This summary is a proposed synthesis of available information for the non-specialist. It does not incorporate all the published data, is inconsistent with some, and reflects the biases of the author. Connexin proteins have a common transmembrane topology, with four alpha-helical transmembrane domains, two extracellular loops, a cytoplasmic loop, and cytoplasmic N- and C-terminal domains. The sequences are most conserved in the transmembrane and extracellular domains, yet many of the key functional differences between connexins are determined by amino-acid differences in these largely conserved domains. Each extracellular loop contains three cysteines with invariant spacing (save one isoform) that are required for channel function. The junctional channel is composed of two end-to-end hemichannels, each of which is a hexamer of connexin subunits. Hemichannels formed by some connexin isoforms can function as well-behaved, single-membrane-spanning channels in plasma membrane. In junctional channels, the cysteines in the extracellular loops form intra-monomer disulfide bonds between the two loops, not intermonomer or inter-hemichannel bonds. The end-to-end homophilic binding between hemichannels is via non-covalent interactions. Mutagenesis studies suggest that the docking region contains beta structures, and may resemble to some degree the beta-barrel structure of porin channels. The two hemichannels that compose a junctional channel are rotationally staggered by approximately 30 degrees relative to each other so that the alpha-helices of each connexin monomer are axially aligned with the alpha-helices of two adjacent monomers in the apposed hemichannel. At present there is a published 3D map with 7.5 A resolution in the plane of the membrane, based on electron cryomicroscopy of 2D crystals of junctional channels formed by C-terminal truncated Cx43. The correspondence between the imaged transmembrane alpha-helices and the known transmembrane amino-acid sequences is a matter of debate. Each of the approximately 20 connexin isoforms produces channels with distinct unitary conductances, molecular permeabilities, and electrical and chemical gating sensitivities. The channels can be heteromeric, and subfamilies among connexins largely determine heteromeric specificity, similar to the specificities within the voltage-dependent potassium channel superfamily. The second extracellular loop contains the primary determinants of the specificity of hemichannel-hemichannel docking (analogous to the tetramerization domain of potassium channels). The 7.5 A map shows that each monomer exposes only two transmembrane alpha-helices to the pore lumen. However the conductance state of the imaged structure and the effects of the C-terminal truncation are unknown, so it is possible that other transmembrane domains contribute to the lumen in other functional states of the channel. In the transmembrane region, SCAM and mutagenesis data suggest that parts of the first three transmembrane alpha-helices are exposed to the lumen. Some of these data are contradictory, but may reflect conformational or isoform differences. There is reason to think that the first part of the N-terminal cytoplasmic domain can line the pore in some conformations. In the extracellular part of junctional channels, the N-terminal portion of the first extracellular loop is exposed to the lumen. The unitary conductances through connexin channels vary over an order of magnitude, from 15 pS to over 300 pS. There is a range of charge selectivities among atomic ions, from slightly anion selective to highly cation selective, which does not correlate with unitary conductance. There appear to be substantial ion-ion interactions within the pore, making the GHK model of assessing selectivities of limited value. Pores formed by different connexins have a range of limiting diameters as assessed by uncharged and charged probes, which also does not correlate with unitary conductance (i.e. some have high conductance but have a narrow limiting diameter, and vice versa). Channels formed by different connexins have different permeabilities to various cytoplasmic molecules. Where it has been assessed, the selectivity among cytoplasmic molecules is substantial and does not correlate in an obvious manner with the size selectivity data derived from fluorescent tracer studies, suggesting there are chemical specificities within the pore that enhance or reduce permeability to specific cytoplasmic molecules, functionally analogous to the ability of some porins to facilitate transport of specific