Abstract
We examined cellular protein processing and functional expression of photoreceptor cyclic nucleotide-gated (CNG) ion channels. In a mammalian cell line, wild type bovine cone photoreceptor channel alpha subunits (bCNGA3) convert from an unglycosylated state, at 90 kDa, to two glycosylated states at 93 and 102 kDa as they transit within the cell to their final location at the plasma membrane. Glycosylation per se is not required to yield functional channels, yet it is a milestone that distinguishes sequential steps in channel protein maturation. CNG ion channels are not gated by membrane voltage although their structure includes the transmembrane S4 motif known to function as the membrane voltage sensor in all voltage-gated ion channels. S4 must be functionally important because its natural mutation in cone photoreceptor CNG channels is associated with achromatopsia, a human autosomal inherited loss of cone function. Point mutation of specific, not all, charged and neutral residues within S4 cause failure of functional channel expression. Cellular channel protein processing fails in every one of the non-functional S4 mutations we studied. Mutant proteins do not reach the 102-kDa glycosylated state and do not arrive at the plasma membrane. They remain trapped within the endoplasmic reticulum and fail to transit out to the Golgi apparatus. Coexpression of cone CNG beta subunit (CNGB3) does not rescue the consequence of S4 mutations in CNGA3. It is likely that an intact S4 is required for proper protein folding and/or assembly in the endoplasmic reticulum membrane.
Highlights
Type bovine cone photoreceptor channel ␣ subunits acetylglucosamine (GlcNAc) to Asn residues in the consensus convert from an unglycosylated state, at 90 kDa, to two glycosylated states at 93 and 102 kDa as they transit within the cell to their final location at the plasma membrane
Various single point mutations in the ␣ subunit of CNG channels of cone photoreceptors have been identified in humans afflicted by achromatopsia, known as total color blindness or rod monochromic, an autosomal, inherited disease characterized by complete loss of cone photoreceptor function [8]
In a mammalian cell line, normal channel proteins are first core glycosylated at asparagine 357, and processed to a heavier glycosylated form as they transit through the cells on their way to the plasma membrane
Summary
Two of the most frequent mutant alleles in achromatopsia are mutations from charged to neutral amino acids in the S4 structural motif of the cone-specific CNG channel ␣ subunit (CNGA3) [8]. Cyclic nucleotide-gated ion channels (CNG) are localized in the plasma membrane of sensory transduction neurons, such as retinal photoreceptor and olfactory sensory cells where they play a central role in signal transduction Nascent channel proteins first attach to endoplasmic residues in S4 change the voltage dependence of gating, whereas mutation of others cause channel functional failure [23,24,25]. CNG channels contain an S4 domain they are not voltage-gated That is, their probability of opening is independent of membrane voltage and depends only on cyclic nucleotide concentration. Mutant channel proteins are not glycosylated and do not reach the surface plasma membrane; they remain trapped within the endoplasmic reticulum, likely misfolded
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