Projection structure of a N-terminal deletion mutant of connexin 26 channel with decreased central pore density

Gated gap junction channels are important cellular conduits for establishing and maintaining intercellular communication. The three-dimensional structure of a mutant human connexin 26 (Cx26M34A) by electron cryocrystallography revealed a plug-like density in the channel pore suggesting that physical blockage of the pore may be one mechanism of closure (Oshima et al. 2007, Proc Natl Acad Sci USA 104: 10034-10039). However, it remains to be determined what part of the sequence contributes to the plug. Here, we present the projection structure of an N-terminus deletion of Cx26M34A missing amino acids 2 to 7 (Cx26M34Adel2-7) crystallized in the same two-dimensional crystal form. A 10 A resolution projection map of Cx26M34Adel2-7 revealed that the plug density was dramatically reduced in comparison with that found in full-length Cx26 channel. The difference map between the deletion and full-length Cx26M34A channels strongly suggests that the N-terminus of connexin contributes to the plug for the physical closure of gap junction channels.     

Analysis of Four Connexin26 Mutant Gap Junctions and Hemichannels Reveals Variations in Hexamer Stability

Connexin26 is a ubiquitous gap junction protein that serves critical homeostatic functions. Four single-site mutations found in the transmembrane helices (M1-M4) cause different types of dysfunctional channels: 1), Cx26T135A in M3 produces a closed channel; 2), Cx26M34A in M1 severely decreases channel activity; 3), Cx26P87L in M2 has been implicated in defective channel gating; and 4), Cx26V84L in M2, a nonsyndromic deafness mutant, retains normal dye coupling and electrophysiological properties but is deficient in IP₃ transfer. These mutations do not affect Cx26 trafficking in mammalian cells, and make normal-appearing channels in baculovirus-infected Sf9 membranes when imaged by negative stain electron microscopy. Upon dodecylmaltoside solubilization of the membrane fraction, Cx26M34A and Cx26V84L are stable as hexamers or dodecamers, but Cx26T135A and Cx26P87L oligomers are not. This instability is also found in Cx26T135A and Cx26P87L hemichannels isolated from mammalian cells. In this work, coexpression of both wild-type Cx26 and Cx26P87L in Sf9 cells rescued P87L hexamer stability. Similarly, in paired Xenopus oocytes, coexpression with wild-type restored function. In contrast, the stability of Cx26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating.     

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

Recent evidence indicatingformation of functional homomeric/heterotypic gap junction channels byconnexin40 (Cx40) and connexin43 (Cx43) raises the question of whetherdata previously interpreted as support for heteromeric channelformation by these connexins might not instead reflect the activity ofhomomeric/heterotypic channels. To address this question and to furthercharacterize the behavior of these channels, we used dual whole cellvoltage-clamp techniques to examine the junctions formed between cellsthat express only Cx40 (Rin40) or Cx43 (Rin43) and compared the results with those obtained when either of these cell types was paired withcells that naturally express both connexins (A7r5 cells). Rin40/Rin43cell pairs formed functional gap junctions that displayed a stronglyasymmetric voltage-dependent gating response. Single-channel eventamplitudes ranged between 34 and 150 pS, with 90- to 130-pS eventspredominating. A7r5/Rin43 and A7r5/Rin40 cell pairs had voltage-dependent gating responses that varied greatly, with most pairsdemonstrating strong asymmetry. These cell pairs exhibited a variety ofsingle-channel events that were not consistent with homomeric/homotypicCx40 or Cx43 channels or homomeric/heterotypic Cx40/Cx43 channels.These data indicate that Cx40 and Cx43 form homomeric/heterotypic aswell as heteromeric/heterotypic channels that display unique gating andconductance properties.

