Calcium-dependent open/closed conformations and interfacial energy maps of reconstituted hemichannels

Using an atomic force microscope, we have studied three-dimensional molecular topography and calcium-sensitive conformational changes of individual hemichannels. Full-length (non-truncated) Cx43 hemichannels (connexons), when reconstituted in lipid bilayer, appear as randomly distributed individual particles and clusters. They show a lack of preferential orientation of insertion into lipid membrane; in a single bilayer, connexons with protrusion of either the extracellular face or the large non-truncated cytoplasmic face are observed. Extracellular domains of these undocked hemichannels are structurally different from hemichannels in the docked gap junctional plaques examined after their exposure by force dissection or chemical dissection. Calcium induced a reversible change in the extracellular pore diameter. Hemichannels imaged in a physiological buffer with 1.8 mm Ca(+2) had the pore diameter of approximately 1.8 nm, consistent with the closed channel conformation. Reducing Ca(+2) concentration to approximately 1.4, 1, and 0 mm, which changes hemichannels from the closed to open conformation, increased the pore diameter to approximately 2.5 nm for approximately 27, 74, and 100% of hemichannels, respectively. Thus, open/close probability of the hemichannel appears to be [Ca(2+)]-dependent. Computational analysis of the atomic force microscopy phase mode imaging reveals a significantly higher interfacial energy for open hemichannels that results from the interactions between the atomic force microscope probe and the hydrophobic domains. Thus, hydrophobic extracellular domains of connexins regulate calcium-dependent conformational changes.     

Mechanism for modulation of gating of connexin26-containing channels by taurine

The mechanisms of action of endogenous modulatory ligands of connexin channels are largely unknown. Previous work showed that protonated aminosulfonates (AS), notably taurine, directly and reversibly inhibit homomeric and heteromeric channels that contain Cx26, a widely distributed connexin, but not homomeric Cx32 channels. The present study investigated the molecular mechanisms of connexin channel modulation by taurine, using hemichannels and junctional channels composed of Cx26 (homomeric) and Cx26/Cx32 (heteromeric). The addition of a 28-amino acid "tag" to the carboxyl-terminal domain (CT) of Cx26 (Cx26(T)) eliminated taurine sensitivity of homomeric and heteromeric hemichannels in cells and liposomes. Cleavage of all but four residues of the tag (Cx26(Tc)) resulted in taurine-induced pore narrowing in homomeric hemichannels, and restored taurine inhibition of heteromeric hemichannels (Cx26(Tc)/Cx32). Taurine actions on junctional channels were fully consistent with those on hemichannels. Taurine-induced inhibition of Cx26/Cx32(T) and nontagged Cx26 junctional channels was blocked by extracellular HEPES, a blocker of the taurine transporter, confirming that the taurine-sensitive site of Cx26 is cytoplasmic. Nuclear magnetic resonance of peptides corresponding to Cx26 cytoplasmic domains showed that taurine binds to the cytoplasmic loop (CL) and not the CT, and that the CT and CL directly interact. ELISA showed that taurine disrupts a pH-dependent interaction between the CT and the CT-proximal half of the CL. These studies reveal that AS disrupt a pH-driven cytoplasmic interdomain interaction in Cx26-containing channels, causing closure, and that the Cx26CT has a modulatory role in Cx26 function.     

Conformational changes in surface structures of isolated connexin 26 gap junctions

Gap junction channels mediate communication between adjacent cells. Using atomic force microscopy (AFM), we have imaged conformational changes of the cytoplasmic and extracellular surfaces of native connexin 26 gap junction plaques. The cytoplasmic domains of the gap junction surface, imaged at submolecular resolution, form a hexameric pore protruding from the membrane bilayer. Exhibiting an intrinsic flexibility, these cytoplasmic domains, comprising the C-terminal connexin end, reversibly collapse by increasing the forces applied to the AFM stylus. The extracellular connexon surface was imaged after dissection of the gap junction with the AFM stylus. Upon injection of Ca(2+) into the buffer solution, the extracellular channel entrance reduced its diameter from 1.5 to 0.6 nm, a conformational change that is fully reversible and specific among the divalent cations tested. Ca(2+) had a profound effect on the cytoplasmic surface also, inducing the formation of microdomains. Consequently, the plaque height increased by 0.6 nm to 18 nm. This suggests that calcium ions induce conformational changes affecting the structure of both the hemichannels and the intact channels forming cell-cell contacts.     

Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.     

