A Cyclic Nucleotide-Gated Channel Mutation Associated with Canine Daylight Blindness Provides Insight into a Role for the S2 Segment Tri-Asp motif in Channel Biogenesis

Abstract

Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (daylight blindness), suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1–S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-day blindness results in the loss of these inter-helical interactions altering the electrostatic equilibrium within in the S1–S4 bundle. Because residues analogous to Tri-Asp in the voltage-gated Shaker potassium channel family were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions impairs the monomer folding state in D262N mutant CNG channels during biogenesis.

Structure, Dynamics and Implied Gating Mechanism of a Human Cyclic Nucleotide-Gated Channel

“…While this paper was in review, a homology model of the TM domain of the canine CNGA3 channel was published [59] . The model, obtained based on a chimeric Kv1.2/2.1 structural template, corresponds to an open state of the channel, while our model structure represents a closed conformation.…”

“…The black dashed line shows the alternative location of helix S1. In (A) the grey lines show the corresponding boundaries of TM helices derived from the recent model-structure of the canine CNGA3 channel in open conformation [59] ; some of the differences could be associated the conformational changes upon channel opening/closure. (C) The sequence alignment of the MlotK1, CNGA3 and CNGB3 channels used here for modeling the TM region.…”

Large Animal Models of Inherited Retinal Degenerations: A Review

“…The D262N mutation in Cngb3 inspired investigation into the Tri-Asp motif that is conserved in CNG channels [89]. When this mutation was introduced to heterologously expressed CNGA3, it abolished homomeric channel function and was responsible for mislocalization of the protein.…”

“…These experiments show the Tri-Asp motif is necessary for proper cone CNG channel formation, but raises the question why mutations in CNGB3 result in the loss of cone function and achromatopsia when CNGA3 can form a functioning homomeric channel in the absence of CNGB3. Further work in retinal tissue and/or cone photoreceptors will be needed to elucidate this [89].…”

Inherited macular degeneration-associated mutations in CNGB3 increase the ligand sensitivity and spontaneous open probability of cone cyclic nucleotide-gated channels

“…In contrast, mutations in CNGB3 are responsible for 40–50% of the cases (Kohl et al, 2004 ). Although several studies have characterized the functional consequences of cone CNG channels containing disease-associated mutations in CNGA3 and CNGB3 subunits (Peng et al, 2003b ; Okada et al, 2004 ; Tränkner et al, 2004 ; Bright et al, 2005 ; Muraki-Oda et al, 2007 ; Reuter et al, 2008 ; Duricka et al, 2012 ; Liu et al, 2013 ; Tanaka et al, 2014 ), the range of molecular phenotypes associated with these mutations remains incompletely explored.…”