Hexameric Connexons Research Articles

Connexin/Innexin Channels in Cytoplasmic Organelles. Are There Intracellular Gap Junctions? A Hypothesis!

This paper proposes the hypothesis that cytoplasmic organelles directly interact with each other and with gap junctions forming intracellular junctions. This hypothesis originated over four decades ago based on the observation that vesicles lining gap junctions of crayfish giant axons contain electron-opaque particles, similar in size to junctional innexons that often appear to directly interact with junctional innexons; similar particles were seen also in the outer membrane of crayfish mitochondria. Indeed, vertebrate connexins assembled into hexameric connexons are present not only in the membranes of the Golgi apparatus but also in those of the mitochondria and endoplasmic reticulum. It seems possible, therefore, that cytoplasmic organelles may be able to exchange small molecules with each other as well as with organelles of coupled cells via gap junctions.

Connexin-mediated cell communication in the kidney: A potential therapeutic target for future intervention of diabetic kidney disease?: Joan Mott Prize Lecture.

The ability of cells to communicate and synchronise their activity is essential for the maintenance of tissue structure, integrity and function. A family of membrane-bound proteins called connexins are largely responsible for mediating the local transfer of information between cells. Assembled in the cell membrane as a hexameric connexon, they either function as a conduit for paracrine signalling, forming a transmembrane hemi-channel, or, if aligned with connexons on neighbouring cells, form a continuous aqueous pore or gap junction, which allows for the direct transmission of metabolic and electrical signals. Regulation of connexin synthesis and activity is critical to cellular function, and a number of diseases are attributed to changes in the expression and/or function of these important proteins. A link between hyperglycaemia, connexin expression, altered nucleotide concentrations and impaired function highlights a potential role for connexin-mediated cell communication in complications of diabetes. In the diabetic kidney, glycaemic injury is the leading cause of end-stage renal failure, reflecting multiple aetiologies including glomerular hyperfiltration, albuminuria, increased deposition of extracellular matrix and tubulointerstitial fibrosis. Loss of connexin-mediated cell-to-cell communication in diabetic nephropathy may represent an early sign of disease progression, but our understanding of the process remains severely limited. This review focuses on recent evidence demonstrating that glucose-evoked changes in connexin-mediated cell communication and associated purinergic signalling may contribute to the pathogenesis of kidney disease in diabetes, highlighting the tantalising potential of targeting these proteins as a novel therapeutic intervention.

Mind the gap: connexins and cell-cell communication in the diabetic kidney.

Connexins, assembled as a hexameric connexon, form a transmembrane hemichannel that provides a conduit for paracrine signalling of small molecules and ions to regulate the activity and function of adjacent cells. When hemichannels align and associate with similar channels on opposing cells, they form a continuous aqueous pore or gap junction, allowing the direct transmission of metabolic and electrical signals between coupled cells. Regulation of gap junction synthesis and channel activity is critical for cell function, and a number of diseases can be attributed to changes in the expression/function of these important proteins. Diabetic nephropathy is associated with several complex metabolic and inflammatory responses characterised by defects at the molecular, cellular and tissue level. In both type 1 and type 2 diabetes, glycaemic injury of the kidney is the leading cause of end-stage renal failure, a consequence of multiple aetiologies, including increased deposition of extracellular matrix, glomerular hyperfiltration, albuminuria and tubulointerstitial fibrosis. In diabetic nephropathy, loss of connexin mediated cell-cell communication within the nephron may represent an early sign of disease; however, our current knowledge of the role of connexins in the diabetic kidney is sparse. This review highlights recent evidence demonstrating that maintenance of connexin-mediated cell-cell communication could benefit region-specific renal function in diabetic nephropathy and suggests that these proteins should be viewed as a tantalising novel target for therapeutic intervention.

489 New animal model in colorectal cancer

489 New animal model in colorectal cancer

Assessment of Gap Junctional Intercellular Communication

Gap junctions are important plasma membrane organelles through which most cells in normal tissues communicate with each other. They exist in two neighboring cells and each cell contributes half of the structure. One gap junction consists of two hexameric connexons that dock with each other to create a channel. Six of the basic subunits referred to as connexins form a connexon. Less than one hundred to several thousand gap junction channels cluster together in the plane of the membrane. The gap junction channels serve as a regulated conduit for the intercellular exchange of small molecules. Maintenance of the integrity of gap junctional intercellular communication (GJIC) is important and required for normal electrical coupling, homeostasis, and embryogenesis. Aberrations of gap junctions have been related to human diseases such as cancer, cardiac arrhythmia, Charcot-Marie-tooth disease, and visceroatrial heterotaxia syndrome. This unit describes methods for measuring gap junctional intercellular communication using primary mouse hepatocytes as a model. Focus is only on functional evaluation based on dye coupling. Other methods, such as intracellular calcium waves and dual patch clamp, have been used to measure gap junctional communication, but these are not described in this unit.

