DIFFUSIONAL PROPERTIES OF CX43, CX45 AND CX40 HEMICHANNELS AND GAP JUNCTION CHANNELS

Choby Cecile, Weingart Robert.
Dept of Physiology, University of Bern.

In multicellular organisms, tissue homeostasis is controlled by an exchange of hormonal, chemical and electrical signals via specialized mechanisms. For example, in heart the synchronization of the mechanical activity involves current flow from cell to cell and diffusion of signalling molecules via gap junctions channels (GJC). GJC are composed of two hemichannels (HC) inserted into the membranes of adjacent cells, thus providing a direct pathway for the exchange of information between cells. Each HC consists of 6 subunits (connexins, Cx) forming a central pore. Each type of channel exhibits specific electrical and diffusional properties. In cardiac tissue, Cx40, Cx43 and Cx45 are the most prominent connexins. The goal of this project is to determine the permeability of single GJC and HC made of Cx40, Cx43 and Cx45 using transfected HeLa cells. For this purpose, we measure the diffusion of dye (flux) and the flow of current (membrane and channel conductance) using a video-imaging system and a dual or single voltage-clamp technique, respectively. The following dyes are examined: Lucifer yellow (LY; mol.wt., MW=457), Alexa-350 (A-350; MW=349) and Alexa-594 (A-594; MW=758) The properties of GJC are examined using cell pairs. A patch pipette filled with fluorescent dye is brought into contact with one cell and the diffusion of dye in both cells is measured as a function of time. The diffusion profiles allow the determination of the flux through GJC. The junctional conductance between the two cells is measured simultaneously via a patch pipette placed on each cell. The properties of HC are explored using single cells. Cells are dye-loaded and -unloaded via HC opened up in zero-calcium solution. In the case of GJC, the incidence of diffusion of LY per channel follows the sequence Cx40>Cx43>Cx45; A-350 follows the inverse sequence while A-594 diffuses through Cx43 GHC only. Moreover, the three Cx examined show a different pattern of diffusion delays for LY and A-350. In the case of HC, the permeability of LY per channel corresponds to the sequence Cx43=Cx45 (Cx40 not tested); for A-350 it obeys the sequence Cx43<Cx45 (Cx40 not tested). A-594 passes through Cx45 HC only.The results indicate distinct differences in permeability between channels consisting of Cx40, Cx43 and Cx45, but also between HC and GJC. The data obtained are crucial for the understanding of the determinant of channel permeation (electric charge, molecular size and geometry) underlying the transfer of biological compounds through cardiac gap junctions.
Supported by the SNSF (31-67230.01) and EU (QLRT-1999-00515).