title: GCN4 enhances the stability of the pore domain of potassium channel KcsA.
abstract: The prokaryotic potassium channel from Streptomyces lividans, KcsA, is the first channel that has a known crystal structure of the transmembrane domain. The crystal structure of its soluble C-terminal domain, however, still remains elusive. Biophysical and electrophysiological studies have previously implicated the essential roles of the C-terminal domain in pH sensing and in vivo channel assembly. We examined this functional assignment by replacing the C-terminal domain with an artificial tetramerization domain, GCN4-LI. The expression of KcsA is completely abolished when its C-terminal domain is deleted, but it can be rescued by fusion with GCN4-LI. The secondary and quaternary structures of the hybrid channel are very similar to those of the wild-type channel according to CD and gel-filtration analyses. The thermostability of the hybrid channel at pH 8 is similar to that of the wild-type but is insensitive to pH changes. This supports the notion that the pH sensor of KcsA is located in the C-terminal domain. The result obtained in the present study is in agreement with the proposed functions of the C-terminal domain and we show that the channel assembly role of the C-terminal domain can be substituted with a non-native tetrameric motif. Because tetramerization domains are found in different families of potassium channels and their presence often enhances the expression of channels, replacement of the elusive C-terminal domains with a known tetrameric scaffold could potentially assist the expression of other potassium channels.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, Hamilton, Canada.
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Yuchi
authors.forename: Victor P T
authors.initials: VP
authors.lastname: Pau
authors.forename: Daniel S C
authors.initials: DS
authors.lastname: Yang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D050976:Basic-Leucine Zipper Transcription Factors; D002850:Chromatography, Gel; D002918:Chymotrypsin; D004268:DNA-Binding Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D015221:Potassium Channels / Q000737:chemistry / Q000502:physiology*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D048170:Streptomyces lividans / Q000502:physiology*; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D015221:Potassium Channels; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C107790:prokaryotic potassium channel; D002918:Chymotrypsin