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The number of cells expressing Hp-ECPN was counted in 7�C10 larvae that had been injected with Hp-ecpn-MASO or standard control-MASO. The above results were analyzed by Student t-test. Significance was set at P?cAMP inhibitor software SOSUI (classification and secondary structure prediction of membrane proteins, http://bp.nuap.nagoya-u.ac.jp/sosui/) predicted that the amino terminus was located on the extracellular side of the plasma membrane and the carboxyl terminus on the cytoplasmic side (Fig.?1B). The open-access proteomics database ExPasy PROSITE (http://www.expasy.ch/prosite/) predicted that the ORF contained a G-protein-coupled receptor domain (Asn83-Tyr333) with two cysteine residues (Cys139 and Cys216) that potentially form a disulfide bond and probably confer conformational stability, and an opsin-retinal binding site (Phe317-Tyr333). According to the TMHMM Server v. 2.0, which predicts transmembrane helices in proteins (http://www.cbs.dtu.dk/services/TMHMM/), Dipivefrine Hp-ECPN has seven transmembrane domains: Leu69-Phe91, Ser103-Ala125, Thr140-Ile162, Ala182-Trp204, Ile231-Leu253, Phe282-Phe304, and Ala314-Leu336. Lys323, which is located in the retinal binding site in the seventh transmembrane domain, corresponds to Lys296 of bovine rhodopsin (bovine-Rh) and Lys294 of fugu Tmt-opsin (Fig.?1A, rectangle). These lysine residues are the sites of Schiff base linkage with 11-cis-retinal. An acidic residue in the CX-5461 clinical trial third transmembrane domain (e.g., Glu113 in bovine-Rh, Glu151 in Ciona Op1) acts as the counterion to the protonated Schiff base, and is conserved in vertebrate visual opsins, whereas the corresponding residue in invertebrate visual opsins is aromatic (e.g., Tyr113 in squid-Rh; Nathans 1990; Terakita et?al. 2004). Thus, Tyr142 in the third transmembrane domain of Hp-ECPN might be the conserved counterion to the protonated Schiff base (Fig.?1A, diamond). A potential N-glycosylation site was predicted at the amino terminus (Asn2), which was exposed to the cell surface (Fig.?1B). A palmitoylation motif (Cys349 and Cys350) was found in the carboxyl terminus, which, in addition, was enriched for Ser and Thr. However, no G-protein-activating Glu (Asp)/Arg/Tyr (Trp) motif was found in Hp-ECPN. Overall, the amino acid sequence was similar to that of mouse-encephalopsin (mouse-ECPN) and S.?purpuratus opsin1 (Sp-Opsin1). The Phylogenetic analysis based on the total amino acid sequences of opsins of invertebrates and vertebrates placed Hp-ECPN in the encephalopsin subfamily (Fig.