title: Basis for intracellular retention of a human mutant of the retinal rod channel alpha subunit.
abstract: A mutant of the a subunit of the retinal rod cyclic GMP-gated channel, [Arg654(1-bp del)], corresponding to a truncated alphaR654Dstop subunit, was previously described in patients with retinitis pigmentosa: when expressed in HEK-293 cells, this mutated a subunit was retained inside the cell, but had normal channel activity in one case where it reached the plasma membrane, indicating that the mechanism of targeting is altered by the mutation, but not the function of the channel. The corresponding mutants of the bovine rod channel (alphaR656D stop), and of the closely related olfactory neuron channel (alphaR632Dstop) alpha subunits were expressed in Xenopus oocytes and their activity was analyzed by patch-clamp. Like their human homologue, these two channels have no activity, and we show that their GFP fusion proteins are accumulated into intracellular compartments. The truncation alone or the R/D mutation alone do not prevent or modify channel activity, indicating that neither the R656 residue nor the C-terminal domain downstream of R656 is necessary for homomeric channel targeting and function. Several mutations of R656 and of the preceding residues in the R656Dstop mutant disclose that the motif responsible for the absence of channel activity is an endoplasmic reticulum retention signal (KXKXXstop) in which the nature of the residues in positions -1 and -4 is determinant.
authors.affiliation: Département de Biologie Moléculaire et Structurale, Laboratoire de Biophysique Moléculaire at Cellulaire (URA CNRS 520), C.E.A.-Grenoble, Grenoble, France.
authors.forename: N
authors.initials: N
authors.lastname: Mallouk
authors.forename: M
authors.initials: M
authors.lastname: Ildefonse
authors.forename: F
authors.initials: F
authors.lastname: Pagès
authors.forename: M
authors.initials: M
authors.lastname: Ragno
authors.forename: N
authors.initials: N
authors.lastname: Bennett
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006152:Cyclic GMP / Q000235:genetics*; D054815:Cyclic Nucleotide-Gated Cation Channels; D004247:DNA / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007473:Ion Channels / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D018034:Olfactory Receptor Neurons / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D012174:Retinitis Pigmentosa / Q000235:genetics; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D014981:Xenopus
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D018076:DNA, Complementary; D007473:Ion Channels; D012333:RNA, Messenger; D004247:DNA; D006152:Cyclic GMP