substrates. For example, heteromeric channels with different stoichiometries or arrangements of isoforms can distinguish among second messengers. The differences in permeability to cytoplasmic molecules have biological consequences; in most cases one connexin cannot fully substitute for another. Voltage and chemical gating mechanisms largely operate within each hemichannel, though there is evidence for inter-hemichannel allosteric effects as well. There are at least two distinct gating mechanisms. One (Vj-gating) is a voltage-driven mechanism that governs rapid transitions between conducting states. Its voltage sensor involves charges in the first several positions of the cytoplasmic N-terminal domain and possibly in the N-terminal part of the first extracellular loop, which may both be exposed to the lumen of the pore in some states. The polarity of Vj-gating sensitivity is connexin-specific, closing with depolarization for some connexins and with hyperpolarization for others. The polarity can be reversed by point mutations at the second position. The lower conductance states induced by Vj-gating correspond to physical restrictions of the pore, and thus restricted or eliminated molecular permeation. Since the channels are not fully closed by Vj-gating, it can be seen as a way to eliminate molecular signaling while leaving electrical signaling operational. A second, independent gating mechanism mediates slow transitions (approximately 10-30 ms) into and out of non-conducting state(s). These transitions can occur in response to voltage ('loop gating'), chemical factors such as pH and lipophiles ('chemical gating'), and the docking of two hemichannels (sometimes called the 'docking gate'). These slow transitions may reflect a common structural change induced by these several effectors (electrical, chemical and homodimerization). Alternatively, they could reflect distinct gating processes responding to one or more of these effectors, that are indistinguishable at the single-channel level and have yet to be resolved mechanistically. The slow or loop gate closes with hyperpolarization. As a result, where Vj-gating closes with depolarization, individual hemichannels can close in response to both polarities of voltage (but only to a subconductance state for the Vj-gating polarity). Because of this, it is difficult to assign a macroscopic voltage sensitivity, or its modification due to mutagenesis, chemical modification or heteromeric interactions, to one or the other of these very distinct voltage-sensitive processes. This distinction can be made reliably only at the single-channel level. The Vj-gating voltage sensor and the loop-gating voltage sensor appear to be independent structures, since the Vj-gating voltage sensitivity can modified without effect on loop gating. For some connexins, certain modifications of the C-terminal domain seem to interfere with the operation of the Vj-gate while leaving loop gating unaffected. In some connexins, but not all, the chemical sensitivity to pH can involve interactions between regions of the C-terminal domain and cytoplasmic loop. Whether these regions exert their effects directly by physically blocking the pore, or by allosteric mechanisms (which may be more consistent with the relatively long time-course of closure) is not clear. For several connexins, truncation of the C-terminal domain eliminates the pH sensitivity, and co-expressing the domain with the truncated connexin restores the pH sensitivity. This has a functional resemblance to the particle-receptor mechanism for N-type inactivation of Shaker channels. What is being protonated is not clear, and may involve cytoplasmic factors, such as endogenous aminosulfonates. For other connexins, the action of pH does not involve the C-terminal domain and seems due to direct protonation of connexin. PKC phosphorylation of serine(s) in the C-terminal domain can affect the substate occupancy of at least one connexin. Phosphorylation of series in the C-terminal domain by MAP kinase appears to facilitate an interaction between it and an unknown receptor domain to eliminate coupling. This process has yet to be studied at the single-channel level. It also has a functional analogy to the particle-receptor model of channel inactivation. Both MAP kinase phosphorylation-induced and pH-induced inhibition can be mediated in truncated connexins by the corresponding free peptide. However, the relation between these two mechanisms are unexplored, as are specific mechanisms of direct endogenous regulation of connexin channel activity. (ABSTRACT TRUNCATED)