     

Stoichiometry of transjunctional voltage-gating polarity reversal by a negative charge substitution in the amino terminus of a connexin32 chimera

Gap junctions are intercellular channels formed by the serial, head to head arrangement of two hemichannels. Each hemichannel is an oligomer of six protein subunits, which in vertebrates are encoded by the connexin gene family. All intercellular channels formed by connexins are sensitive to the relative difference in the membrane potential between coupled cells, the transjunctional voltage (Vj), and gate by the separate action of their component hemichannels (Harris, A.L., D.C. Spray, and M.V. Bennett. 1981. J. Gen. Physiol. 77:95-117). We reported previously that the polarity of Vj dependence is opposite for hemichannels formed by two closely related connexins, Cx32 and Cx26, when they are paired to form intercellular channels (Verselis, V.K., C.S. Ginter, and T.A. Bargiello. 1994. Nature. 368:348-351). The opposite gating polarity is due to a difference in the charge of the second amino acid. Negative charge substitutions of the neutral asparagine residue present in wild-type Cx32 (Cx32N2E or Cx32N2D) reverse the gating polarity of Cx32 hemichannels from closure at negative Vj to closure at positive Vj. In this paper, we further examine the mechanism of polarity reversal by determining the gating polarity of a chimeric connexin, in which the first extracellular loop (E1) of Cx32 is replaced with that of Cx43 (Cx43E1). The resulting chimera, Cx32*Cx43E1, forms conductive hemichannels when expressed in single Xenopus oocytes and intercellular channels in pairs of oocytes (Pfahnl, A., X.W. Zhou, R. Werner, and G. Dahl. 1997. Pflügers Arch. 433:733-779). We demonstrate that the polarity of Vj dependence of Cx32*Cx43E1 hemichannels in intercellular pairings is the same as that of wild-type Cx32 hemichannels and is reversed by the N2E substitution. In records of single intercellular channels, Vj dependence is characterized by gating transitions between fully open and subconductance levels. Comparable transitions are observed in Cx32*Cx43E1 conductive hemichannels at negative membrane potentials and the polarity of these transitions is reversed by the N2E substitution. We conclude that the mechanism of Vj dependence of intercellular channels is conserved in conductive hemichannels and term the process Vj gating. Heteromeric conductive hemichannels comprised of Cx32*Cx43E1 and Cx32N2E*Cx43E1 subunits display bipolar Vj gating, closing to substates at both positive and negative membrane potentials. The number of bipolar hemichannels observed in cells expressing mixtures of the two connexin subunits coincides with the number of hemichannels that are expected to contain a single oppositely charged subunit. We conclude that the movement of the voltage sensor in a single connexin subunit is sufficient to initiate Vj gating. We further suggest that Vj gating results from conformational changes in individual connexin subunits rather than by a concerted change in the conformation of all six subunits.     

Conformational changes in a pore-forming region underlie voltage-dependent “loop gating” of an unapposed connexin hemichannel

The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA–biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA–biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent “loop-gating” mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd²⁺ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd²⁺ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.     

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.     

Voltage-dependent conformational changes in connexin channels

Channels formed by connexins display two distinct types of voltage-dependent gating, termed V(j)- or fast-gating and loop- or slow-gating. Recent studies, using metal bridge formation and chemical cross-linking have identified a region within the channel pore that contributes to the formation of the loop-gate permeability barrier. The conformational changes are remarkably large, reducing the channel pore diameter from 15 to 20? to less than 4?. Surprisingly, the largest conformational change occurs in the most stable region of the channel pore, the 3(10) or parahelix formed by amino acids in the 42-51 segment. The data provide a set of positional constraints that can be used to model the structure of the loop-gate closed state. Less is known about the conformation of the V(j)-gate closed state. There appear to be two different mechanisms; one in which conformational changes in channel structure are linked to a voltage sensor contained in the N-terminus of Cx26 and Cx32 and a second in which the C-terminus of Cx43 and Cx40 may act either as a gating particle to block the channel pore or alternatively to stabilize the closed state. The later mechanism utilizes the same domains as implicated in effecting pH gating of Cx43 channels. It is unclear if the two V(j)-gating mechanisms are related or if they represent different gating mechanisms that operate separately in different subsets of connexin channels. A model of the V(j)-closed state of Cx26 hemichannel that is based on the X-ray structure of Cx26 and electron crystallographic structures of a Cx26 mutation suggests that the permeability barrier for V(j)-gating is formed exclusively by the N-terminus, but recent information suggests that this conformation may not represent a voltage-closed state. Closed state models are considered from a thermodynamic perspective based on information from the 3.5? Cx26 crystal structure and molecular dynamics (MD) simulations. The applications of computational and experimental methods to define the path of allosteric molecular transitions that link the open and closed states are discussed. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Specificity of the connexin W3/4 locus for functional gap junction formation

