Expression of a bovine rhodopsin gene in Xenopus oocytes: demonstration of light-dependent ionic currents.

Abstract

Xenopus oocytes express a gene encoding bovine rhodopsin as well as its SP6 RNA polymerase-derived transcripts and total retinal mRNA. The opsin produced is in unglycosylated (30 kDa) and two glycosylated (35 kDa and 41 kDa) forms. Incubation of the cells expressing the above proteins with 11-cis-retinal generates rhodopsin, which was purified by iminunoaffinity chromatography. The purified protein shows the expected UV/visible absorption spectrum and characteristic light-dependent activation of the rod outer segment GTP binding protein. Oocytes expressing rhodopsin exhibit light-dependent ionic currents that are detected by voltage-clamp techniques.The process of visual transduction in rod photoreceptor cells begins with photon absorption by rhodopsin, 11-cis to all-trans isomerization of its retinal chromophore, and relaxation of the protein through a series of metastable photointermediates (1). One of the intermediates, metarhodopsin II, is believed to activate a guanine nucleotide binding protein (G protein)-transducin (GT)-and thus initiate an enzymatic cascade that results in hyperpolarization of the rod cell (2). Similar interactions between membrane-bound receptors and G proteins occur in many signal transduction systems (3). Sequence homology between the receptors (4) on the one hand and the G proteins (5) on the other hand suggests that the mechanism of interaction between these two classes of proteins has been conserved. However, the structural basis underlying receptor-mediated G-protein activation remains unclear. The visual system provides a highly attractive model for the study of this interaction.As an approach to rhodopsin structure-function studies, we have expressed a synthetic rhodopsin gene (6) in mammalian cells and have shown that the rhodopsin isolated after addition of 11-cis-retinal to the cells is active in vitro (7,8). Xenopus oocytes offer the possibility of an alternative expression system in which recombinant proteins can be assayed in situ, often by electrophysiological recordings (9). Furthermore, oocytes contain G protein-regulated ionic conductances (9, 10), and coupling to these could form the basis of an assay for rhodopsin. Indeed, a number of cloned receptor genes expressed in oocytes have been assayed by their ability to activate endogenous channels (11)(12)(13). In this report we demonstrate that a bovine opsin gene is expressed by Xenopus oocytes, and the opsin produced is active in vitro. Further, we show that rhodopsin in oocytes produces light-dependent ionic currents that are detected by voltageclamp techniques.

Mechanisms of spectral tuning in the RH2 pigments of Tokay gecko and American chameleon

“…Similarly, the full-length medaka 2-A opsin cDNA was obtained by RT-PCR using two primers (Forward: 5′ -ATCCTAGAATTCCACCATGGAGAACGGCACAGAG -3′ and Reverse: 5′ -CGTGGTGTCGACGCAGCAGTAGAGACTTC -3′), which were based on the medaka RH2-A gene (AB223053). The amplified cDNA fragments were cloned into the EcoRI and SalI restriction sites of the expression vector pMT5 (Khorana et al, 1988). The full length RH2 opsin cDNA of American chameleon has been subcloned into pMT5 in our laboratory (Kawamura and Yokoyama, 1998).…”

Characterization of the Xenopus Rhodopsin Gene

“…First, a complete cDNA was constructed by ligating a BamHI-BstEII RACE PCR fragment into pXOP71. Then, an ApoIHindIII fragment, containing nucleotides 111-1153 (encoding amino acids 5-347) of the Xenopus opsin cDNA, was ligated into the EcoRISalI of pMT5 (Khorana et al, 1988) using synthetic nucleotide adaptors. The final product, pMT-XOP, consists of nucleotides 1-11 from the synthetic bovine rhodopsin gene (encoding amino acids 1-4, which are identical in bovine and Xenopus; see Ferretti et al (1986)), nucleotides 111-1153 (encoding amino acids 5-347) of the Xenopus opsin and nucleotides 1000 -1047 (encoding amino acids 334 -348 and the stop codon) from the synthetic bovine rhodopsin gene.…”

Cloning and Functional Expression of Two Families of β-Subunits of the Large Conductance Calcium-activated K+ Channel

“…Solubilization and immunoprecipitation (from batches of 24 -30 oocytes) was carried out as described previously (31), using 1% Triton X-100 as the detergent and a 1/100 dilution of preserum or antiserum. Immunoprecipitated proteins were fractionated by SDSpolyacrylamide gel electrophoresis and visualized by fluorography.…”