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.

Highlights

  • Cyclic nucleotide-gated (CNG) channels in retinal rod and cone photoreceptor outer segments open in response to the binding of intracellular cGMP

  • Cone photoreceptor CNG channels are assembled from both CNGA3 (A3) and CNGB3 (B3) subunits

  • We investigated the relative permeability of Ca2+ to Na+ and we find a,10 fold increase in Ca2+ permeability when the human B3 (hB3) subunit is co-expressed with canine A3 (cA3)

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Summary

Introduction

Cyclic nucleotide-gated (CNG) channels in retinal rod and cone photoreceptor outer segments open in response to the binding of intracellular cGMP. Both Na+ and Ca2+ enter the outer segments through CNG channels; Na+ is the major current carrying ion while Ca2+ is a critical second messenger whose activity is closely monitored by many of the phototransduction enzymes as well as molecules involved in both light and dark adaptation [1]. The intracellular N-terminal regions of CNG channels are involved in trafficking through an interaction with C-termini [3,4]; these domains contain regulatory sites specific to the channel function [5,6,7,8]. Cone photoreceptor CNG channels are assembled from both CNGA3 (A3) and CNGB3 (B3) subunits. The stoichiometry of cone channels is still debated [3,10,11], while the stoichiometry of rod channels is established as 3:1 CNGA1/ CNGB1 [12,13,14]

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