Cytosolic CaZ+ levels are regulated in part by Caz+-pumping ATPases that export Caa+ from the cytoplasm; however, the types and properties of Caz+ pumps in plants are not well understood. We have characterized the kinetic properties of a 120-kD phosphoenzyme (PE) to examine the kinetics of PE formation. PE formation exhibited a K,,, for Ca" of 1 to 2 p~ and a K,,, for ATP of 67 nM. Relative affinities of substrates, determined by competition experiments, were 0.075 NM for ATP, 1 p~ for ADP, 100 p~ for ITP, and 250 p~ for CTP. Thapsigargin and cyclopiazonic acid, specific inhibitors of animal sarcoplasmic/endoplasmic reticulum Ca'+-ATPase, had no effect on PE formation; erythrosin B inhibited with 50% inhibition at <0.1 KM. Calmodulin (1 p~) stimulated PE formation by 25%. The results indicate that the carrot 120-kD CaZ+-ATPase i s similar but not identical to animal plasma membrane-type Ca'+-ATPase and yet is located on endomembranes, such as the endoplasmic reticulum. This type of Caz+ pump may reside on the cortical endoplasmic reticulum, which is thought to play a major role in anchoring the cytoskeleton and in facilitating secretion.

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...

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.