Exploring Ligand Regulation of Ion Channels in the EAG Family

Abstract

Similar to hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, ion channels in the ether-a-go-go (EAG) family contain a cyclic nucleotide-binding domain (CNBD). Evidence is mounting that, despite the presence of the CNBD, vertebrate ion channels in the EAG family are not regulated by direct binding of cyclic nucleotides to the CNBD. To explore why cyclic nucleotides fail to regulate ion channels in the EAG family we sought to crystallize the CNBD region of the channels in the EAG family. The major hurdle with protein crystallization is identification of monodispersed proteins suitable for crystallization trials. To identify monodispersed proteins for our experiments we screened CNBD containing regions from 30 ion channels in the EAG family with Fluorescence-detection Size-Exclusion Chromatography (FSEC). For the FSEC screen the thirty target proteins were fused to the green fluorescence protein (GFP), loaded on a size exclusion column and then passed through a fluorescence detector set to detect the GFP fluorescence. The FSEC screen identified four monodispersed CNBD containing regions. Two of the four candidates formed well diffracting crystals under various conditions. The analysis of the diffraction data should reveal the structural differences between the CNBDs of EAG and HCN channels responsible for the differential modulation by cyclic nucleotides.