Channel Behavior and Voltage Gating of a Cx43 Mutant Simulating Preconditioning.

Background: Ischemic preconditioning induces lateralization and dephosphorylation of Connexin 43 (Cx43). However, the Cx43 protein that remains at intercalated disks may be phosphorylated by casein kinase 1 (CK1) and protein kinase C (PKC), and both kinases provide cardioprotection from further ischemic injury. Here we explore the channel characteristics of a Cx43 mutant mimicking preconditioning by CK1 and PKC phosphorylation. Materials and Methods: Whole-cell patch-clamp recordings were performed in cells expressing the mutant Cx43pc (S325,328,330,368D, S365A-Cx43), and the connexin electrical behavior was analyzed at the single channel and macroscopic level. Results: Cx43pc hemichannels opened readily, whereas gap junctions channels displayed amplitudes between the wild-type and CK1 phosphorylated forms, and weaker voltage gating than either counterpart. Conclusions: Ischemic preconditioning and the ensuing phosphorylation of Cx43 by PKC may render junctional channels insensitive to transjunctional voltages, allowing the preservation of intercellular communication in ischemic conditions.

The Role of Gap Junctions Dysfunction in the Development of Cataracts: From Loss of Cell-to-Cell Transfer to Blurred Vision-Review.

Mutations of lens connexins are linked to congenital cataracts. However, the role of connexin mutations in the development of age-related lens opacification remains largely unknown. Here, we present a focused review of the literature on lens organization and factors associated with cataract development. Several lines of evidence indicate that disturbances of the lens circulation by dysfunctional connexin channels, and/or accumulation of protein damage due to oxidative stress, are key factors in cataract development. Phosphorylation by protein kinase A improves the permeability of connexins channels to small molecules and mitigates the lens clouding induced by oxidative stress. We conclude (1) that connexin channels are central to the lens circulation and (2) that their permeability to antioxidant molecules contributes to the maintenance of lens transparency.

Patch clamp studies on TRPV4-dependent hemichannel activation in lens epithelium.

ATP release from the lens via hemichannels has been explained as a response to TRPV4 activation when the lens is subjected to osmotic swelling. To explore the apparent linkage between TRPV4 activation and connexin hemichannel opening we performed patch-clamp recordings on cultured mouse lens epithelial cells exposed to the TRPV4 agonist GSK1016790A (GSK) in the presence or absence of the TRPV4 antagonist HC067047 (HC). GSK was found to cause a fast, variable and generally large non-selective increase of whole cell membrane conductance evident as a larger membrane current (Im) over a wide voltage range. The response was prevented by HC. The GSK-induced Im increase was proportionally larger at negative voltages and coincided with fast depolarization and the simultaneous disappearance of an outward current, likely a K+ current. The presence of this outward current in control conditions appeared to be a reliable predictor of a cell's response to GSK treatment. In some studies, recordings were obtained from single cells by combining cell-attached and whole-cell patch clamp configurations. This approach revealed events with a channel conductance 180-270 pS following GSK application through the patch pipette on the cell-attached side. The findings are consistent with TRPV4-dependent opening of Cx43 hemichannels.

MicroRNA-mediated downregulation of K+ channels in pulmonary arterial hypertension.

Downregulated expression of K+ channels and decreased K+ currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K+ channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3'-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K+ channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K+ channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K+ currents and downregulated voltage-gated K+ channel 1.5 (KV1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K+ channel function and KV1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca2+-activated K+ (BKCa) channel currents and downregulated BKCa channel ß1 subunit (BKCaß1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BKCa channel activity and BKCaß1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of KV and BKCa channels in PASMC from patients with IPAH.

The lipidated connexin mimetic peptide SRPTEKT-Hdc is a potent inhibitor of Cx43 channels with specificity for the pS368 phospho-isoform.

Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) have been used as reversible, Cx-specific blockers of hemichannel (HCh) and gap junction channel (GJCh) function. These blockers typically require high concentrations (~5 µM, <1 h for HCh; ~100 µM, >1 h for GJCh) to achieve inhibition. We have shown that addition of a hexadecyl (Hdc) lipid tail to the conserved SRPTEKT peptide sequence (SRPTEKT-Hdc) results in a novel, highly efficacious, and potent inhibitor of mechanically induced Ca2+-wave propagation (IC50 64.8 pM) and HCh-mediated dye uptake (IC50 45.0 pM) in Madin-Darby canine kidney cells expressing rat Cx43 (MDCK43). The lack of similar effect on dye coupling (NBD-MTMA) suggested channel conformation-specific inhibition. Here we report that SRPTEKT-Hdc inhibition of Ca2+-wave propagation, dye coupling, and dye uptake depended on the functional configuration of Cx43 as determined by phosphorylation at serine 368 (S368). Ca2+-wave propagation was enhanced in MDCK cells expressing single-site mutants of Cx43 that mimicked (MDCK43-S368D) or favored (MDCK43-S365A) phosphorylation at S368. Furthermore, SRPTEKT-Hdc potently inhibited GJCh-mediated Ca2+-wave propagation (IC50 230.4 pM), dye coupling, and HCh-mediated dye uptake in MDCK43-S368D and -S365A cells. In contrast, Ca2+-wave propagation, dye coupling, and dye uptake were largely unaffected (IC50 12.3 µM) by SRPTEKT-Hdc in MDCK43-S368A and -S365D cells, mutations that mimic or favor dephosphorylation at S368. Together, these data indicate that SRPTEKT-Hdc is a potent inhibitor of physiological Ca2+-wave signaling mediated specifically by the pS368 phosphorylated form of Cx43.

Cx43 Channel Gating and Permeation: Multiple Phosphorylation-Dependent Roles of the Carboxyl Terminus.

Connexin 43 (Cx43), a gap junction protein seemingly fit to support cardiac impulse propagation and synchronic contraction, is phosphorylated in normoxia by casein kinase 1 (CK1). However, during cardiac ischemia or pressure overload hypertrophy, this phosphorylation fades, Cx43 abundance decreases at intercalated disks and increases at myocytes' lateral borders, and the risk of arrhythmia rises. Studies in wild-type and transgenic mice indicate that enhanced CK1-phosphorylation of Cx43 protects from arrhythmia, while dephosphorylation precedes arrhythmia vulnerability. The mechanistic bases of these Cx43 (de)phosphoform-linked cardiac phenotypes are unknown. We used patch-clamp and dye injection techniques to study the channel function (gating, permeability) of Cx43 mutants wherein CK1-targeted serines were replaced by aspartate (Cx43-CK1-D) or alanine (Cx43-CK1-A) to emulate phosphorylation and dephosphorylation, respectively. Cx43-CK1-D, but not Cx43-CK1-A, displayed high Voltage-sensitivity and variable permselectivity. Both mutants showed multiple channel open states with overall increased conductivity, resistance to acidification-induced junctional uncoupling, and hemichannel openings in normal external calcium. Modest differences in the mutant channels' function and regulation imply the involvement of dissimilar structural conformations of the interacting domains of Cx43 in electrical and chemical gating that may contribute to the divergent phenotypes of CK1-(de)phospho-mimicking Cx43 transgenic mice and that may bear significance in arrhythmogenesis.

Determinants of Cx43 Channel Gating and Permeation: The Amino Terminus.

Separate connexin domains partake in proposed gating mechanisms of gap junction channels. The amino-terminus (NT) domains, which contribute to voltage sensing, may line the channel's cytoplasmic-facing funnel surface, stabilize the channel's overall structure through interactions with the transmembrane domains and each other, and integrate to form a compound particle to gate the channel closed. Interactions of the carboxyl-terminus (CT) and cytoplasmic loop (CL) domains underlie voltage- and low pH-triggered channel closure. To elucidate potential cooperation of these gating mechanisms, we replaced the Cx43NT with the Cx37NT (chimera Cx43(∗)NT37), leaving the remainder of the Cx43 sequence, including the CT and CL, unchanged. Compared to wild-type Cx43 (Cx43WT), Cx43(∗)NT37 junctions exhibited several functional alterations: extreme resistance to halothane- and acidification-induced uncoupling, absence of voltage-dependent fast inactivation, longer channel open times, larger unitary channel conductances, low junctional dye permeability/permselectivity, and an overall cation selectivity more typical of Cx37WT than Cx43WT junctions. Together, these results suggest a cohesive model of channel function wherein: 1) channel conductance and size selectivity are largely determined by pore diameter, whereas charge selectivity results from the NT domains, and 2) transition between fully open and (multiple) closed states involves global changes in structure of the pore-forming domains transduced by interactions of the pore-forming domains with either the NT, CT, or both, with the NT domains forming the gate of the completely closed channel.

