Free energy and kinetics of cAMP permeation through connexin26 via applied voltage and milestoning.

The connexin family is a diverse group of highly regulated wide-pore channels permeable to biological signaling molecules. Despite the critical roles of connexins in mediating selective molecular signaling in health and disease, the basis of molecular permeation through these pores remains unclear. Here, we report the thermodynamics and kinetics of binding and transport of a second messenger, adenosine-3',5'-cyclophosphate (cAMP), through a connexin26 hemichannel (Cx26). First, inward and outward fluxes of cAMP molecules solvated in KCl solution were obtained from 4 µs of ± 200 mV simulations. These fluxes data yielded a single-channel permeability of cAMP and cAMP/K+ permeability ratio consistent with experimentally measured values. The results from voltage simulations were then compared with the potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP without voltage, obtained from a total of 16.5 µs of Voronoi-tessellated Markovian milestoning simulations. Both the voltage simulations and the milestoning simulations revealed two cAMP-binding sites, for which the binding constants KD and dissociation rates koff were computed from PMF and MFPTs. The protein dipole inside the pore produces an asymmetric PMF, reflected in unequal cAMP MFPTs in each direction once within the pore. The free energy profiles under opposite voltages were derived from the milestoning PMF and revealed the interplay between voltage and channel polarity on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and nonlocal pore diameter in a position-dependent manner.

Mechanism of gating by calcium in connexin hemichannels.

Aberrant opening of nonjunctional connexin hemichannels at the plasma membrane is associated with many diseases, including ischemia and muscular dystrophy. Proper control of hemichannel opening is essential to maintain cell viability and is achieved by physiological levels of extracellular Ca2+, which drastically reduce hemichannel activity. Here we examined the role of conserved charged residues that form electrostatic networks near the extracellular entrance of the connexin pore, a region thought to be involved in gating rearrangements of hemichannels. Molecular dynamics simulations indicate discrete sites for Ca2+ interaction and consequent disruption of salt bridges in the open hemichannels. Experimentally, we found that disruption of these salt bridges by mutations facilitates hemichannel closing. Two negatively charged residues in these networks are putative Ca2+ binding sites, forming a Ca2+-gating ring near the extracellular entrance of the pore. Accessibility studies showed that this Ca2+-bound gating ring does not prevent access of ions or small molecules to positions deeper into the pore, indicating that the physical gate is below the Ca2+-gating ring. We conclude that intra- and intersubunit electrostatic networks at the extracellular entrance of the hemichannel pore play critical roles in hemichannel gating reactions and are tightly controlled by extracellular Ca2+ Our findings provide a general mechanism for Ca2+ gating among different connexin hemichannel isoforms.

Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization.

Gap junctions, composed of connexins, mediate electrical coupling and impulse propagation in the working myocardium. In the human heart, the spatio-temporal regulation and distinct functional properties of the three dominant connexins (Cx43, Cx45, and Cx40) suggests non-redundant physiological roles for each isoform. There are substantial differences in gating properties, expression, and trafficking among these isoforms, however, little is known about the determinants of these different phenotypes. To gain insight regarding these determinants, we focused on the carboxyl-terminal (CT) domain because of its importance in channel regulation and large degree of sequence divergence among connexin family members. Using in vitro biophysical experiments, we identified a structural feature unique to Cx45: high affinity (KD~100nM) dimerization between CT domains. In this study, we sought to determine if this dimerization occurs in cells and to identify the biological significance of the dimerization. Using a bimolecular fluorescence complementation assay, we demonstrate that the CT domains dimerize at the plasma membrane. By inhibiting CT dimerization with a mutant construct, we show that CT dimerization is necessary for proper Cx45 membrane localization, turnover, phosphorylation status, and binding to protein partners. Furthermore, CT dimerization is needed for normal intercellular communication and hemichannel activity. Altogether, our results demonstrate that CT dimerization is a structural feature important for correct Cx45 function. This study is significant because discovery of how interactions mediated by the CT domains can be modulated would open the door to strategies to ameliorate the pathological effects of altered connexin regulation in the failing heart.

Endothelial cAMP deactivates ischemia-reperfusion-induced microvascular hyperpermeability via Rap1-mediated mechanisms.

