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 α
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 α (CNGA3) and β (CNGB3) channel subunits.
For α, the gene product is 1956 bp long, the mRNA 3.4 kb, and the
protein 74 kDa. For β, 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 Ca2+-binding glutamate in the pore loop are absolutely
the same in mammalian and nonmammalian species.