Extracellular domains play different roles in gap junction formation and docking compatibility

GJ (gap junction) channels mediate direct intercellular communication and play an important role in many physiological processes. Six connexins oligomerize to form a hemichannel and two hemichannels dock together end-to-end to form a GJ channel. Connexin extracellular domains (E1 and E2) have been shown to be important for the docking, but the molecular mechanisms behind the docking and formation of GJ channels are not clear. Recent developments in atomic GJ structure and functional studies on a series of connexin mutants revealed that E1 and E2 are likely to play different roles in the docking. Non-covalent interactions at the docking interface, including hydrogen bonds, are predicted to form between interdocked extracellular domains. Protein sequence alignment analysis on the docking compatible/incompatible connexins indicate that the E1 domain is important for the formation of the GJ channel and the E2 domain is important in the docking compatibility in heterotypic channels. Interestingly, the hydrogen-bond forming or equivalent residues in both E1 and E2 domains are mutational hot spots for connexin-linked human diseases. Understanding the molecular mechanisms of GJ docking can assist us to develop novel strategies in rescuing the disease-linked connexin mutants.

Asparagine 175 of Connexin32 Is a Critical Residue for Docking and Forming Functional Heterotypic Gap Junction Channels with Connexin26

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn175 of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.

Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45

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 ( Pγ). All homotypic channels were permeable to the anionic monovalent dye Alexa Fluor-350 (AF350), but mCx30.2 channels exhibited a significantly lower Pγ 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 Pγ values that were intermediate to the corresponding homotypic channels. Pγ values of mCx30.2/Cx40, mCx30.2/Cx43, or mCx30.2/Cx45 heterotypic channels for AF350 were similar and approximately twofold higher than Pγ 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 ∼400 Da. The ratio of single-channel conductance to permeability for AF350 was ∼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.

Human connexin26 and connexin30 form functional heteromeric and heterotypic channels

Mutations in GJB2 and GJB6, the genes that encode the human gap junction proteins connexin26 (Cx26) and connexin30 (Cx30), respectively, cause hearing loss. Cx26 and Cx30 are both expressed in the cochlea, leading to the potential formation of heteromeric hemichannels and heterotypic gap junction channels. To investigate their interactions, we expressed human Cx26 and Cx30 individually or together in HeLa cells. When they were expressed together, Cx26 and Cx30 appeared to interact directly (by their colocalization in gap junction plaques, by coimmunoprecipitation, and by fluorescence resonance energy transfer). Scrape-loading cells that express either Cx26 or Cx30 demonstrated that Cx26 homotypic channels robustly transferred both cationic and anionic tracers, whereas Cx30 homotypic channels transferred cationic but not anionic tracers. Cells expressing both Cx26 and Cx30 also transferred both cationic and anionic tracers by scrape loading, and the rate of calcein (an anionic tracer) transfer was intermediate between their homotypic counterparts by fluorescence recovery after photobleaching. Fluorescence recovery after photobleaching also showed that Cx26 and Cx30 form functional heterotypic channels, allowing the transfer of calcein, which did not pass the homotypic Cx30 channels. Electrophysiological recordings of cell pairs expressing different combinations of Cx26 and/or Cx30 demonstrated unique gating properties of cell pairs expressing both Cx26 and Cx30. These results indicate that Cx26 and Cx30 form functional heteromeric and heterotypic channels, whose biophysical properties and permeabilities are different from their homotypic counterparts.

Virtual cloning, functional expression, and gating analysis of human connexin31.9

We have identified a novel gap junction gene by searching the human genome sequence database that encodes a protein designated as connexin31.9 (Cx31.9). Cx31.9 was most homologous to human Cx32.4 and did not cluster with either the purported α- or β-connexin subfamilies. Expression of Cx31.9 was detected by RT-PCR in human mRNA from several tissues including cerebral cortex, heart, liver, lung, kidney, spleen, and testis. A partial Cx31.9 sequence was also represented in the human Expressed Sequence Tag database. Cx31.9 formed intercellular channels in both paired Xenopus oocytes and transfected neuroblastoma N2A cells that were distinguished by an apparent low unitary conductance (12–15 pS) and a remarkable insensitivity to transjunctional voltage. In contrast, Cx31.9 channels were gated by cytoplasmic acidification or exposure to halothane like other connexins. Cx31.9 was able to form heterotypic channels with the highly voltage-sensitive Xenopus Cx38 (XenCx38), which provides an opportunity to study gating in heterotypic channels formed by hemichannels (connexons) composed of connexins with widely divergent properties. Thus Cx31.9 is a novel human connexin that forms channels with unique functional properties.