Triarylmethanes - a New Class of Connexin Blockers

Connexins (Cx) are a family of proteins with 4 transmembrane regions, which are encoded by 21 genes in humans and which form hexameric connexons (= hemichannels). These connexons can either function as transmembrane ion channels or assemble into gap junctions that directly mediate signaling between adjacent cells by allowing the passage of ions, metabolites and signaling molecules up to 1 kd in mass. Both gap junctions and hemichannels play important roles in many tissues and have therefore been proposed as potential new targets for the treatment of epilepsy, cardiac arrhythmia and cancer. However, there are no specific and potent pharmacological tools to study the physiological as well as the pathophysiological role of connexins. The existing connexin modulators are either of low potency or cross-react with other ion channels. In order to identify potent and selective connexin blockers we screened a small library of compounds containing pharmacophores known to modulate other ion channels. From this library, we identified four new small molecule chemotypes including triarlymethanes (TRAMs) like clotrimazole and benzimidazoles like astemizole that inhibit Cx50 channels in a sub-type specific manner with IC50 values in the range of 1-10 μM while having little or no effect on those formed by Cx46, Cx36 and Cx32. We are currently exploring the structure activity relationship (SAR) of TRAMs for Cx50 inhibition and have recently identified T66 (N-[(2-chlorophenyl)(diphenyl)methyl]-N-(1,3-thiazol-2yl)amine), which exhibits an IC50 of 3 μM. In general, the SAR of the Cx50 inhibiting TRAMs significantly differs from the SAR of KCa3.1 blocking TRAMs. We propose T66 and its derivatives as novel pharmacological tool compounds that may be used to study the physiological role of connexins.

Incorporation of connexins into plasma membranes and gap junctions.

Gap junctions are polymeric assemblies of aligned pairs of interacting hexameric connexon hemichannel units facilitating direct intercellular communication. The principal process leading to assembly of gap junctions involves the cotranslational insertion of connexin (Cx) proteins into the endoplasmic reticulum, followed by their rapid oligomeric association into homo- or heteromeric connexons that are trafficked via the Golgi apparatus to the plasma membrane. Oligomerisation is a high-fidelity process that determines connexon channel stoichiometry and conductance characteristics. A large number of mutations in Cx26 and Cx32 detected in genetic diseases have emphasised the requirement for precise oligomerisation of connexins into hexameric connexons that traffic to the plasma membrane. Mutations in Cx43 are rare, and in the cardiovascular system, where it is the dominant connexin, disease changes are linked to its abundance and to gap junction remodelling. Connexins with short carboxyl tails may also be post-translationally inserted as oligomeric channels directly into plasma membranes. This mechanism of channel assembly is highly dependent on microtubule integrity and may allow cells to rapidly modulate gap junctional cross talk.

Projection structure of full length connexin 43 by electron cryo-crystallography.

We previously used electron cryo-crystallography to determine the three-dimensional structure of recombinant gap junction channels formed by a C-terminal truncation mutant of Cx43 (11). The dodecameric channel is formed by the end-to-end docking of two hexameric connexons, each comprised of 24 transmembrane alpha-helices. We have now generated two-dimensional crystals of the recombinant, full-length channel, as well as crystals in which the C-tail has been completely removed by trypsin digestion. Projection density maps at 7.5 A resolution closely resemble our previous analysis of the C-terminal truncation mutant (9). A difference map between the full length and trypsin-treated channels suggests that there are small but significant shifts in protein density upon removal of the C-tail.

Assembly of gap junction channels: mechanism, effects of calmodulin antagonists and identification of connexin oligomerization determinants.

The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell-free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin-binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin-binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin-binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Delta215, when transiently expressed in COS-7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C-terminal sequence of Cx32 incorporating the calmodulin-binding site was required for the formation of hetero-oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS-7 cells. The results indicate that amino-acid sequences in the third transmembrane domain and a calmodulin-binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.

Transmembrane alpha-helices in the gap junction membrane channel: systematic search of packing models based on the pair potential function.

Recent progress in the field of electron cryo-microscopy and image analysis has shown that there is an overwhelming need to interpret medium resolution (5 to 10 A) three-dimensional maps. Traditional methods of fitting amino acid residues into electron density using molecular modeling programs must be supplemented with further analysis. We have used a potential of mean force (PMF) method, derived from Boltzmann statistics in protein structure, to generate models for the packing of alpha-helices, using pairwise potentials between amino acid residues. The approach was tested using the three-dimensional map of a recombinant cardiac gap junction membrane channel provided by electron cryo-crystallography (Unger et al., 1997; 1999a, 1999b) which had a resolution of 7.5 A in the membrane plane and 21 A in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which was consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. Based on the three-dimensional map and the amino acid sequence for the 4 transmembrane domains determined by hydropathy analysis, we used the modeling utility SymServ (Macke et al., 1998) to build hexameric connexons with 24 transmembrane alpha-helices. Canonical alpha-helices were aligned to the axes of the rods of density and translated along the density so that the center of masses coincided. The PMF function was used to evaluate 162,000 conformations for each of the 24 possible alpha-helical packing models. Since the different packing models yielded different energy distributions, the pair potential function appears to be a promising tool for evaluating the packing of alpha-helices in membrane proteins. The analysis will be refined by energy calculations based on the expectations that the outer boundary of the channel will be formed by hydrophobic residues in contact with the lipids.