The N-terminal (NT) domain of the connexins forms an essential transjunctional voltage (V_j) sensor and pore-forming domain that when truncated, tagged, or mutated often leads to formation of a nonfunctional channel. The NT domain is relatively conserved among the connexins though the α- and δ-group connexins possess a G2 residue not found in the β- and γ-group connexins. Deletion of the connexin40 G2 residue (Cx40G2Δ) affected the V_j gating, increased the single channel conductance (γ_j), and decreased the relative K⁺/Cl^? permeability (P_K/P_Cl) ratio of the Cx40 gap junction channel. The conserved α/β-group connexin D2/3 and W3/4 loci are postulated to anchor the NT domain within the pore via hydrophilic and hydrophobic interactions with adjacent connexin T5 and M34 residues. Cx40D3N and D3R mutations produced limited function with progressive reductions in V_j gating and noisy low γ_j gap junction channels that reduced the γ_j of wild-type Cx40 channels from 150 pS to < 50 pS when coexpressed. Surprisingly, hydrophobic Cx40 W4F and W4Y substitution mutations were not compatible with function despite their ability to form gap junction plaques. These data are consistent with minor and major contributions of the G2 and D3 residues to the Cx40 channel pore structure, but not with the postulated hydrophobic W4 intermolecular interactions. Our results indicate an absolute requirement for an amphipathic W3/4 residue that is conserved among all α/β/δ/γ-group connexins. We alternatively hypothesize that the connexin D2/3-W3/4 locus interacts with the highly conserved FIFR M1 motif to stabilize the NT domain within the pore.     

Site-directed mutagenesis reveals putative regions of protein interaction within the transmembrane domains of connexins

Through cysteine-scanning mutagenesis, the authors have compared sites within the transmembrane domains of two connexins, one from the alpha-class (Cx50) and one from the beta-class (Cx32), where amino acid substitution disrupts the function of gap junction channels. In Cx32, 11 sites resulted in no channel function, or an aberrant voltage gating phenotype referred to as "reverse gating," whereas in Cx50, 7 such sites were identified. In both connexins, the sites lie along specific faces of transmembrane helices, suggesting that these may be sites of transmembrane domain interactions. In Cx32, one broad face of the M1 transmembrane domain and a narrower, polar face of M3 were identified, including one site that was shown to come into close apposition with M4 in the closed state. In Cx50, the same face of M3 was identified, but sensitive sites in M1 differed from Cx32. Many fewer sites in M1 disrupted channel function in Cx50, and those that did were on a different helical face to the sensitive sites in Cx32. A more in depth study of two sites in M1 and M2 of Cx32 showed that side-chain length or branching are important for maintenance of normal channel behavior, consistent with this being a site of transmembrane domain interaction.     

Projection structure of channelrhodopsin-2 at 6 Å resolution by electron crystallography

Channelrhodopsin-2 (ChR2) is the prototype of a new class of light-gated ion channels that is finding widespread applications in optogenetics and biomedical research. We present a  6-Å projection map of ChR2, obtained by cryo-electron microscopy of two-dimensional crystals grown from pure, heterologously expressed protein. The map shows that ChR2 is the same dimer with non-crystallographic 2-fold symmetry in three different membrane crystals. This is consistent with biochemical analysis, which shows a stable dimer in detergent solution. Comparison to the projection map to bacteriorhodopsin indicates a similar structure of seven transmembrane alpha helices. Based on the projection map and sequence alignments, we built a homology model of ChR2 that potentially accounts for light-induced channel gating. Although a monomeric channel is not ruled out, comparison to other membrane channels and transporters suggests that the ChR2 channel is located at the dimer interface on the 2-fold axis, lined by transmembrane helices 3 and 4.     

