Single mutations in the S4 domain of cone photoreceptor cGMP–gated channels, characteristic of human achromatopsia, cause functional channel failure

Abstract

Abstract: : Purpose:cGMP–gated ion channels in retinal photoreceptors (CNG channels) are activated by cGMP, but are not voltage–dependent. Yet, their structure includes an S4 motif consisting of tandem repeats of the R(or K)XX sequence within the 4th transmembrane helix. S4 is characteristic of every known voltage–gated ion channel and mutations in S4 in these channels alter their voltage–dependent function. Human achromatopsia is a congenital loss of cone function and a frequent mutation in this patient population is a single amino acid substitution in S4 of cone CNGA3 channels. We investigated the functional consequence of point mutations in S4 of bovine CNGA3 cone channels by assessing the electrical properties and cell protein processing pattern of mammalian cultured cells (tsA–201) transformed with wt or mutant channel cDNA. Methods: We measured membrane currents under voltage–clamp and assayed the consequence of sudden intracellular delivery of 8–cpt–cGMP by flash uncaging the caged nucleotide. We studied cell protein processing using HA–tagged channel proteins and following their subcellular localization with target–specific fluorescent labels under high–resolution confocal microscopy. Results: Wt CNGA3 cDNA drives the expression of functional channels that sustain large cGMP–dependent membrane currents in transformed tsA–201 cells. Wt channel proteins exist first in a 90 KDa state that is core glycosylated into a 93 KDa state, and then further processed into a final 102 kDa state. Glycosylation is a milestone of normal channel protein processing, but it is not strictly required for function since non–glycosylated proteins also yield functional channels. Neutralization of each and every charged amino acid in S4 prevents formation of the 102 kDa protein states and causes failure of channel function. Conservative mutations of some, but not all of the neutral amino acids in S4 also cause channel failure. Mutant channel proteins are improperly processed and do not transit from the ER into the Golgi apparatus. Conclusions: Point mutations in the S4 cone CNGA3 channel prevent proper protein trafficking within cells. Mutant proteins are core glycosylated, but incompletely processed. They remain trapped in the ER and fail to form functional channels at the plasma membrane. This class of mutations occurs in achromatopsia and probably causes the loss of cone function characteristic of this congenital disease.

Keywords: photoreceptors • ion channels • protein structure/function