Homology Models of the Trimeric CNG Channel C-Leucine Zipper Domains Offer Insight about the Olfactory CNG Channel Subunit Stoichiometry

Abstract

CNG channels in rod, cone and olfactory sensory neurons are tetrameric proteins composed of A-type and B-type subunits. The subunit compositions of rod and cone photoreceptors are 3:1 CNGA1:CNGB1 and CNGA3:CNGB3, respectively. A parallel 3-helix coiled-coil domain in the carboxy-terminal leucine zipper (CLZ) region of CNGA1 constrains the channel to incorporate a single CNGB1 subunit. High-resolution crystal structures of soluble trimeric CLZ domains from CNGA1 and CNGA3 were similar and support the idea that the trimeric CLZ domain governs rod and cone subunit stoichiometry (Shuart et al. 2011 doi:10.1038/ncomms1466). By contrast, olfactory neurons have a subunit composition of 2:1:1 of CNGA2:CNGA4:CNGB1b. We used the X-ray structures of the CNGA1 and CNGA3 CLZ domains to construct a homo-trimeric CNGA2 and a hetero-trimeric CNGA2:CNGA4 (2:1) model using probablistic protein design. Once the homology models were constructed, the energies of the structures were minimized to equilibrate the systems. The potential energies of the systems after equilibration serve as a baseline reference point to quantify the overall stability of each structure. The potential energies from CNGA1, CNGA2, CNGA3 and CNGA2/CNGA4 were compared, and the heterotrimer was shown to have a more favorable structure than the homotrimers. We then performed energetic calculations using robust classical methods. The results reinforced the hypothesis that the heterotrimeric CNGA2/CNGA4 CLZ structure is the thermodynamically favored configuration. This approach was used to examine achromatopsia-associated mutations in the CLZ region of the cone CNGA3 channel subunit. The results and implications from the modeling will be presented in the context of achromatopsia pathophysiology.