Chemical gating of gap junction channels

Chemical gating of gap junction channels is a complex phenomenon that may involve intra- and intermolecular interactions among connexin domains and a cytosolic molecule (calmodulin?) that may function as channel plug. This article focuses on the methodology we have employed for studying the molecular basis of chemical gating by lowered cytosolic pH. Our approach has combined molecular genetics and biophysics, using exposure to 100% CO(2) for assaying chemical gating efficiency. Chimeras of connexin 32 (Cx32) and connexin 38 (Cx38) and Cx32 mutants modified at residues of the cytoplasmic loop, the initial C-terminus domain, or both have been expressed in Xenopus oocytes, and channel expression and gating have been tested electrophysiologically by double voltage clamp. In addition, various channel forms, including homotypic, heterotypic, and heteromeric channel combinations, have been evaluated for chemical gating sensitivity.     

The M34A mutant of Connexin26 reveals active conductance states in pore-suspending membranes

Connexin26 (Cx26) is a member of the connexin family, the building blocks for gap junction intercellular channels. These dodecameric assemblies are involved in gap junction-mediated cell–cell communication allowing the passage of ions and small molecules between two neighboring cells. Mutations in Cx26 lead to the disruption of gap junction-mediated intercellular communication with consequences such as hearing loss and skin disorders. We show here that a mutant of Cx26, M34A, forms an active hemichannel in lipid bilayer experiments. A comparison with the Cx26 wild-type is presented. Two different techniques using micro/nano-structured substrates for the formation of pore-suspending lipid membranes are used. We reconstituted the Cx26 wild-type and Cx26M34A into artificial lipid bilayers and observed single channel activity for each technique, with conductance levels of around 35, 70 and 165 pS for the wild-type. The conductance levels of Cx26M34A were found at around 45 and 70 pS.     

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 (V(j))-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.     

Gap Junction Assembly: Multiple Connexin Fluorophores Identify Complex Trafficking Pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Structure and closure of connexin gap junction channels

Connexin gap junctions comprise assembled channels penetrating two plasma membranes for which gating regulation is associated with a variety of factors, including voltage, pH, Ca²⁺, and phosphorylation. Functional studies have established that various parts of the connexin peptides are related to channel closure and electrophysiology studies have provided several working models for channel gating. The corresponding structural models supporting these findings, however, are not sufficient because only small numbers of closed connexin structures have been reported. To fully understand the gating mechanisms, the channels should be visualized in both the open and closed states. Electron crystallography and X-ray crystallography studies recently revealed three-dimensional structures of connexin channels in a couple of states in which the main difference is the conformation of the N-terminal domain, which have helped to clarify the structure in regard to channel closure. Here the closure models for connexin gap junction channels inferred from structural and functional studies are described in the context of each domain of the connexin protein associated with gating modulation.     

Gap Junction Assembly: Multiple Connexin Fluorophores Identify Complex Trafficking Pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Gap junction assembly: multiple connexin fluorophores identify complex trafficking pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Altered permeability and modulatory character of connexin channels during mammary gland development

Abrupt developmental changes occur in structural form and function of connexin (Cx) channels in the mouse mammary gland. Microarray study shows that the principal connexin isoform in epithelial cells during pregnancy is Cx26, up-regulated and persisting from the virgin. After parturition, there is rapid induction of Cx32. In epithelial plasma membranes, size exclusion chromatography reveals that Cx32 organizes initially with Cx26 as heteromeric (Cx26-Cx32) hemichannels and later in heteromeric and homomeric Cx32 channels. Dramatic alterations of connexin channel function following these developmental changes in channel composition are characterized using native channels reconstituted into liposomes. Changes to channel stoichiometry increase the allowable physical size limits of permeant after parturition; the new Cx32 channels are wider than channels containing Cx26. Most remarkably, heteromeric Cx26-Cx32 channels are selectively permeability to adenosine 3',5' cyclic phosphate (cAMP), guanosine 3',5' cyclic phosphate (cGMP), and inositol 1,4,5-triphosphate (IP(3)), whereas homomeric channels are not. Homomeric Cx26 and heteromeric channels with high Cx26/Cx32 stoichiometry are also inhibited by taurine, an osmolyte playing a key role in milk protein synthesis. Taurine effect is reduced where heteromeric channels contain Cx32 > Cx26 and eliminated when channels contain only Cx32. Connexin channel stoichiometry, permeability, and chemical gating character change in precisely the desired fashion after parturition to maximize molecular and electrical coupling to support coordinated milk secretion.     

