The above results suggest that the CLZ domain has an important role in constraining the stoichiometry of rod CNG channels to 3:1 CNGA1:CNGB1 subunits. Previously, it has been shown that a protein fragment containing the CLZ domain from cone CNGA3 forms homotypic trimers in solution 8 . To determine whether the CLZ domain from CNGA1 would also form trimers, and to localize the region involved, we screened a number of carboxy-terminal fragments of both CNGA1 and CNGA3 using fluorescence-detection size-exclusion chromatography (FSEC) (Supplementary Fig. S3) 28 . The protein fragments were expressed in bacteria as N-terminal (NGFP) or C-terminal (CGFP) fusions to GFP. After cell lysis, the crude supernatant was then loaded on a size-exclusion chromatography system with an inline fluorescence detector. All the expressing fragments that contained the CLZ domain showed an appreciable component that eluted at 13 mL, consistent with the formation of a trimer (Fig. 5a) . To verify the composition of the high molecular weight component, samples of the purified protein, following cleavage of GFP, were analysed by light scattering size-exclusion chromatography (LS-SEC) (Fig. 5b) 29, 30 . For both CNGA1 and CNGA3, the molecular weights determined from LS-SEC were within about 1% of the predicted molecular weights for a trimer (Table 1) . Similar results with smaller fragments revealed a minimal fragment, corresponding to the CLZ domain, that formed trimers in both CNGA1 (amino acids 621-667) and CNGA3 (626-672) subunits.