Molecular biology. The bovine CNGA1 complementary DNA used was described previously 43, 44 and contains a C-terminal FLAG epitope (DYKDDYK). The bovine CNGB1 cDNA was a kind gift from Dr. R. Molday 14 and the human CNGA3 cDNA was a kind gift from Dr. K.W. Yau 45 . CNGA1-∆CLZ (CNGA1-∆609-693) and the CNGB1-∆N (CNGB1-∆2-765) were made as described 21 . CNGB1-∆N had a further manipulation, where its internal NcoI restriction site was removed via silent mutation and re-inserted at the initial methionine codon using a PCRbased method. Improved monomeric versions of eCFP and eYFP (mCerulean and mCitrine) were employed in the generation of all fluorescent protein fusions with CNG subunits used for FRET experiments. cDNAs for mCerulean and mCitrine were kindly provided by Dr D Piston 46 and Dr RY Tsien 47 , respectively. All constructs were confirmed with fluorescence-based automated sequencing. cDNAs were subcloned into the high-expression pGEMHE vector 48 for expression in Xenopus oocytes. Vectors were linearized and complementary RNA (cRNA) was transcribed using the mMessage mMachine kit (Ambion). For co-injection experiments, relative amounts of RNA were quantified on agarose gels with a densitometric method. CNGA1 and CNGB1 were injected at an RNA ratio of 1:4 that has previously been The CnGA1 subunits (red) initially assemble into trimers through homotypic association of their carboxy-terminal CLZ domains into a parallel three helix coiled coil. subsequently, a CnGB1 subunit (green), if present, is preferentially incorporated into the remaining slot of the channel tetramer due to a high affinity association with CnGA1 subunits. (b) Deletion of the CLZ domain in CnGA1 allows CnGA1 subunits to assemble with CnGB1 subunits. This produces channels with multiple CnGB1 subunits and a large dominant negative effect of CnGB1.