brief communications
Selective transfer of endogenous metabolites through gap junctions composed of different connexins Gary S. Goldberg*†‡, Paul D. Lampe§ and Bruce J. Nicholson* *Biological Sciences, Cooke Hall, State University of New York at Buffalo, Buffalo, New York 14260, USA §Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA †Present address: Experimental Pathology and Chemotherapy, National Cancer Center Research Institute, 5-1-1 Tsukiji Chuo-ku, Tokyo 104, Japan ‡e-mail:
[email protected].
ap junctions, the only channels that allow direct exchange of small metabolites between cells, are composed of a family of integral membrane proteins, called connexins in vertebrates. Different connexins have been implicated in a diverse array of biological processes and diseases1, indicating that gap-junction properties may vary with connexin composition. For example, channels composed of different connexins have different conductances2 and permeabilities to ions3 and fluorescent dyes4,5. However, although the permeability of gap junctions to some nucleotides6, cyclic AMP7, calcium8 and possibly inositol-1,4,5-trisphosphate8 has been documented, the comparative selectivities of different connexins for biologically significant molecules remain an enigma. Here, using new techniques, we show that the rate of permeation of metabolites through gap junctions differs according to connexin composition. One demonstration of the biological consequences of expression of different connexin isotypes is their variable effectiveness in suppressing growth of tumour cells9. Although transfection of C6 glioma cells with connexin-43 (Cx43) suppressed their in vitro growth potential10, Cx32 did not11. Paradoxically, however, Cx32 transfectants are far better coupled to each other, as assayed by calcein-dye transfer (Table 1; for methods see ref. 12). A diffusion constant for each cell interface was obtained from the time course of calcein transfer in two C6 cell transfectants12, using quantitative modelling of dye diffusion through cell monolayers5. Independently, we determined the ratio of calcein permeabilities of Cx43 and Cx32 channels to be 1.08 from quantitative measurements of calcein diffusion between Xenopus oocyte pairs expressing defined numbers of Cx43 or Cx32 channels, calculated from the junctional and single-channel conductance of each connexin (P.A. Weber, Y.I. Chen, J. Nitsche and B.J.N., unpublished observations). Applying the relative calcein permeability of the two connexins to the diffusion constants calculated above for the transfected C6 cell lines produced an estimate of the relative number of channels in the Cx32 versus the Cx43 transfectants of 16:1. Hence, the differential effects of these two connexins on cell growth were related not to their expression level but rather to some specific property of the channels, such as selective permeabilities for endogenous signals. To address this possibility, we have developed techniques with which to ‘capture’, identify and quantify the junctional transfer of endogenous metabolites between cells transfected with Cx43 or Cx32 (ref. 13). ‘Donor’ cells were metabolically labelled overnight with [14C]glucose, fluorescently stained with DiI, and plated with unlabelled ‘receiver’ cells at a 1:6.25 ratio to yield confluent monolayers. After allowing 2 h for plating and establishment of cell contact and communication, we separated donors and receivers from each other by fluorescence-activated cell sorting (FACS). Potential transjunctional molecules, which travelled from the donors to the receivers, were then resolved from larger components of the cellular lysates by filtration (selecting material of relative molecular mass