Structural determinants and proliferative consequences of connexin 37 hemichannel function in insulinoma cells.

Connexin (Cx) 37 suppresses vascular and cancer cell proliferation. The C terminus and a channel able to function are necessary, and neither by itself is sufficient, for Cx37 to mediate growth suppression. Cx37 supports transmembrane and intercellular signaling by forming functional hemichannels (HCs) and gap junction channels (GJCs), respectively. Here we determined whether Cx37 with HC, but not GJC, functionality would suppress proliferation of rat insulinoma (Rin) cells comparably to wild-type Cx37 (Cx37-WT). We mutated extracellular loop residues hypothesized to compromise HC docking but not HC function (six cysteines mutated to alanine, C54A,C61A,C65A, C187A,C192A,C198A (designated as C6A); N55I; and Q58L). All three mutants trafficked to the plasma membrane and formed protein plaques comparably to Cx37-WT. None of the mutants formed functional GJCs, and Cx37-C6A did not form functional HCs. Cx37-N55I and -Q58L formed HCs with behavior and permeation properties similar to Cx37-WT (especially Q58L), but none of the mutants suppressed Rin cell proliferation. The data indicate that determinants of Cx37 HC function differ from other Cxs and that HC functions with associated HC-supported protein-protein interactions are not sufficient for Cx37 to suppress Rin cell proliferation. Together with previously published data, these results suggest that Cx37 suppresses Rin cell proliferation only when in a specific conformation achieved by interaction of the C terminus with a Cx37 pore-forming domain able to open as a GJC.

Structural basis for the selective permeability of channels made of communicating junction proteins.

The open state(s) of gap junction channels is evident from their permeation by small ions in response to an applied intercellular (transjunctional/transchannel) voltage gradient. That an open channel allows variable amounts of current to transit from cell-to-cell in the face of a constant intercellular voltage difference indicates channel open/closing can be complete or partial. The physiological significance of such open state options is, arguably, the main concern of junctional regulation. Because gap junctions are permeable to many substances, it is sensible to inquire whether and how each open state influences the intercellular diffusion of molecules as valuable as, but less readily detected than current-carrying ions. Presumably, structural changes perceived as shifts in channel conductivity would significantly alter the transjunctional diffusion of molecules whose limiting diameter approximates the pore's limiting diameter. Moreover, changes in junctional permeability to some molecules might occur without evident changes in conductivity, either at macroscopic or single channel level. Open gap junction channels allow the exchange of cytoplasmic permeants between contacting cells by simple diffusion. The identity of such permeants, and the functional circumstances and consequences of their junctional exchange presently constitute the most urgent (and demanding) themes of the field. Here, we consider the necessity for regulating this exchange, the possible mechanism(s) and structural elements likely involved in such regulation, and how regulatory phenomena could be perceived as changes in chemical vs. electrical coupling; an overall reflection on our collective knowledge of junctional communication is then applied to suggest new avenues of research. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.

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.

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.

Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43.

