title: Green fluorescent protein changes the conductance of connexin 43 (Cx43) hemichannels reconstituted in planar lipid bilayers.
abstract: In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.
authors.affiliation: Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Carnarius
authors.forename: Mohamed
authors.initials: M
authors.lastname: Kreir
authors.forename: Marcel
authors.initials: M
authors.lastname: Krick
authors.forename: Christoph
authors.initials: C
authors.lastname: Methfessel
authors.forename: Volker
authors.initials: V
authors.lastname: Moehrle
authors.forename: Oliver
authors.initials: O
authors.lastname: Valerius
authors.forename: Andrea
authors.initials: A
authors.lastname: Brüggemann
authors.forename: Claudia
authors.initials: C
authors.lastname: Steinem
authors.forename: Niels
authors.initials: N
authors.lastname: Fertig
mesh_terms: D000818:Animals; D018031:Connexin 43 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D010843:Pichia / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D018031:Connexin 43; D007473:Ion Channels; D008051:Lipid Bilayers; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins