Synthesis and assembly of connexins in vitro into homomeric and heteromeric functional gap junction hemichannels.
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ABSTRACT: The biogenesis of connexins and their assembly into functional gap junction hemichannels (connexons) was studied with the use of a cell-free transcription/translation system. Velocity sedimentation on sucrose gradients showed that a small proportion of connexin (Cx) 26 and Cx32 that were co-translationally translocated into microsomes were oligomers of Cx26 and Cx32. Chemical cross-linking studies showed that these corresponded to hexameric connexons. Reconstitution of connexons synthesized in vitro into liposomes induced permeability properties consistent with the view that open gap junction hemichannels were produced. By using an immunoprecipitation approach, a simultaneous translation of Cx26 and Cx32 incorporated into microsomes resulted in homomeric connexons. However, supplementation of the translation system in vitro with liver Golgi membranes produced heteromeric connexons constructed of Cx32 and Cx26, and also resulted in an increased oligomerization especially of Cx32. All of the connexins analysed were inserted co-translationally into canine pancreatic microsomal membranes. In addition, Cx26 and Cx43, but not Cx32, were also inserted into microsomal membranes post-translationally. Analysis of various connexin constructs in which the cytoplasmic carboxy tails were transposed, the cytoplasmic tail of Cx43 was truncated or a reporter protein, aequorin, was attached to the C-terminus showed that tail length was not the major determinant of the post-translational membrane insertion of connexins.
Project description:Since most cells in the heart co-express multiple connexins, we studied the possible heteromeric interactions between connexin30.2 and connexin40, connexin43 or connexin45 in transfected cells. Double-label immunofluorescence microscopy showed that connexin30.2 extensively co-localized with each co-expressed connexin at appositional membranes. When Triton X-100 solubilized connexons were affinity purified from co-expressing cells, connexin30.2 was isolated together with connexin40, connexin43, or connexin45. Co-expression of connexin30.2 with connexin40, connexin43, or connexin45 did not significantly reduce total junctional conductance. Gap junction channels in cells co-expressing connexin30.2 with connexin43 or connexin45 exhibited voltage-dependent gating intermediate between that of either connexin alone. In contrast, connexin30.2 dominated the voltage-dependence when co-expressed with connexin40. Our data suggest that connexin30.2 can form heteromers with the other cardiac connexins and that mixed channel formation will influence the gating properties of gap junctions in cardiac regions that co-express these connexins.
Project description:To study the assembly of gap junctions, connexin--green-fluorescent-protein (Cx--GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26-- and Cx32--GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32--GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx--GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.
Project description:Genetic diseases demonstrate that the normal function of CNS myelin depends on connexin32 (Cx32) and Cx47, gap junction (GJ) proteins expressed by oligodendrocytes. GJs couple oligodendrocytes and astrocytes (O/A channels) as well as astrocytes themselves (A/A channels). Because astrocytes express different connexins (Cx30 and Cx43), O/A channels must be heterotypic, whereas A/A channels may be homotypic or heterotypic. Using electrophysiological and immunocytochemical approaches, we found that Cx47/Cx43 and Cx32/Cx30 efficiently formed functional channels, but other potential heterotypic O/A and A/A pairs did not. These results suggest that Cx30/Cx30 and Cx43/Cx43 channels mediate A/A coupling, and Cx47/Cx43 and Cx32/Cx30 channels mediate O/A coupling. Furthermore, Cx47/Cx43 and Cx32/Cx30 channels have distinct macroscopic and single-channel properties and different dye permeabilities. Finally, Cx47 mutants that cause Pelizaeus-Merzbacher-like disease do not efficiently form functional channels with Cx43, indicating that disrupted Cx47/Cx43 channels cause this disease.
Project description:Gap junctions are cellular contact sites composed of clustered connexin transmembrane proteins that act in dual capacities as channels for direct intercellular exchange of small molecules and as structural adhesion complexes known as gap junction nexuses. Depending on the connexin isoform, the cluster of channels (the gap junction plaque) can be stably or fluidly arranged. Here we used confocal microscopy and mutational analysis to identify the residues within the connexin proteins that determine gap junction plaque stability. We found that stability is altered by changing redox balance using a reducing agent-indicating gap junction nexus stability is modifiable. Stability of the arrangement of connexins is thought to regulate intercellular communication by establishing an ordered supramolecular platform. By identifying the residues that establish plaque stability, these studies lay the groundwork for exploration of mechanisms by which gap junction nexus stability modulates intercellular communication.
Project description:Gap junction channels and hemichannels formed by concatenated connexins were analyzed. Monomeric (hCx26, hCx46), homodimeric (hCx46-hCx46, hCx26-hCx26), and heterodimeric (hCx26-hCx46, hCx46-hCx26) constructs, coupled to GFP, were expressed in HeLa cells. Confocal microscopy showed that the tandems formed gap junction plaques with a reduced plaque area compared to monomeric hCx26 or hCx46. Dye transfer experiments showed that concatenation allows metabolic transfer. Expressed in Xenopus oocytes, the inside-out patch-clamp configuration showed single channels with a conductance of about 46 pS and 39 pS for hemichannels composed of hCx46 and hCx26 monomers, respectively, when chloride was replaced by gluconate on both membrane sides. The conductance was reduced for hCx46-hCx46 and hCx26-hCx26 homodimers, probably due to the concatenation. Heteromerized hemichannels, depending on the connexin-order, were characterized by substates at 26 pS and 16 pS for hCx46-hCx26 and 31 pS and 20 pS for hCx26-hCx46. Because of the linker between the connexins, the properties of the formed hemichannels and gap junction channels (e.g., single channel conductance) may not represent the properties of hetero-oligomerized channels. However, should the removal of the linker be successful, this method could be used to analyze the electrical and metabolic selectivity of such channels and the physiological consequences for a tissue.
Project description:An Arabidopsis thaliana library constructed in the pBluescript expression vector lambda ZAP II (Stratagene) was screened with three affinity-purified antibodies raised against (i) rat liver connexin 32, (ii) a polypeptide from soybean root cells that migrates with a molecular mass of 29 kDa in SDS/polyacrylamide gels and is immunologically related to rat liver connexin, and (iii) a synthetic peptide corresponding to a sequence in rat liver connexin 32. A single clone was obtained whose gene product demonstrated immunological crossreactivity with all three reagents. The cDNA from this clone contained 1171 base pairs and coded for a protein of 280 amino acids with a calculated molecular mass of 32,339 Da (migrates as a 29-kDa polypeptide in SDS/polyacrylamide gels). The sequence homology observed between the 32-kDa polypeptide of Arabidopsis and connexin 32 from rat liver, in conjunction with observed similarities in predicted number and distribution of hydrophobic domains, sites for posttranslational modification, and basic pI, provides strong evidence that the biological range for connexin-type proteins may now be considered to include the plant kingdom.
Project description:Ca2+/calmodulin (Ca2+/CaM) interaction with connexins (Cx) is well-established; however, the mechanistic basis of regulation of gap junction function by Ca2+/CaM is not fully understood. Ca2+/CaM is predicted to bind to a domain in the C-terminal portion of the intracellular loop (CL2) in the vast majority of Cx isoforms and for a number of Cx-s this prediction has proved correct. In this study, we investigate and characterise both Ca2+/CaM and apo-CaM binding to selected representatives of each of the α, β and γ connexin family to develop a better mechanistic understanding of CaM effects on gap junction function. The affinity and kinetics Ca2+/CaM and apo-CaM interactions of CL2 peptides of β-Cx32, γ-Cx35, α-Cx43, α-Cx45 and α-Cx57 were investigated. All five Cx CL2 peptides were found to have high affinity for Ca2+/CaM with dissociation constants (Kd(+Ca)) from 20 to 150 nM. The limiting rate of binding and the rates of dissociation covered a broad range. In addition, we obtained evidence for high affinity Ca2+-independent interaction of all five peptides with CaM, consistent with CaM remaining anchored to gap junctions in resting cells. However, for the α-Cx45 and α-Cx57 CL2 peptides, Ca2+-dependent association at resting [Ca2+] of 50-100 nM is indicated in these complexes as one of the CaM Ca2+ binding sites displays high affinity with Kd of 70 and 30 nM for Ca2+, respectively. Furthermore, complex conformational changes were observed in peptide-apo-CaM complexes with the structure of CaM compacted or stretched by the peptide in a concentration dependent manner suggesting that the CL2 domain may undergo helix-to-coil transition and/or forms bundles, which may be relevant in the hexameric gap junction. We demonstrate inhibition of gap junction permeability by Ca2+/CaM in a dose dependent manner, further cementing Ca2+/CaM as a regulator of gap junction function. The motion of a stretched CaM-CL2 complex compacting upon Ca2+ binding may bring about the Ca2+/CaM block of the gap junction pore by a push and pull action on the CL2 C-terminal hydrophobic residues of transmembrane domain 3 (TM3) in and out of the membrane.
