Gap junction protein connexin-43 interacts directly with microtubules

Gap junctions are specialized cell-cell junctions that mediate intercellula r communication. They are composed of connexin proteins, which form transme mbrane channels for small molecules [1, 2]. The C-terminal tail of connexin -43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull- down experiments using the C-terminal tail of Cx43 fused to glutathione-S-t ransferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is s pecific in that it is not observed with three other connexins. We establish ed that a 35-amino acid juxtamembrane region in the Cx43 tail, which contai ns a presumptive tubulin binding motif, is necessary and sufficient for mic rotubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted ce lls. However, intact microtubules are dispensable for the regulation of Cx4 3 gap-junctional communication. Our findings suggest that, in addition to i ts well-established role as a channel-forming protein, Cx43 can anchor micr otubule distal ends to gap junctions and thereby might influence the proper ties of microtubules; in contacted cells.