Biosynthesis and structural composition of gap junction intercellular membrane channels

Gap junction channels assemble as dodecameric complexes, in which a hexameric connexon (hemichannel) in one plasma membrane docks end-to-end with a connexon in the membrane of a closely apposed cell to provide direct cell-to-cell communication. Synthesis, assembly, and trafficking of the gap junction channel subunit proteins referred to as connexins, largely appear to follow the general secretory pathway for membrane proteins. The connexin subunits can assemble into homo-, as well as distinct hetero-oligomeric connexons. Assembly appears to be based on specific signals located within the connexin polypeptides. Plaque formation by the clustering of gap junction channels in the plane of the membrane, as well as channel degradation are poorly understood processes that are topics of current research. Recently, we tagged connexins with the autofluorescent reporter green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants and combined this reporter technology with single, and dual-color, high resolution deconvolution microscopy, computational volume rendering, and time-lapse microscopy to examine the detailed organization, structural composition, and dynamics of gap junctions in live cells. This technology provided for the first time a realistic, three-dimensional impression of gap junctions as they appear in the plasma membranes of adjoining cells, and revealed an excitingly detailed structural organization of gap junctions never seen before in live cells. Here, I summarize recent progress in areas encompassing the synthesis, assembly and structural composition of gap junctions with a special emphasis on the recent results we obtained using cell-free translation/ membrane-protein translocation, and autofluorescent reporters in combination with live-cell deconvolution microscopy.

Expression, Two-Dimensional Crystallization, and Electron Cryo-crystallography of Recombinant Gap Junction Membrane Channels

We used electron cryo-microscopy and image analysis to examine frozen-hydrated, two-dimensional (2D) crystals of a recombinant, 30-kDa C-terminal truncation mutant of the cardiac gap junction channel formed by 43-kDa α 1 connexin. To our knowledge this is the first example of a structural analysis of a membrane protein that has been accomplished using microgram amounts of starting material. The recombinant α 1 connexin was expressed in a stably transfected line of baby hamster kidney cells and spontaneously assembled gap junction plaques. Detergent treatment with Tween 20 and 1,2-diheptanoyl- sn-phosphocholine resulted in well-ordered 2D crystals. A three-dimensional density (3D) map with an in-plane resolution of ∼7.5 Å revealed that each hexameric connexon was formed by 24 closely packed rods of density, consistent with an α-helical conformation for the four transmembrane domains of each connexin subunit. In the extracellular gap the aqueous channel was bounded by a continuous wall of protein that formed a tight electrical and chemical seal to exclude exchange of substances with the extracellular milieu.

Synthesis and assembly of connexins in vitro into homomeric and heteromeric functional gap junction hemichannels

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.

Synthesis and assembly of connexins in vitro into homomeric and heteromeric functional gap junction hemichannels

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.

799 Chronic inhibition of phosphatases 1 and 2A in rat brain — A non-transgenic model of Alzheimer's disease

799 Chronic inhibition of phosphatases 1 and 2A in rat brain — A non-transgenic model of Alzheimer's disease

Editorial Board

Editorial Board

Modeling connexon skewing in gap junction lattices

Micrographs of negatively stained gap junction lattices recorded by low irradiation methods show skewed, hexameric connexons. Differences in appearance among reconstructed images illustrate variation in staining of the connexon channel and the space between connexons. Consistently observed stain concentration at six symmetrically related sites ∽3.4nm from the connexon center, 8° to the right or left of the [1,1] lattice vector outlines the skewed connexons. Fig. 1 shows that the connexon can be represented at the 2.5nm resolution of the data by a simple model with four distinct domains: 1) a circular stain excluding subunit of 1.1nm radius located ∽2.2nm from the six-fold axis at an angle of 8° to the [1,0] lattice vector; 2) a stain accessible core ∽2nm in diameter; 3) a preferential staining region centered at ∽3.4nm between the stain excluding lobes of the connexon model; 4) a variable stain excluding region on the three-fold axis. Adjustment of the weight of the stain features in this model with only small changes in dimensions can account for a wide range of image appearance. The R-factor comparing the Fourier transform of the connexon image averaged from 21 micrographs (Fig. 1a) with that of a single specimen showing a densely stained center is 33%; the models fitted to these images correspond with R-factors of about 10%.