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Emerging issues of connexin channels: biophysics fills the gap.

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct inter...

Gap junctions: structure and function (review)

Gap junctions are assemblies of intercellular channels that regulate a variety of physiologic and developmental processes through the exchange of small ions and signaling molecules. These channels consist of connexin family proteins that allow for diversity of channel composition and conductance properties. The human connexin 43 gene, or GJA1, is located at human chromosome 6q22-q23 within the candidate region for the oculodentodigital dysplasia locus. This autosomal dominant syndrome presents with craniofacial (ocular, nasal, and dental) and limb dysmorphisms, spastic paraplegia, and neurodegeneration. Syndactyly type III and conductive deafness can occur in some cases, and cardiac abnormalities are observed in rare instances. We found mutations in the GJA1 gene in all 17 families with oculodentodigital dysplasia that we screened. Sixteen different missense mutations and one codon duplication were detected. These mutations may cause misassembly of channels or alter channel conduction properties. Expression patterns and phenotypic features of gja1 animal mutants, reported elsewhere, are compatible with the pleiotropic clinical presentation of oculodentodigital dysplasia.

Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia.

Gap junctions form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the regular rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are expressed in the heart: connexin43 (Cx43), Cx40 and Cx45 are found in distinctive combinations and relative quantities in different, functionally-specialized subsets of cardiac myocyte. Mutations in genes that encode connexins have only rarely been identified as being a cause of human cardiac disease, but remodelling of connexin expression and gap junction organization are well documented in acquired adult heart disease, notably ischaemic heart disease and heart failure. Remodelling may take the form of alterations in (i) the distribution of gap junctions and (ii) the amount and type of connexins expressed. Heterogeneous reduction in Cx43 expression and disordering in gap junction distribution feature in human ventricular disease and correlate with electrophysiologically identified arrhythmic changes and contractile dysfunction in animal models. Disease-related alterations in Cx45 and Cx40 expression have also been reported, and some of the functional implications of these are beginning to emerge. Apart from ventricular disease, various features of gap junction organization and connexin expression have been implicated in the initiation and persistence of the most common form of atrial arrhythmia, atrial fibrillation, though the disparate findings in this area remain to be clarified. Other major tasks ahead focus on the Purkinje/working ventricular myocyte interface and its role in normal and abnormal impulse propagation, connexin-interacting proteins and their regulatory functions, and on defining the precise functional properties conferred by the distinctive connexin co-expression patterns of different myocyte types in health and disease.

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Remodelling of gap junctions and connexin expression in diseased myocardium

Hypoplastic Left Heart Syndrome : Current Considerations and Expectations

Gap junction channels formed by the connexin43 protein are considered to play crucial roles in development and function because they allow the direct cell-to-cell exchange of molecules that mediate multiple signaling events. Previous results have shown that connexin43 channels are intricately gated by phosphorylation and that disruption of this regulation gives rise to severe heart malformations and defects of laterality in human, chick and frog. Here we report the identification of connexin43 gene mutations that represent a minor population of connexin43 alleles, which could be reliably detected by using denaturing gradient gel electrophoresis (DGGE) to visualize normal and mutant DNAs that were separately sequenced. In contrast, sequencing of total PCR products without DGGE-pre-selection failed to consistently identify these mutations. Forty-six controls and 20 heart transplant recipients were examined in this study. In the latter group, 14 children had hypoplastic left heart syndrome (HLHS) in which connexin43 gene defects were detected in eight. The remaining six transplant patients with HLHS and all controls showed no defects. All eight HLHS children with gene defects had the same four substitutions: two that were silent polymorphisms, and two that were missense, replacing arginine codons at positions 362 and 376 with codons for glutamines. All four of these substitutions are identical to the nucleotide sequence of the connexin43 pseudogene, suggesting the possibility of an illicit recombination. A breakpoint region was identified 5' to the mutation site in a 63bp domain that is 100% identical in the gene and pseudogene. Results from in vitro phosphorylation indicate that the absence of arginines 362 and 376 completely abolishes phosphorylation in the connexin43 channel regulation domain suggesting a possible mechanism for the pathologies associated with HLHS.