Heterotypic gap junction channel formation between heteromeric and homomeric Cx40 and Cx43 connexons

Recent evidence indicating formation of functional homomeric/heterotypic gap junction channels by connexin40 (Cx40) and connexin43 (Cx43) raises the question of whether data previously interpreted as support for heteromeric channel formation by these connexins might not instead reflect the activity of homomeric/heterotypic channels. To address this question and to further characterize the behavior of these channels, we used dual whole cell voltage-clamp techniques to examine the junctions formed between cells that express only Cx40 (Rin40) or Cx43 (Rin43) and compared the results with those obtained when either of these cell types was paired with cells that naturally express both connexins (A7r5 cells). Rin40/Rin43 cell pairs formed functional gap junctions that displayed a strongly asymmetric voltage-dependent gating response. Single-channel event amplitudes ranged between 34 and 150 pS, with 90- to 130-pS events predominating. A7r5/Rin43 and A7r5/Rin40 cell pairs had voltage-dependent gating responses that varied greatly, with most pairs demonstrating strong asymmetry. These cell pairs exhibited a variety of single-channel events that were not consistent with homomeric/homotypic Cx40 or Cx43 channels or homomeric/heterotypic Cx40/Cx43 channels. These data indicate that Cx40 and Cx43 form homomeric/heterotypic as well as heteromeric/heterotypic channels that display unique gating and conductance properties.

Opposing gates model for voltage gating of gap junction channels

Gap junctions are intercellular channels that link the cytoplasm of neighboring cells. Because a gap junction channel is composed of two connexons docking head-to-head with each other, the channel voltage-gating profile is symmetrical for homotypic channels made of two identical connexons (hemichannels) and asymmetric for the heterotypic channels made of two different connexons (i.e., different connexin composition). In this study we have developed a gating model that allows quantitative characterization of the voltage gating of homotypic and heterotypic channels. This model differs from the present model in use by integrating, rather than separating, the contributions of the voltage gates of the two member connexons. The gating profile can now be fitted over the entire voltage range, eliminating the previous need for data splicing and fusion of two hemichannel descriptions, which is problematic when dealing with heterotypic channels. This model also provides a practical formula to render quantitative several previously qualitative concepts, including a similarity principle for matching a voltage gate to its host connexon, assignment of gating polarity to a connexon, and the effect of docking interactions between two member connexons in an intact gap junction channel.

Molecular Determinants of Electrical Rectification of Single Channel Conductance in Gap Junctions Formed by Connexins 26 and 32

The fully open state of heterotypic gap junction channels formed by pairing cells expressing connexin 32 (Cx32) with those expressing connexin 26 (Cx26) rectifies in a way that cannot be predicted from the current–voltage (I–V) relation of either homotypic channel. Using a molecular genetic analysis, we demonstrate that charged amino acids positioned in the amino terminus (M1 and D2) and first extracellular loop (E42) are major determinants of the current–voltage relation of the fully open state of homotypic and heterotypic channels formed by Cx26 and Cx32. The observed I–V relations of wild-type and mutant channels were closely approximated by those obtained with the electrodiffusive model of Chen and Eisenberg (Chen, D., and R. Eisenberg. 1993. Biophys. J. 64:1405–1421), which solves the Poisson-Nernst-Plank equations in one dimension using charge distribution models inferred from the molecular analyses. The rectification of the Cx32/Cx26 heterotypic channel results from the asymmetry in the number and position of charged residues. The model required the incorporation of a partial charge located near the channel surface to approximate the linear I–V relation observed for the Cx32*Cx26E1 homotypic channel. The best candidate amino acid providing this partial charge is the conserved tryptophan residue (W3). Incorporation of the partial charge of residue W3 and the negative charge of the Cx32E41 residue into the charge profile used in the Poisson-Nernst-Plank model of homotypic Cx32 and heterotypic Cx26/Cx32 channels resulted in I–V relations that closely resembled the observed I–V relations of these channels. We further demonstrate that some channel substates rectify. We suggest that the conformational changes associated with transjunctional voltage (Vj)-dependent gating to these substates involves a narrowing of the cytoplasmic entry of the channel that increases the electrostatic effect of charges in the amino terminus. The rectification that is observed in the Cx32/Cx26 heterotypic channel is similar although less steep than that reported for some rectifying electrical synapses. We propose that a similar electrostatic mechanism, which results in rectification through the open and substates of heterotypic channels, is sufficient to explain the properties of steeply rectifying electrical synapses.