a cyclic nucleotide channel that has also been recently cloned from this system (Chen et al . 1999), and may resolve the differences in phototransduction between Limulus and Drosophila photoreceptors.…”

“…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.…”

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

“…These channels have properties similar to the light-activated channels in cell-attached patches on the R-lobe [48]. 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.…”