Identification of connexin43 (α1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE)

α1 connexin (connexin43) is the dominant gap junction protein of the developing and mature heart where it forms channels that mediate intercellular electrical and metabolic coupling events that are critical for heart function. α1 connexin channels are rapidly and reversibly gated by actions of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), and disruption of consensus sites for these phosphorylations are associated with severe heart malformations. However, there have been no reports on the relative activities of PKA or PKC in early heart formation. Nor has the presence and phosphorylation state of α1 connexin been documented in these same developmental stages. To begin these studies, we used hearts from 8.5–18.5 dpc (days postcoitus) mouse embryos, postpartum pups, and adults. Membrane or supernatant fractions were used for immunoblots to assess the amounts and distribution of α1 connexin protein and each protein kinase. Phosphotransferase assays were done to document the endogenous activities of PKA and PKC. Three species of α1 connexin at 44, 46, and 49 kDa were evident in 8.5- and 9.5-dpc embryos and adult hearts, but the 49-kDa band was not consistently found in 10.5 dpc or embryos through 18.5 dpc, although it was robust in adult heart. The amount of PKA was minimal in 8.5-dpc hearts but rose thereafter and was maximal by 10.5 dpc and remained stable throughout development. Catalytic activity of this enzyme was minimal in 8.5-dpc hearts then rose thereafter and was maximal by 10.5 dpc of development. PKCδ was confined mainly to membrane fractions, whereas PKCϵ had supernatant- and membrane-associated forms. Both enzyme isoforms showed large fluctuations throughout development. In 8.5- and 9.5-dpc hearts, PKC catalytic activity was maximal but, by 10.5 dpc, activity dramatically declined and remained low thereafter. The results demonstrate that α1 connexin is present at the heart tube stage (8.5 dpc) of development onward and provide evidence suggesting that channels formed by this protein are dynamically regulated by PKA and PKC, especially in 8.5- and 9.5-day embryonic hearts, which are crucial times for heart formation and left/right patterning in general. © 2001 Wiley-Liss, Inc.

α1 connexin (connexin43) gap junctions and activities of cAMP‐dependent protein kinase and protein kinase C in developing mouse heart

Although there is general agreement that gap junction channels formed by the connexin43 (Cx43; alpha 1) protein most likely have important roles during heart development, evidence to support this view has been equivocal. Lacking this information, it is difficult to understand the basis of heart malformations found in the Cx43 knockout mice and in children with a severe form of visceroatrial heterotaxia that coincides with missense mutations of the Cx43 gene. To address this issue we used a combination of western blots to follow the emergence of Cx43 in heart, and in vitro and in vivo phosphorylation to assess the effect of mutation on Cx43 phosphorylation. We evaluated the activity ratios of cAMP-dependent protein kinase and protein kinase C in hearts of 8.5-day-old mouse embryos through to birth. The results demonstrate that Cx43 is present in the native phosphorylated species in day 8.5 hearts and thereafter. Further, the activities of cAMP-dependent protein kinase and protein kinase C are mirror images of each other during the 8.5-10.5 days of early heart development. From these results we conclude that Cx43 gap junction channels are present and capable of being regulated by day 8.5 of embryonic heart development.

Misregulation of connexin43 gap junction channels and congenital heart defects.

In an attempt to compare the regulation of chick connexin43 channels to those of mammalian connexin43, we found that the nucleotide sequence reported for chick connexin43 differs from that of the chick connexin gene by two codons that had been entered as histidine49 (H49) and valine50 (V50) (accession no. M29003), but are in fact glutamine49 (Q49) and serine50 (S50). Neuro2A cells were transfected with corrected wild-type (Q49/S50) chick connexin43 (accession no. AF233738), the double-replacement Q49H/S50V connexin43, or the single replacement of Q49H or S50V. All clones had gap junctions in membrane based on immunocytochemistry and immunoblots of the triton-resistant membrane fraction. Wild-type transfectants had three conductance states with a predominant channel conductance of 85 &#45 5 pS. Cells producing the Q49H-Cx43 or the double-replacement Q49H/S50V-Cx43 protein had no detectable connexin43 channels. In contrast, cells expressing S50V-Cx43 gap junctions had channels with reduced conductances (75 ...

William H. Fletcher

Papers

Identification of connexin43 (α1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE)

α1 connexin (connexin43) gap junctions and activities of cAMP‐dependent protein kinase and protein kinase C in developing mouse heart

Misregulation of connexin43 gap junction channels and congenital heart defects.

The highly conserved Gln49 and Ser50 of mammalian connexin43 are present in chick connexin43 and essential for functional gap junction channels.