title: Purification and functional reconstitution of N- and C-halves of the MscL channel.
abstract: MscL is a mechanosensitive channel gated by membrane tension in the lipid bilayer alone. Its structure, known from x-ray crystallography, indicates that it is a homopentamer. Each subunit comprises two transmembrane segments TM1 and TM2 connected by a periplasmic loop. The closed pore is lined by five TM1 helices. We expressed in Escherichia coli and purified two halves of the protein, each containing one of the transmembrane segments. Their electrophysiological activity was studied by the patch-clamp recording upon reconstitution in artificial liposomes. The TM2 moiety had no electrophysiological activity, whereas the TM1 half formed channels, which were not affected by membrane tension and varied in conductance between 50 and 350 pS in 100 mM KCl. Coreconstitution of the two halves of MscL however, yielded mechanosensitive channels having the same conductance as the native MscL (1500 pS), but exhibiting increased sensitivity to pressure. Our results confirm the current view on the functional role of TM1 and TM2 helices in the MscL gating and emphasize the importance of helix-helix interactions for the assembly and functional properties of the channel protein. In addition, the results indicate a crucial role of the periplasmic loop for the channel mechanosensitivity.
authors.affiliation: Unité Mixte de Recherche Centre National de la Recherche Scientifique 8619, Université Paris-Sud, 91405, Orsay Cedex, France.
authors.forename: Kyu-Ho
authors.initials: KH
authors.lastname: Park
authors.forename: Catherine
authors.initials: C
authors.lastname: Berrier
authors.forename: Boris
authors.initials: B
authors.lastname: Martinac
authors.forename: Alexandre
authors.initials: A
authors.lastname: Ghazi
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry*; D015640:Ion Channel Gating / Q000502:physiology*; D007473:Ion Channels / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D018408:Patch-Clamp Techniques; D011994:Recombinant Proteins / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D007473:Ion Channels; D008081:Liposomes; C086596:MscL protein, E coli; D011994:Recombinant Proteins