Approaches to reduce excessive edema due to the microvascular hyperpermeability that occurs during ischemia-reperfusion (I/R) are needed to prevent muscle compartment syndrome. We tested the hypothesis that cAMP-activated mechanisms actively restore barrier integrity in postischemic striated muscle. We found, using I/R in intact muscles and hypoxia-reoxygenation (H/R, an I/R mimic) in human microvascular endothelial cells (HMVECs), that hyperpermeability can be deactivated by increasing cAMP levels through application of forskolin. This effect was seen whether or not the hyperpermeability was accompanied by increased mRNA expression of VEGF, which occurred only after 4 h of ischemia. We found that cAMP increases in HMVECs after H/R, suggesting that cAMP-mediated restoration of barrier function is a physiological mechanism. We explored the mechanisms underlying this effect of cAMP. We found that exchange protein activated by cAMP 1 (Epac1), a downstream effector of cAMP that stimulates Rap1 to enhance cell adhesion, was activated only at or after reoxygenation. Thus, when Rap1 was depleted by small interfering RNA, H/R-induced hyperpermeability persisted even when forskolin was applied. We demonstrate that 1) VEGF mRNA expression is not involved in hyperpermeability after brief ischemia, 2) elevation of cAMP concentration at reperfusion deactivates hyperpermeability, and 3) cAMP activates the Epac1-Rap1 pathway to restore normal microvascular permeability. Our data support the novel concepts that 1) different hyperpermeability mechanisms operate after brief and prolonged ischemia and 2) cAMP concentration elevation during reperfusion contributes to deactivation of I/R-induced hyperpermeability through the Epac-Rap1 pathway. Endothelial cAMP management at reperfusion may be therapeutic in I/R injury.NEW & NOTEWORTHY Here, we demonstrate that 1) stimulation of cAMP production deactivates ischemia-reperfusion-induced hyperpermeability in muscle and endothelial cells; 2) VEGF mRNA expression is not enhanced by brief ischemia, suggesting that VEGF mechanisms do not activate immediate postischemic hyperpermeability; and 3) deactivation mechanisms operate via cAMP-exchange protein activated by cAMP 1-Rap1 to restore integrity of the endothelial barrier.

Computational Studies of Molecular Permeation through Connexin26 Channels.

A signal property of connexin channels is the ability to mediate selective diffusive movement of molecules through plasma membrane(s), but the energetics and determinants of molecular movement through these channels have yet to be understood. Different connexin channels have distinct molecular selectivities that cannot be explained simply on the basis of size or charge of the permeants. To gain insight into the forces and interactions that underlie selective molecular permeation, we investigated the energetics of two uncharged derivatized sugars, one permeable and one impermeable, through a validated connexin26 (Cx26) channel structural model, using molecular dynamics and associated analytic tools. The system is a Cx26 channel equilibrated in explicit membrane/solvent, shown by Brownian dynamics to reproduce key conductance characteristics of the native channel. The results are consistent with the known difference in permeability to each molecule. The energetic barriers extend through most of the pore length, rather than being highly localized as in ion-specific channels. There is little evidence for binding within the pore. Force decomposition reveals how, for each tested molecule, interactions with water and the Cx26 protein vary over the length of the pore and reveals a significant contribution from hydrogen bonding and interaction with K(+). The flexibility of the pore width varies along its length, and the tested molecules have differential effects on pore width as they pass through. Potential sites of interaction within the pore are defined for each molecule. The results suggest that for the tested molecules, differences in hydrogen bonding and entropic factors arising from permeant flexibility substantially contribute to the energetics of permeation. This work highlights factors involved in selective molecular permeation that differ from those that define selectivity among atomic ions.

Connexin 43 is involved in the generation of human-induced pluripotent stem cells.

Although somatic cells can be successfully programmed to create pluripotent stem cells by ectopically expressing defined transcriptional factors, reprogramming efficiency is low and the reprogramming mechanism remains unclear. Previous reports have shown that almost all human connexin (CX) isoforms are expressed by human embryonic stem (hES) cells and that gap junctional intercellular communication (GJIC) is important for ES cell survival and differentiation. However, the CX expression profiles in human induced pluripotent stem (iPS) cells and the role of CXs in the process of reprogramming back to iPS cells remains unknown. Here, we determined the expression levels of most forms of CX in human embryonic fibroblasts (hEFs) and in the hEF-derived iPS cells. A scrape loading/dye transfer assay showed that human iPS cells contained functional gap junctions (GJs) that could be affected by pharmacological inhibitors of GJ function. We found that CX43 was the most dramatically upregulated CX following reprogramming. Most importantly, the ectopic expression of CX43 significantly enhanced the reprogramming efficiency, whereas shRNA-mediated knockdown of endogenous CX43 expression greatly reduced the efficiency. In addition, we found that CX43 overexpression or knockdown affected the expression of E-CADHERIN, a marker of the mesenchymal-to-epithelial transition (MET), during reprogramming. In conclusion, our data indicate that CX43 expression is important for reprogramming and may mediate the MET that is associated with the acquisition of pluripotency.