Roles of Met-34, Cys-64, and Arg-75 in the assembly of human connexin 26. Implication for key amino acid residues for channel formation and function

Connexins form a family of membrane proteins that assemble into communication channels and directly connect the cytoplasms of adjoining cells. Malfunctioning of connexin channels often cause disease, such as the mutations M34T and R75W in human connexin 26, which are associated with hereditary deafness. Another residue known to be essential for normal channel activity in the connexin is Cys-64. To obtain structural and functional insights of connexin 26, we studied the roles of these three residues by expressing mutant connexins in insect Sf9 and HeLa cells. The M34T and M34A mutants both formed gap junction plaques, but dye transfer assays showed that the M34A mutant had a significantly reduced permeability, suggesting that for proper channel function a side chain of adequate size is required at this position. We propose that Met-34 is located in the innermost helix of the channel, where it ensures a fully open channel structure via interactions with other transmembrane helices. Gap junction channels formed by the R75W and R75D mutants dissociated upon solubilization in dodecyl maltoside, whereas the R75A mutant remained hexameric. All gap junctions formed by Arg-75 mutants also showed only negligible activity in dye transfer experiments. These results suggest that residue Arg-75 plays a role in subunit interactions needed to retain a functional and stable connexin hexamer. The C64S mutant was suggested to be defective in oligomerization and/or protein folding even in the presence of wild-type connexin.     

The role of a conserved proline residue in mediating conformational changes associated with voltage gating of Cx32 gap junctions.

We have explored the role of a proline residue located at position 87 in the second transmembrane segment (TM2) of gap junctions in the mechanism of voltage-dependent gating of connexin32 (Cx32). Substitution of this proline (denoted Cx32P87) with residues G, A, or V affects channel function in a progressive manner consistent with the expectation that a proline kink (PK) motif exists in the second transmembrane segment (TM2) of this connexin. Mutations of the preceding threonine residue T86 to S, A, C, V, N, or L shift the conductance-voltage relation of wild-type Cx32, such that the mutated channels close at smaller transjunctional voltages. The observed shift in voltage dependence is consistent with a reduction in the open probability of the mutant hemichannels at a transjunctional voltage (Vj) of 0 mV. In both cases in which kinetics were examined, the time constants for reaching steady state were faster for T86N and T86A than for wild type at comparable voltages, suggesting that the T86 mutations cause the energetic destabilization of the open state relative to the other states of the channel protein. The structural underpinnings of the observed effects were explored with Monte Carlo simulations. The conformational space of TM2 helices was found to differ for the T86A, V, N, and L mutants, which produce a less bent helix ( approximately 20 degrees bend angle) compared to the wild type, which has a approximately 37 degrees bend angle. The greater bend angle of the wild-type helix reflects the propensity of the T86 residue to hydrogen bond with the backbone carbonyl of amino acid residue I82. The relative differences in propensity for hydrogen bonding of the mutants relative to the wild-type threonine residue in the constructs we studied (T86A, V, N, L, S, and C) correlate with the shift in the conductance-voltage relation observed for T86 mutations. The data are consistent with a structural model in which the open conformation of the Cx32 channel corresponds to a more bent TM2 helix, and the closed conformation corresponds to a less bent helix. We propose that the modulation of the hydrogen-bonding potential of the T86 residue alters the bend angle of the PK motif and mediates conformational changes between open and closed channel states.