Abstract
Numerous missense mutations in cyclic nucleotide-gated (CNG) channels cause achromatopsia and retinitis pigmentosa, but the underlying pathogenic mechanisms are often unclear. We investigated the structural basis and molecular/cellular effects of R410W, an achromatopsia-associated, presumed loss-of-function mutation in human CNGA3. Cryo-EM structures of the Caenorhabditis elegans TAX-4 CNG channel carrying the analogous mutation, R421W, show that most apo channels are open. R421, located in the gating ring, interacts with the S4 segment in the closed state. R421W disrupts this interaction, destabilizes the closed state, and stabilizes the open state. CNGA3_R410W/CNGB3 and TAX4_R421W channels are spontaneously active without cGMP and induce cell death, suggesting cone degeneration triggered by spontaneous CNG channel activity as a possible cause of achromatopsia. Our study sheds new light on CNG channel allosteric gating, provides an impetus for a reevaluation of reported loss-of-function CNG channel missense disease mutations, and has implications for mutation-specific treatment of retinopathy.
Highlights
Numerous missense mutations in cyclic nucleotide-gated (CNG) channels cause achromatopsia and retinitis pigmentosa, but the underlying pathogenic mechanisms are often unclear
Our results show that R421W opens TAX-4 in the absence of cyclic guanosine monophosphate (cGMP), that R421W/R410W increases the spontaneous activity of TAX-4 and human CNGA3/CNGB3 channels, and that expression of the R410W mutant channel causes cell death
It is located in the A’ α helix of the gating ring, a crucial structural element sandwiched between the transmembrane domain (TMD) and the cyclic nucleotide-binding domain (CNBD) (Fig. 1b)
Summary
Numerous missense mutations in cyclic nucleotide-gated (CNG) channels cause achromatopsia and retinitis pigmentosa, but the underlying pathogenic mechanisms are often unclear. Numerous inherited mutations in both rod and cone CNG channel genes have been associated with degenerative visual disorders such as retinitis pigmentosa and achromatopsia (ACHM)[1,3,5] These mutations include frame-shift, deletion, insertion and missense mutations, and both loss-of-function (LOF) and gain-of-function (GOF) mutations have been reported[5–24]. Three-dimensional (3D) cryo-EM structures of eukaryotic CNG channels in apo closed state and cGMP-bound open state have been solved, including homotetrameric C. elegans TAX-425,26, human CNGA1 channels[27], and human CNGA1/ CNGB1 channels[28]. These structures illustrate the conformational changes associated with ligand activation of the channels and provide a framework for testing the involvement and importance of various structural elements in this process. As proper characterization of DAMs may be instrumental for devising suitable therapeutic treatments of mutation-specific visual disorders, our findings call for a reevaluation of some previously characterized missense DAMs
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