Gap junction channel structure in the early 21st century: facts and fantasies.

Gap junction channels connect the cytoplasms of adjacent cells through the end-to-end docking of single-membrane structures called connexons, formed by a ring of six connexin monomers. Each monomer contains four transmembrane alpha-helices, for a total of 24 alpha-helices in a connexon. The fundamental structure of the connexon pore is probably similar in unpaired connexons and junctional channels, and for channels formed by different connexin isoforms. Nevertheless, variability in results from structurally focused mutagenesis and electrophysiological studies raise uncertainty about the specific assignments of the transmembrane helices. Mapping of human mutations onto a suggested C(alpha) model predicts that mutations that disrupt helix-helix packing impair channel function. An experimentally determined structure at atomic resolution will be essential to confirm and resolve these concepts.

Connexin Gap Junction Channels and Hemichannels: Insights from High-Resolution Structures.

Connexins (Cxs) are a family of integral membrane proteins, which function as both hexameric hemichannels (HCs) and dodecameric gap junction channels (GJCs), behaving as conduits for the electrical and molecular communication between cells and between cells and the extracellular environment, respectively. Their proper functioning is crucial for many processes, including development, physiology, and response to disease and trauma. Abnormal GJC and HC communication can lead to numerous pathological states including inflammation, skin diseases, deafness, nervous system disorders, and cardiac arrhythmias. Over the last 15 years, high-resolution X-ray and electron cryomicroscopy (cryoEM) structures for seven Cx isoforms have revealed conservation in the four-helix transmembrane (TM) bundle of each subunit; an αß fold in the disulfide-bonded extracellular loops and inter-subunit hydrogen bonding across the extracellular gap that mediates end-to-end docking to form a tight seal between hexamers in the GJC. Tissue injury is associated with cellular Ca2+ overload. Surprisingly, the binding of 12 Ca2+ ions in the Cx26 GJC results in a novel electrostatic gating mechanism that blocks cation permeation. In contrast, acidic pH during tissue injury elicits association of the N-terminal (NT) domains that sterically blocks the pore in a "ball-and-chain" fashion. The NT domains under physiologic conditions display multiple conformational states, stabilized by protein-protein and protein-lipid interactions, which may relate to gating mechanisms. The cryoEM maps also revealed putative lipid densities within the pore, intercalated among transmembrane α-helices and between protomers, the functions of which are unknown. For the future, time-resolved cryoEM of isolated Cx channels as well as cryotomography of GJCs and HCs in cells and tissues will yield a deeper insight into the mechanisms for channel regulation. The cytoplasmic loop (CL) and C-terminal (CT) domains are divergent in sequence and length, are likely involved in channel regulation, but are not visualized in the high-resolution X-ray and cryoEM maps presumably due to conformational flexibility. We expect that the integrated use of synergistic physicochemical, spectroscopic, biophysical, and computational methods will reveal conformational dynamics relevant to functional states. We anticipate that such a wealth of results under different pathologic conditions will accelerate drug discovery related to Cx channel modulation.

Molecular dynamics simulations of the Cx26 hemichannel: insights into voltage-dependent loop-gating.

Loop-gating is one of two voltage-dependent mechanisms that regulate the open probability of connexin channels. The loop-gate permeability barrier is formed by a segment of the first extracellular loop (E1) (the parahelix) and appears to be accompanied by straightening of the bend angle between E1 and the first transmembrane domain (TM1). Here, all-atom molecular dynamics simulations are used to identify and characterize interacting van der Waals and electrostatic networks that stabilize the parahelices and TM1/E1 bend angles of the open Cx26 hemichannel. Dynamic fluctuations in an electrostatic network in each subunit are directly linked to the stability of parahelix structure and TM1/E1 bend angle in adjacent subunits. The electrostatic network includes charged residues that are pore-lining and thus positioned to be voltage sensors. We propose that the transition to the closed state is initiated by voltage-driven disruption of the networks that stabilize the open-state parahelix configuration, allowing the parahelix to protrude into the channel pore to form the loop-gate barrier. Straightening of the TM1/E1 bend appears to be a consequence of the reorganization of the interacting networks that accompany the conformational change of the parahelix. The electrostatic network extends across subunit boundaries, suggesting a concerted gating mechanism.