Conductance and permeability of the residual state of connexin43 gap junction channels

We used cell lines expressing wild-type connexin43 and connexin43 fused with the enhanced green fluorescent protein (Cx43-EGFP) to examine conductance and perm-selectivity of the residual state of Cx43 homotypic and Cx43/Cx43-EGFP heterotypic gap junction channels. Each hemichannel in Cx43 cell-cell channel possesses two gates: a fast gate that closes channels to the residual state and a slow gate that fully closes channels; the transjunctional voltage (V(j)) closes the fast gate in the hemichannel that is on the relatively negative side. Here, we demonstrate macroscopically and at the single-channel level that the I-V relationship of the residual state rectifies, exhibiting higher conductance at higher V(j)s that are negative on the side of gated hemichannel. The degree of rectification increases when Cl(-) is replaced by Asp(-) and decreases when K(+) is replaced by TEA(+). These data are consistent with an increased anionic selectivity of the residual state. The V(j)-gated channel is not permeable to monovalent positively and negatively charged dyes, which are readily permeable through the fully open channel. These data indicate that a narrowing of the channel pore accompanies gating to the residual state. We suggest that the fast gate operates through a conformational change that introduces positive charge at the cytoplasmic vestibule of the gated hemichannel, thereby producing current rectification, increased anionic selectivity, and a narrowing of channel pore that is largely responsible for reducing channel conductance and restricting dye transfer. Consequently, the fast V(j)-sensitive gating mechanism can serve as a selectivity filter, which allows electrical coupling but limits metabolic communication.     

Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation

pH is a potent modulator of gap junction (GJ) mediated cell-cell communication. Mechanisms proposed for closure of GJ channels by acidification include direct actions of H+ on GJ proteins and indirect actions mediated by soluble intermediates. Here we report on the effects of acidification on connexin (Cx)46 cell-cell channels expressed in Neuro-2a cells and Cx46 hemichannels expressed in Xenopus oocytes. Effects of acidification on hemichannels were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the exposed hemichannel face. Both types of Cx46 channel were found to be sensitive to cytoplasmic pH, and two effects were evident. A rapid and reversible closure was reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible loss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at pH 6.4. Hemichannels remained sensitive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was maintained near resting ( approximately 10(-7) M) levels. Thus, Cx46 hemichannel pH gating does not depend on cytoplasmic intermediates or a rise in [Ca2+]. Rapid application of low pH to the cytoplasmic face of open hemichannels resulted in a minimum latency to closure near zero, indicating that Cx46 hemichannels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible from the cytoplasmic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+ permeation through the pore to reach an internal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is located directly on Cx46 near the pore entrance on the cytoplasmic side.     

Modulation of enrofloxacin binding in OmpF by Mg2+ as revealed by the analysis of fast flickering single-porin current

The anti-malarial drug quinine and its quaternary derivative N-benzylquininium (BQ(+)) have been shown to inhibit gap junction (GJ) channels with specificity for Cx50 over its closely related homologue Cx46. Here, we examined the mechanism of BQ(+) action using undocked Cx46 and Cx50 hemichannels, which are more amenable to analyses at the single-channel level. We found that BQ(+) (300 μM-1 mM) robustly inhibited Cx50, but not Cx46, hemichannel currents, indicating that the Cx selectivity of BQ(+) is preserved in both hemichannel and GJ channel configurations. BQ(+) reduced Cx50 hemichannel open probability (P(o)) without appreciably altering unitary conductance of the fully open state and was effective when added from either extracellular or cytoplasmic sides. The reductions in P(o) were dependent on BQ(+) concentration with a Hill coefficient of 1.8, suggesting binding of at least two BQ(+) molecules. Inhibition by BQ(+) was voltage dependent, promoted by hyperpolarization from the extracellular side and conversely by depolarization from the cytoplasmic side. These results are consistent with binding of BQ(+) in the pore. Substitution of the N-terminal (NT) domain of Cx46 into Cx50 significantly impaired inhibition by BQ(+). The NT domain contributes to the formation of the wide cytoplasmic vestibule of the pore and, thus, may contribute to the binding of BQ(+). Single-channel analyses showed that BQ(+) induced transitions that did not resemble pore block, but rather transitions indistinguishable from the intrinsic gating events ascribed to loop gating, one of two mechanisms that gate Cx channels. Moreover, BQ(+) decreased mean open time and increased mean closed time, indicating that inhibition consists of an increase in hemichannel closing rate as well as a stabilization of the closed state. Collectively, these data suggest a mechanism of action for BQ(+) that involves modulation loop gating rather than channel block as a result of binding in the NT domain.     

