| Summary: | The cyclic nucleotide-gated (CNG) cation channel of rod photoreceptors plays an
important role in the perception of light. The channel gating is the final step in the visual
transduction cascade. The cloning (Kaupp et al., 1989) of what is now known to be the
CNG channel α subunit facilitated its molecular characterization. A heterologous
expression system was developed to examine molecular aspects of the α and β subunits
of the CNG channel. Expression in mammalian cells helped resolve confusion regarding
the JVL of the rod CNG channel a subunit. Although the cDNA codes for an 80 kDa
protein, the a subunit exists in the rod outer segment as a 63 kDa protein suggesting the
existence of a processing mechanism. PCR was used to construct cDNA clones which
code for both the 63 and 80 kDa forms of the a subunit of the rod CNG channel. A
polyclonal antibody, generated to specifically label the 80 kDa α subunit, was used in
conjunction with a monoclonal antibody specific for the 63 kDa α subunit in labeling
studies. These antibodies were shown to be mono-specific, capable of differentiating
between the two size forms of the a subunit. Immunohistochemical studies
demonstrated that the 63 kDa α subunit is the predominant species in the rod outer
segment while the 80 kDa form is present in very low quantities. Rod outer segment
(ROS) purification on sucrose gradients also showed that the 80 kDa form co-sediments
with the 63 kDa α subunit indicating that the two size forms coexist in the outer
segment. Examining the α subunits of the photoreceptors of another species
demonstrated similar processing of the CNG α subunit polypeptides, however, an
olfactory a subunit did not appear to undergo similar processing.
The cloned CNG channel β subunit cDNA codes for a protein with a predicted
molecular mass of 155 kDa (Korschen et al, 1995). Heterologous expression of the β
subunit cDNA yielded a 240 kDa protein, positively identifying the 240 kDa protein in
ROS which co-purifies with the a subunit as the CNG β subunit. Construction and expression of a truncated form of the (3 subunit demonstrated that it is the glutamic acidrich
N-terminal portion of the (3 subunit that is responsible for its anomalous migration
on an SDS gel. Co-expression of both subunits in mammalian cells indicated that the 3
subunit was not responsible for the processing of the α subunit. As seen for the subunits
of the native CNG channel the heterologously expressed α and β subunits coimmunoprecipitated.
The subunit interaction was not dependent on the 92 N terminal
amino acids of the 80 kDa α subunit or on the glutamic acid-rich portion of the P
subunit. A method for reconstitution of the heterologously expressed CNG channel was
also developed. The expressed a subunit reconstituted alone did not generate functional
cGMP-gated channels. Reconstitution of the heterologously expressed channel complex
comprising the 80 kDa α and complete β subunits generated functional channels.
Compared to the native CNG channel, 10% of the heterologously expressed channel
complex exhibited cGMP-gated channel activity. This is the first example of
reconstitution of a heterologously expressed cation channel into lipid vesicles for Ca²⁺ efflux measurements. The system presented here will be useful to further define CNG α
and β subunit interactions and to carry out structure-function studies on the channel
using a biochemical efflux assay.
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