Expression of connexin 40 (Cx40) and Cx43 in cardiovascular tissues varies as a function of age, injury, and development with unknown consequences on the selectivity of junctional communication and its acute regulation. We investigated the PKC-dependent regulation of charge selectivity in junctions composed of Cx43, Cx40, or both by simultaneous assessment of junctional permeance rate constants (B(dye)) for dyes of similar size but opposite charge, N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; +1) and Alexa 350 (-1). The ratio of dye rate constants (B(NBD-M-TMA)/B(Alexa 350)) indicated that Cx40 junctions are cation selective (10.7 +/- 0.5), whereas Cx43 junction are nonselective (1.22 +/- 0.14). In coexpressing cells, a broad range of junctional selectivities was observed with mean cation selectivity increasing as the Cx40 to Cx43 expression ratio increased. PKC activation reduced or eliminated dye permeability of Cx43 junctions without altering their charge selectivity, had no effect on either permeability or charge selectivity of Cx40 junctions, and significantly increased the cation selectivity of junctions formed by coexpressing cells (approaching charge selectivity of Cx40 junctions). Junctions composed of Cx43 truncated at residue 257 (Cx43tr) were also not charge selective, but when Cx43tr was coexpressed with Cx40, a broad range of junctional selectivities that was unaffected by PKC activation was observed. Thus, whereas the charge selectivities of homomeric/homotypic Cx43 and Cx40 junctions appear invariant, the selectivities of junctions formed by cells coexpressing Cx40 and Cx43 vary considerably, reflecting both their relative expression levels and phosphorylation-dependent regulation. Such regulation could represent a mechanism by which coexpressing cells such as vascular endothelium and atrial cells regulate acutely the selective intercellular communication mediated by their gap junctions.

Selectivity of connexin 43 channels is regulated through protein kinase C-dependent phosphorylation.

Coordinated contractile activation of the heart and resistance to ischemic injury depend, in part, on the intercellular communication mediated by Cx43-composed gap junctions. The function of these junctions is regulated at multiple levels (assembly to degradation) through phosphorylation at specific sites in the carboxyl terminus (CT) of the Cx43 protein. We show here that the selective permeability of Cx43 junctions is regulated through protein kinase C (PKC)-dependent phosphorylation at serine 368 (S368). Selective permeability was measured in several Cx43-expressing cell lines as the rate constant for intercellular dye diffusion relative to junctional conductance. The selective permeability of Cx43 junctions under control conditions was quite variable, as was the open-state behavior of the comprising channels. Coexpression of the CT of Cx43 as a distinct protein, treatment with a PKC inhibitor, or mutation of S368 to alanine, all reduced (or eliminated) phosphorylation at S368, reduced the incidence of 55- to 70-pS channels, and reduced by 10-fold the selective permeability of the junctions for a small cationic dye. Because PKC activation during preischemic conditioning is cardioprotective during subsequent ischemic episodes, we examined no-flow, ischemic hearts for Cx43 phosphorylated at S368 (pS368). Consistent with early activation of PKC, pS368-Cx43 was increased in ischemic hearts; despite extensive lateralization of total Cx43, pS368-Cx43 remained predominantly at intercalated disks. Our data suggest that the selectivity of gap junction channels at intercalated disks is increased early in ischemia.

Quantification of gap junction selectivity.

Gap junctions, which are essential for functional coordination and homeostasis within tissues, permit the direct intercellular exchange of small molecules. The abundance and diversity of this exchange depends on the number and selectivity of the comprising channels and on the transjunctional gradient for and chemical character of the permeant molecules. Limited knowledge of functionally significant permeants and poor detectability of those few that are known have made it difficult to define channel selectivity. Presented herein is a multifaceted approach to the quantification of gap junction selectivity that includes determination of the rate constant for intercellular diffusion of a fluorescent probe (k2-DYE) and junctional conductance (gj) for each junction studied, such that the selective permeability (k2-DYE/gj) for dyes with differing chemical characteristics or junctions with differing connexin (Cx) compositions (or treatment conditions) can be compared. In addition, selective permeability can be correlated using single-channel conductance when this parameter is also measured. Our measurement strategy is capable of detecting 1) rate constants and selective permeabilities that differ across three orders of magnitude and 2) acute changes in that rate constant. Using this strategy, we have shown that 1) the selective permeability of Cx43 junctions to a small cationic dye varied across two orders of magnitude, consistent with the hypothesis that the various channel configurations adopted by Cx43 display different selective permeabilities; and 2) the selective permeability of Cx37 vs. Cx43 junctions was consistently and significantly lower.