title: Atomic force microscopy shows connexin26 hemichannel clustering in purified membrane fragments.
abstract: Connexin proteins form hexameric assemblies known as hemichannels. When docked to form gap junction (GJ) channels, hemichannels play a critical role in cell-cell communication and cellular homeostasis, but often are functional entities on their own in unapposed cell membranes. Defects in the Connexin26 (Cx26) gene are the major cause of hereditary deafness arising from dysfunctional hemichannels in the cochlea. Structural studies of Cx26 hemichannels properly trafficked and inserted in plasma membranes, including their clustering that forms a plaque-like feature in whole gap junctions, are limited. We used atomic force microscopy (AFM) to study the surface topography of Cx26 hemichannels using two different membrane preparations. Rat Cx26 containing appended carboxy terminal V5 and hexahistidine tags were expressed in baculovirus/Sf9 cell systems. The expressed Cx26 proteins form hemichannels in situ in Sf9 cells that were then purified either as (1) Sf9 membrane fragments containing Cx26 hemichannels or (2) solubilized hemichannels. The latter were subsequently reconstituted in liposomes. AFM images of purified membrane fragments showed clusters of protein macromolecular structures in the membrane that at higher magnification corresponded to Cx26 hemichannels. Hemichannels reconstituted into DOPC bilayers displayed two populations of channel heights likely resulting from differences in orientations of inserted hemichannels. Hemichannels in the protein rich portions of purified membranes also showed a reduced channel height above the bilayer compared to membranes with reconstituted hemichannels perhaps due to reduced AFM probe access to the lipid bilayer. These preparations of purified membranes enriched for connexin hemichannels that have been properly trafficked and inserted in membranes provide a platform for high-resolution AFM imaging of the structure, interconnexon interactions, and cooperativity of properly trafficked and inserted noncrystalline connexin hemichannels.
authors.affiliation: Department of Bioengineering, ‡National Center for Microscopy and Imaging Research, §Department of Aerospace and Mechanical Engineering, ∥Department of Neurosciences, and ⊥Materials Science Program, University of California San Diego , 9500 Gillman Drive, La Jolla, California 92093, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Meckes
authors.forename: Cinzia
authors.initials: C
authors.lastname: Ambrosi
authors.forename: Heather
authors.initials: H
authors.lastname: Barnard
authors.forename: Fernando Teran
authors.initials: FT
authors.lastname: Arce
authors.forename: Gina E
authors.initials: GE
authors.lastname: Sosinsky
authors.forename: Ratnesh
authors.initials: R
authors.lastname: Lal
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D000072259:Connexin 26; D017630:Connexins / Q000737:chemistry* / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D008051:Lipid Bilayers / Q000378:metabolism; D018625:Microscopy, Atomic Force; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D055550:Protein Stability / Q000187:drug effects; D051381:Rats; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D017630:Connexins; D003902:Detergents; C000607044:Gjb2 protein, rat; D008051:Lipid Bilayers; D010446:Peptide Fragments; D000072259:Connexin 26