Structural Studies of Heteromeric Connexin26/30 Hemichannels via Atomic Force Microscopy Imaging

Abstract

Connexins (Cx) form hexameric hemichannels that mediate release of small molecules, including ATP and glutamate, to mediate paracrine communication and signaling. There are ∼20 vertebrate connexins, and most cells seems to express more than one isoform. Biochemical and functional data demonstrate that hemichannels can be heteromeric - composed of more than one connexin isoform and that the gating and permeability properties are modulated by the heteromericity. However, the subunit stoichiometry and arrangement within heteromeric hemichannels is essentially unexplored. Heteromeric Cx26/30 channels were immunopurified from HeLa cells expressing a haemagglutinin tagged Cx26 (Cx26-HA) and Cx30. Presence of the heteromeric Cx26/30 hemichannels was confirmed by mass spectrometry analysis. Purified samples were imaged by air tapping mode atomic force microscopy (AFM). Molecular volumes of the Cx26/30 hemichannel complexes showed a particle population centered at 337±10 nm3, which is within the volume range expected for heteromeric Cx hexamers based on their molecular weights. Ongoing studies will visualize Cx26/30 hemichannels decorated with isoform-specific anti-haemagglutinin antibodies by AFM to determine both the number of each subunit in the complex and its subunit arrangement. Funded by Millennium Science Initiative P10-035F/Fondecyt 1120169/Anillo ACT 1108 grants.