Kinetics of protein-protein interactions of connexins: use of enzyme linked sorbent assays

Determination of the protein-protein interactions of connexins has become a rapidly expanding field of research. While there are multiple methods of determining the identity of binding partners, determination of the strengths of interactions is not as simple. Here we describe the use of the in vitro method Enzyme Linked Sorbent Assay (ELSA) to compare binding affinities of known protein partners for Connexin43. We used the binding of Cx43 Carboxyl Terminal domain to the PDZ-2 domain of Zonula Occludens-1 and to the SH3 domain of c-Src. In the ELSA assay we found that while the binding of the SH3 domain of c-Src is pH-dependent, the interaction of the PDZ domain of ZO-1 is not. These data confirm findings using Surface Plasmon Resonance (1) and indicate that ELSA can be a useful tool in determining the kinetics of protein-protein interactions.     

Connexin family members target to lipid raft domains and interact with caveolin-1

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.     

Mechanisms of glutamate release from astrocytes: gap junction "hemichannels", purinergic receptors and exocytotic release

Neuronal exocytotic release of glutamate at synapses involves a highly specialized vesicular apparatus, consisting of a variety of proteins connected to the vesicles or required for vesicular fusion to the presynaptic membrane. Astrocytes also release glutamate, and recent evidence indicates that this release can modify neuronal function. Several mechanisms have been proposed for astrocytic release of glutamate under pathological conditions, such as reversal of glutamate transporters and opening of volume sensitive ion channels. In this review we limit our discussion to findings supporting the exocytotic release of glutamate, as well as two new pathways implicated in this release, the ionotropic (P2X) purinergic receptors and gap junction hemichannels.     

Properties of gap junction channels formed by Cx46 alone and in combination with Cx50

Gap junctions formed of connexin46 (Cx46) and connexin50 (Cx50) in lens fiber cells are crucial for maintaining lens transparency. We determined the functional properties of homotypic Cx46, heterotypic Cx46/Cx50, and heteromeric Cx46/Cx50 channels in a communication-deficient neuroblastoma (N2A) cell line, using dual whole-cell recordings. N2A cultures were stably and/or transiently transfected with Cx46, Cx50, and green fluorescent protein (EGFP). The macroscopic voltage sensitivity of homotypic Cx46 conformed to the two-state model (Boltzmann parameters: G(min) = 0.11, V(0) = +/- 48.1 mV, gating charge = 2). Cx46 single channels showed a main-state conductance of 140 +/- 8 pS and multiple subconductance states ranging from < or =10 pS to 60 pS. Conservation of homotypic properties in heterotypic Cx46/Cx50 cell pairs allowed the determination of a positive relative gating polarity for the dominant gating mechanisms in Cx46 and Cx50. Observed gating properties were consistent with a second gating mechanism in Cx46 connexons. Moreover, rectification was observed in heterotypic cell pairs. Some cell pairs in cultures simultaneously transfected with Cx46 and Cx50 exhibited junctional properties not observed in other preparations, suggesting the formation of heteromeric channels. We conclude that different combinations of Cx46 and Cx50 within gap junction channels lead to unique biophysical properties.     

Gap junction expression and cell proliferation in differentiating cultures of Cx43 KO mouse hepatocytes

Primary cultures of adult mouse hepatocytes are shown here to reexpress differentiated hepatocyte features following treatment with 2% DMSO and 10(-7) M glucagon. To examine the roles of gap junctional communication during hepatocyte growth and differentiation, we have compared treated and untreated hepatocytes from connexin (Cx)32-deficient [Cx32 knockout (KO)] and wild-type mice. In untreated cultures, DNA replication of Cx32 KO hepatocytes was markedly higher than of wild types. Although Cx26 mRNA levels remained high at all time points in wild-type and Cx32 KO hepatocytes, Cx32 mRNA and protein in wild-type hepatocytes underwent a marked decline, which recovered in 10-day treated cultures. Increased levels of Cx26 protein and junctional conductance were observed in Cx32 KO hepatocytes at 96 h in culture, a time when cell growth rate was high. Treatment with DMSO/glucagon highly reinduced Cx26 expression in Cx32 KO hepatocytes, and such treatment reinduced expression of both Cx32 and Cx26 expression in wild types. Dye transfer was not observed following Lucifer yellow injection into DMSO/glucagon-treated Cx32 KO hepatocytes, whereas the spread was extensive in wild types. Nevertheless, high junctional conductance values were observed in treated cells from both genotypes. These studies provide a method by which the differentiated phenotype can be obtained in cultured mouse hepatocytes and provide in vitro evidence that expression of gap junctions formed of Cx32 are involved in the regulation of growth of mouse hepatocytes.