Cloning and molecular characterization of cGMP-gated ion channels from rod and cone photoreceptors of striped bass ( M. saxatilis ) retina.
Vertebrate photoreceptors respond to light with changes in membrane conductance that reflect the activity of cyclic-nucleotide gated channels (CNG channels). The functional features of these channels differ in rods and cones; to understand the basis of these differences we cloned CNG channels from the retina of striped bass, a fish from which photoreceptors can be isolated and studied electrophysiologically. Through a combination of experimental approaches, we recovered and sequenced three full-length cDNA clones. We made unambiguous assignments of the cellular origin of the clones through single photoreceptor RT-PCR. Synthetic peptides derived from the sequence were used to generate monospecific antibodies which labeled intact, unfixed photoreceptors and confirmed the cellular assignment of the various clones. In rods, we identified the channel alpha subunit gene product as 2040 bp in length, transcribed into two mRNA 1.8 kb and 2.9 kb in length and translated into a single 96-kDa protein. In cones we identified both alpha (CNGA3) and beta (CNGB3) channel subunits. For alpha, the gene product is 1956 bp long, the mRNA 3.4 kb, and the protein 74 kDa. For beta, the gene product is 2265 bp long and the mRNA 3.3 kb. Based on deduced amino acid sequence, we developed a phylogenetic map of the evolution of vertebrate rod and cone CNG channels. Sequence comparison revealed channels in striped bass, unlike those in mammals, are likely not N-linked-glycosylated as they are transported within the photoreceptor. Also bass cone channels lack certain residues that, in mammals, can be phosphorylated and, thus, affect the cGMP sensitivity of gating. On the other hand, functionally critical residues, such as positively charged amino acids within the fourth transmembrane helix (S4) and the Ca(2+)-binding glutamate in the pore loop are absolutely the same in mammalian and nonmammalian species.
Paillart
Christophe
C
Department of Physiology, School of Medicine, University of California at San Francisco, San Francisco, California 94143, USA.
Zhang
Kai
K
Rebrik
Tatiana I
TI
Baehr
Wolfgang
W
Korenbrot
Juan I
JI
D000595:Amino Acid Sequence; D000818:Animals; D001492:Bass / Q000502:physiology*; D015152:Blotting, Northern / Q000379:methods; D015139:Blotting, Southern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D019295:Computational Biology / Q000379:methods; D006152:Cyclic GMP / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels; D015723:Gene Library; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010802:Phylogeny; D012333:RNA, Messenger / Q000096:biosynthesis; D012160:Retina / Q000166:cytology; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D012333:RNA, Messenger; D006152:Cyclic GMP