Project description:We examined the permeabilities of homotypic and heterotypic gap junction (GJ) channels formed of rodent connexins (Cx) 30.2, 40, 43, and 45, which are expressed in the heart and other tissues, using fluorescent dyes differing in net charge and molecular mass. Combining fluorescent imaging and electrophysiological recordings in the same cell pairs, we evaluated the single-channel permeability (P(gamma)). All homotypic channels were permeable to the anionic monovalent dye Alexa Fluor-350 (AF(350)), but mCx30.2 channels exhibited a significantly lower P(gamma) than the others. The anionic divalent dye Lucifer yellow (LY) remained permeant in Cx40, Cx43, and Cx45 channels, but transfer through mCx30.2 channels was not detected. Heterotypic channels generally exhibited P(gamma) values that were intermediate to the corresponding homotypic channels. P(gamma) values of mCx30.2/Cx40, mCx30.2/Cx43, or mCx30.2/Cx45 heterotypic channels for AF(350) were similar and approximately twofold higher than P(gamma) values of mCx30.2 homotypic channels. Permeabilities for cationic dyes were assessed only qualitatively because of their binding to nucleic acids. All homotypic and heterotypic channel configurations were permeable to ethidium bromide and 4,6-diamidino-2-phenylindole. Permeability for propidium iodide was limited only for GJ channels that contain at least one mCx30.2 hemichannel. In summary, we have demonstrated that Cx40, Cx43, and Cx45 are permeant to all examined cationic and anionic dyes, whereas mCx30.2 demonstrates permeation restrictions for molecules with molecular mass over approximately 400 Da. The ratio of single-channel conductance to permeability for AF(350) was approximately 40- to 170-fold higher for mCx30.2 than for Cx40, Cx43, and Cx45, suggesting that mCx30.2 GJs are notably more adapted to perform electrical rather than metabolic cell-cell communication.
Project description:This review is based in part on a roundtable discussion session: "Physiological roles for heterotypic/heteromeric channels" at the 2013 International Gap Junction Conference (IGJC 2013) in Charleston, South Carolina. It is well recognized that multiple connexins can specifically co-assemble to form mixed gap junction channels with unique properties as a means to regulate intercellular communication. Compatibility determinants for both heteromeric and heterotypic gap junction channel formation have been identified and associated with specific connexin amino acid motifs. Hetero-oligomerization is also a regulated process; differences in connexin quality control and monomer stability are likely to play integral roles to control interactions between compatible connexins. Gap junctions in oligodendrocyte:astrocyte communication and in the cardiovascular system have emerged as key systems where heterotypic and heteromeric channels have unique physiologic roles. There are several methodologies to study heteromeric and heterotypic channels that are best applied to either heterologous expression systems, native tissues or both. There remains a need to use and develop different experimental approaches in order to understand the prevalence and roles for mixed gap junction channels in human physiology.
Project description:Connexins are a family of transmembrane proteins that form gap junction channels. These proteins undergo both proteasomal and lysosomal degradation, mechanisms that serve to regulate connexin levels. Our previous work described CIP75 [connexin43 (Cx43)-interacting protein of 75 kDa], a protein involved in proteasomal degradation, as a novel Cx43-interacting protein. We have discovered two additional connexins, connexin40 (Cx40) and connexin45 (Cx45), that interact with CIP75. Nuclear magnetic resonance (NMR) analyses identified the direct interaction of the CIP75 UBA domain with the carboxyl-terminal (CT) domains of Cx40 and Cx45. Reduction in CIP75 by shRNA in HeLa cells expressing Cx40 or Cx45 resulted in increased levels of the connexins. Furthermore, treatment with trafficking inhibitors confirmed that both connexins undergo endoplasmic reticulum-associated degradation (ERAD), and that CIP75 preferentially interacts with the connexin proteins bound for proteasomal degradation from the ER. In addition, we have also discovered that CIP75 interacts with ER-localized Cx32 in a process that is likely mediated by Cx32 ubiquitination. Thus, we have identified novel interacting connexin proteins of CIP75, indicating a role for CIP75 in regulating the levels of connexins in general, through proteasomal degradation.