Monocyte-endothelial adhesion is modulated by Cx43-stimulated ATP release from monocytes.

Adhesion of circulating monocytes to vascular endothelial cells is a crucial event in development of vascular inflammatory conditions, including atherosclerosis. We investigated the roles of connexin43 (Cx43) and ATP release on monocyte-endothelial adhesion. Cx43 function and expression were manipulated by connexin channel inhibitors, overexpression and siRNA. Connexin channel inhibitors rapidly decreased ATP release from U937 monocytes and increased adhesion to human umbilical vein endothelial cells (HUVEC). Monocyte ATP release correlated with Cx43 expression, not with Cx37 expression. Exogenous adenosine (ADO) or ATP decreased adhesion, and inhibition of ATP conversion to ADO increased adhesion. We infer that monocyte Cx43 channel activity causes ATP release, likely via Cx43-containing hemichannels, and that ATP decreases adhesion via conversion to ADO. Inhibition of HUVEC connexin channel activity did not affect ATP release or adhesion. In contrast, expression of Cx43 protein in U937 cells enhanced adhesion. Thus, Cx43 channel function and expression have opposite effects: Cx43 channel function in monocytes, but not in HUVEC, rapidly decreases adhesion via ATP release and conversion to ADO, whereas Cx43 expression itself enhances adhesion. These studies suggest that local regulation of monocyte Cx43 activity within the vasculature can dynamically modulate the monocyte-endothelial adhesion that is an initiating event in vascular inflammatory pathologies, with the baseline adhesion set by Cx43 expression levels. This balance of rapid and tonic influences may be crucial in development of vascular pathologies.

Cisplatin and oxaliplatin inhibit gap junctional communication by direct action and by reduction of connexin expression, thereby counteracting cytotoxic efficacy.

Cisplatin [cis-diamminedichloroplatinum(II)]/oxaliplatin [1,2-diamminocyclohexane(trans-1)oxolatoplatinum(II)] toxicity is enhanced by functional gap junctions between treated cells, implying that inhibition of gap junctions may decrease cytotoxic activity of these platinum-based agents. This study investigates the effect of gap junction modulation by cisplatin/oxaliplatin on cytotoxicity in a transformed cell line. The effects were explored using junctional channels expressed in transfected HeLa cells and purified hemichannels. Junctional channels showed a rapid, dose-dependent decrease in dye coupling with exposure to cisplatin/oxaliplatin. With longer exposure, both compounds also decreased connexin expression. Both compounds inhibit the activity of purified connexin hemichannels, over the same concentration range that they inhibit junctional dye permeability, demonstrating that inhibition occurs by direct interaction of the drugs with connexin protein. Cisplatin/oxaliplatin reduced the clonogenic survival of HeLa cells at low density and high density in a dose-dependent manner, but to a greater degree at high density, consistent with a positive effect of gap junctional intercellular communication (GJIC) on cytotoxicity. Reduction of GJIC by genetic or pharmacological means decreased cisplatin/oxaliplatin toxicity. At low cisplatin/oxaliplatin concentrations, where effects on connexin channels are minimal, the toxicity increased with increased cell density. However, higher concentrations strongly inhibited GJIC, and this counteracted the enhancing effect of greater cell density on toxicity. The present results indicate that inhibition of GJIC by cisplatin/oxaliplatin decreases their cytotoxicity. Direct inhibition of GJIC and reduction of connexin expression by cisplatin/oxaliplatin may thereby compromise the effectiveness of these compounds and be a factor in the development of resistance to this class of chemotherapeutic agents.

Post-translational modifications of connexin26 revealed by mass spectrometry.