Extracellular loop cysteine mutant of cx37 fails to suppress proliferation of rat insulinoma cells

Although a functional pore domain is required for connexin 37 (Cx37)–mediated suppression of rat insulinoma (Rin) cell proliferation, it is unknown whether functional hemichannels would be sufficient or if Cx37 gap junction channels are required for growth suppression. To test this possibility, we targeted extracellular loop cysteines for mutation, expecting that the mutated protein would retain hemichannel, but not gap junction channel, functionality. Cysteines at positions 61 and 65 in the first extracellular loop of Cx37 were mutated to alanine and the mutant protein (Cx37-C61,65A) expressed in Rin cells. Although the resulting iRin37-C61,65A cells expressed the mutant protein comparably to Cx37 wild-type (Cx37-WT)–expressing Rin cells (iRin37), Cx37-C61,65A expression did not suppress the proliferation of Rin cells. As expected, iRin37-C61,65A cells did not form functional gap junction channels. However, functional hemichannels also could not be detected in iRin37-C61,65A cells by either dye uptake or electrophysiological approaches. Thus, failure of Cx37-C61,65A to suppress the proliferation of Rin cells is consistent with previous data demonstrating the importance of channel functionality to Cx37’s growth-suppressive function. Moreover, failure of the Cx37-C61,65A hemichannel to function, even in low external calcium, emphasizes the importance of extracellular loop cysteines not only in hemichannel docking but also in determining the ability of the hemichannel to adopt a closed configuration that can open in response to triggers, such as low external calcium, effective at opening Cx37-WT hemichannels.     

Gating of Connexin Channels by transjunctional-voltage: Conformations and models of open and closed states

Voltage is an important physiologic regulator of channels formed by the connexin gene family. Connexins are unique among ion channels in that both plasma membrane inserted hemichannels (undocked hemichannels) and intercellular channels (aggregates of which form gap junctions) have important physiological roles. The hemichannel is the fundamental unit of gap junction voltage-gating. Each hemichannel displays two distinct voltage-gating mechanisms that are primarily sensitive to a voltage gradient formed along the length of the channel pore (the transjunctional voltage) rather than sensitivity to the absolute membrane potential (V_m or V_i-o). These transjunctional voltage dependent processes have been termed V_j- or fast-gating and loop- or slow-gating. Understanding the mechanism of voltage-gating, defined as the sequence of voltage-driven transitions that connect open and closed states, first and foremost requires atomic resolution models of the end states. Although ion channels formed by connexins were among the first to be characterized structurally by electron microscopy and x-ray diffraction in the early 1980′s, subsequent progress has been slow. Much of the current understanding of the structure-function relations of connexin channels is based on two crystal structures of Cx26 gap junction channels. Refinement of crystal structure by all-atom molecular dynamics and incorporation of charge changing protein modifications has resulted in an atomic model of the open state that arguably corresponds to the physiologic open state. Obtaining validated atomic models of voltage-dependent closed states is more challenging, as there are currently no methods to solve protein structure while a stable voltage gradient is applied across the length of an oriented channel. It is widely believed that the best approach to solve the atomic structure of a voltage-gated closed ion channel is to apply different but complementary experimental and computational methods and to use the resulting information to derive a consensus atomic structure that is then subjected to rigorous validation. In this paper, we summarize our efforts to obtain and validate atomic models of the open and voltage-driven closed states of undocked connexin hemichannels.This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

The connexin 43 C-terminus: A tail of many tales

Connexins are chordate gap junction channel proteins that, by enabling direct communication between the cytosols of adjacent cells, create a unique cell signalling network. Gap junctional intercellular communication (GJIC) has important roles in controlling cell growth and differentiation and in tissue development and homeostasis. Moreover, several non-canonical connexin functions unrelated to GJIC have been discovered. Of the 21 members of the human connexin family, connexin 43 (Cx43) is the most widely expressed and studied. The long cytosolic C-terminus (CT) of Cx43 is subject to extensive post-translational modifications that modulate its intracellular trafficking and gap junction channel gating. Moreover, the Cx43 CT contains multiple domains involved in protein interactions that permit crosstalk between Cx43 and cytoskeletal and regulatory proteins. These domains endow Cx43 with the capacity to affect cell growth and differentiation independently of GJIC. Here, we review the current understanding of the regulation and unique functions of the Cx43 CT, both as an essential component of full-length Cx43 and as an independent signalling hub. We highlight the complex regulatory and signalling networks controlled by the Cx43 CT, including the extensive protein interactome that underlies both gap junction channel-dependent and -independent functions. We discuss these data in relation to the recent discovery of the direct translation of specific truncated forms of Cx43. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Voltage-dependent gating of the Cx32*43E1 hemichannel: Conformational changes at the channel entrances

