Connexin Family Members Target to Lipid Raft Domains and Interact with Caveolin-1

Abstract

Lipid rafts are cholesterol-sphingolipid-rich microdomains that function as platforms for membrane trafficking and signal transduction. Caveolae are specialized lipid raft domains that contain the structural proteins known as the caveolins. Connexins are a family of transmembrane proteins that self-associate to form cell-cell connections known as gap junctions and that are linked to cytosolic proteins, forming a protein complex or Nexus. To determine the extent to which these intracellular compartments intersect, we have systematically evaluated whether connexins are associated with lipid rafts and caveolin-1. We show that connexin 43 (Cx43) colocalizes, cofractionates, and coimmunoprecipitates with caveolin-1. A mutational analysis of Cx43 reveals that the hypothesized PDZ- and presumptive SH2/SH3-binding domains within the Cx43 carboxyl terminus are not required for this targeting event or for its stable interaction with caveolin-1. Furthermore, Cx43 appears to interact with two distinct caveolin-1 domains, i.e., the caveolin-scaffolding domain (residues 82-101) and the C-terminal domain (135-178). We also show that other connexins (Cx32, Cx36, and Cx46) are targeted to lipid rafts, while Cx26 and Cx50 are specifically excluded from these membrane microdomains. Interestingly, recombinant coexpression of Cx26 with caveolin-1 recruits Cx26 to lipid rafts, where it colocalizes with caveolin-1. This trafficking event appears to be unique to Cx26, since the other connexins investigated in this study do not require caveolin-1 for targeting to lipid rafts. Our results provide the first evidence that connexins interact with caveolins and partition into lipid raft domains and indicate that these interactions are connexin specific.

Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

“…This lack of total PKC␥ activation is most likely due to the fact that cells have a rapid and robust PKC␥ turn-off mechanism including, among other things, dephosphorylation of PKC␥ by phosphatase (41). In brain tissues PKC␥ appears to prevent brain ischemia and is suggested as a target for ischemic preconditioning (40,(42)(43)(44). However, the role of PKC␥ in ischemic preconditioning was not clearly understood until recently (1, 2), when we demonstrated that PKC␥ is involved in gap junction control.…”

Gating and regulation of connexin 43 (Cx43) hemichannels

“…Transfection with connexin cDNAs can induce expression of other genes (32), and Cx43 has binding partners that associate with it (e.g., ZO-1 and caveolin-1) (33,34). Thus, we were concerned that expression of Cx43 could induce surface expression of other channels that were responsible for the currents that we were identifying as hemichannel-mediated.…”

New roles for astrocytes: Gap junction hemichannels have something to communicate

“…The evidence for open hemichannels can be briefly summarized (and criticized [16][17][18][19].) The permeability and/or currents are inhibited by gap-junction blockers [10][11][12][13][14] (although most of the blockers are of dubious specificity).…”

“…Conversely, cells from connexin knockout animals do not show these properties, even though wild-type cells do [12,14]. (However, connexin transfection can induce changes in the expression of many other genes [15], and connexins have binding partners and might transport other channel molecules to the cell surface [16][17][18][19].) The permeability and/or currents are inhibited by gap-junction blockers [10][11][12][13][14] (although most of the blockers are of dubious specificity).…”