Voltage is an important parameter that regulates the open probability of both intercellular channels (gap junctions) and undocked hemichannels formed by members of the connexin gene family. All connexin channels display two distinct voltage-gating processes, termed loop- or slow-gating and V_j- or fast-gating, which are intrinsic hemichannel properties. Previous studies have established that the loop-gate permeability barrier is formed by a large conformational change that reduces pore diameter in a region of the channel pore located at the border of the first transmembrane domain and first extracellular loop (TM1/E1), the parahelix (residues 42–51). Here, we use cadmium metal bridge formation to measure conformational changes reported by substituted cysteines at loci demarcating the intracellular (E109 and L108) and extracellular (Q56) entrance of hemichannels formed by the Cx32 chimera (Cx32*43E1). The results indicate that the intracellular pore entrance narrows from ∼15 Å to ∼10 Å with loop-gate but not apparently with V_j-gate closure. The extracellular entrance does not appear to undergo large conformational changes with either voltage-gating process. The results presented here combined with previous studies suggest that the loop-gate permeability is essentially focal, in that conformational changes in the parahelix but not the intracellular entrance are sufficient to prevent ion flux.     

Intracellular calcium changes trigger connexin 32 hemichannel opening

Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration ([Ca2+]i) triggers hemichannel opening, making use of peptides that are identical to a short amino-acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)-expressing cells showed that an increase in [Ca2+]i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on [Ca2+]i elevation within a window situated around 500 nM. Our results indicate that [Ca2+]i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.     

Emerging issues of connexin channels: biophysics fills the gap

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)     

Influence of V5/6-His Tag on the Properties of Gap Junction Channels Composed of Connexin43, Connexin40 or Connexin45

HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V _j-sensitive gating of I _j (V _j, gap junction voltage; I _j, gap junction current), (2) contribution and (3) kinetics of I _j deactivation and (4) single-channel conductance. The first three reflect alterations of fast V _j gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological properties.     

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size.     

Connexin channels, connexin mimetic peptides and ATP release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP(3) elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.     

Two-dimensional kinetics of inter-connexin interactions from single-molecule force spectroscopy

Gap junction channels are intercellular channels that form by docking the extracellular loops of connexin protein subunits. While the structure and function of gap junctions as intercellular channels have been characterized using different techniques, the physics of the inter-connexin interaction remain unknown. Moreover, as far as we know, the capacity of gap junction channels to work as adhesion complexes supporting pulling forces has not yet been quantitatively addressed. We report the first quantitative characterization of the kinetics and binding strength of the interaction of a short peptide mimicking extracellular loop 2 of Cx26 with membrane-reconstituted Cx26, combining the imaging and force spectroscopy capabilities of atomic force microscopy. The fast dissociation rate inferred a dynamic bond, while the slow association rate reflected the reduced flexibility and small size of extracellular loops. Our results propose the gap junction channel as an adhesion complex that associates slowly and dissociates fast at low force but is able to support important pulling forces in its native, hexameric form.     

Phosphorylation of connexin 50 by protein kinase A enhances gap junction and hemichannel function

Phosphorylation of connexins is an important mechanism regulating gap junction channels. However, the role(s) of connexin (Cx) phosphorylation in vivo are largely unknown. Here, we showed by mass spectrometry that Ser-395 in the C terminus of chicken Cx50 was phosphorylated in the lens. Ser-395 is located within a PKA consensus site. Analyses of Cx50 phosphorylation by two-dimensional thin layer chromatography tryptic phosphopeptide profiles suggested that Ser-395 was targeted by PKA in vivo. PKA activation increased both gap junction dye coupling and hemichannel dye uptake in a manner not involving increases in total Cx50 expression or relocation to the cell surface or gap junctional plaques. Single channel recordings indicated PKA enhanced transitions between the closed and ～200-pS open state while simultaneously reducing transitions between this open state and a ～65-pS subconductance state. The mutation of Ser-395 to alanine significantly attenuated PKA-induced increases in dye coupling and uptake by Cx50. However, channel records indicated that phosphorylation at this site was unnecessary for enhanced transitions between the closed and ～200-pS conductance state. Together, these results suggest that Cx50 is phosphorylated in vivo by PKA at Ser-395 and that this event, although unnecessary for PKA-induced alterations in channel conductance, promotes increased dye permeability of Cx50 channels, which plays an important role in metabolic coupling and transport in lens fibers.     

Connexin Channels, Connexin Mimetic Peptides and ATP Release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP₃ elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.