Gap junctions play important roles in auditory function and skin biology; mutations in the Cx26 (connexin26) gene are the predominant cause of inherited non-syndromic deafness and cause disfiguring skin disorders. Mass spectrometry (MS) was used to identify PTMs (post-translational modifications) of Cx26 and to determine whether they occur at sites of disease-causing mutations. Cx26 was isolated from transfected HeLa cells by sequential immunoaffinity and metal chelate chromatography using a tandem C-terminal haemagglutinin epitope and a (His-Asn)6 sequence. In-gel and in-solution enzymatic digestions were carried out in parallel with trypsin, chymotrypsin and endoproteinase GluC. Peptides were fractionated using a reversed-phase matrix by stepwise elution with increasing concentrations of organic solvent. To improve detection of low-abundance peptides and to maximize sequence coverage, MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry; MS) and MALDI-TOF/TOF-MS/MS (matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry; MS/MS) spectra were acquired from each elution step using an Applied Biosystems 4800 tandem mass spectrometer. Acquisition, processing and interpretation parameters were optimized to improve ionization and fragmentation of hydrophobic peptides. MS and MS/MS coverage of Cx26 was significantly above that reported for other membrane proteins: 71.3% by MS, with 29.9% by MS/MS. MS coverage was 92.6% if peptides resulting from in-source collisions and/or partial enzymatic cleavages were considered. A variety of putative PTMs of Cx26 were identified, including acetylation, hydroxylation, gamma-carboxyglutamation, methylation and phosphorylation, some of which are at sites of deafness-causing mutations. Knowledge of the PTMs of Cx26 will be instrumental in understanding how alterations in the cellular mechanisms of Cx26 channel biogenesis and function lead to losses in auditory function and disfiguring skin disorders.

Connexin channels and phospholipids: association and modulation.

BACKGROUND: For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood. RESULTS: Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred. CONCLUSION: This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.

A novel voltage-clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels.

Large-pore channels permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, their mechanisms of molecular permeation and selectivity remain mostly unexplored. Combining fluorescence microscopy and electrophysiology, we developed a novel technique that allows kinetic analysis of molecular permeation through connexin and CALHM1 channels in Xenopus oocytes rendered translucent. Using this methodology, we found that (1) molecular flux through these channels saturates at low micromolar concentrations, (2) kinetic parameters of molecular transport are sensitive to modulators of channel gating, (3) molecular transport and ionic currents can be differentially affected by mutation and gating, and (4) N-terminal regions of these channels control transport kinetics and permselectivity. Our methodology allows analysis of how human disease-causing mutations affect kinetic properties and permselectivity of molecular signaling and enables the study of molecular mechanisms, including selectivity and saturability, of molecular transport in other large-pore channels.

The connexin26 human mutation N14K disrupts cytosolic intersubunit interactions and promotes channel opening.

A group of human mutations within the N-terminal (NT) domain of connexin 26 (Cx26) hemichannels produce aberrant channel activity, which gives rise to deafness and skin disorders, including keratitis-ichthyosis-deafness (KID) syndrome. Structural and functional studies indicate that the NT of connexin hemichannels is folded into the pore, where it plays important roles in permeability and gating. In this study, we explore the molecular basis by which N14K, an NT KID mutant, promotes gain of function. In macroscopic and single-channel recordings, we find that the N14K mutant favors the open conformation of hemichannels, shifts calcium and voltage sensitivity, and slows deactivation kinetics. Multiple copies of MD simulations of WT and N14K hemichannels, followed by the Kolmogorov-Smirnov significance test (KS test) of the distributions of interaction energies, reveal that the N14K mutation significantly disrupts pairwise interactions that occur in WT hemichannels between residue K15 of one subunit and residue E101 of the adjacent subunit (E101 being located at the transition between transmembrane segment 2 [TM2] and the cytoplasmic loop [CL]). Double mutant cycle analysis supports coupling between the NT and the TM2/CL transition in WT hemichannels, which is disrupted in N14K mutant hemichannels. KS tests of the α carbon correlation coefficients calculated over MD trajectories suggest that the effects of the N14K mutation are not confined to the K15-E101 pairs but extend to essentially all pairwise residue correlations between the NT and TM2/CL interface. Together, our data indicate that the N14K mutation increases hemichannel open probability by disrupting interactions between the NT and the TM2/CL region of the adjacent connexin subunit. This suggests that NT-TM2/CL interactions facilitate Cx26 hemichannel closure.

Ca2+-dependent inactivation of CaV1.2 channels prevents Gd3+ block: does Ca2+ block the pore of inactivated channels?

Lanthanide gadolinium (Gd(3+)) blocks Ca(V)1.2 channels at the selectivity filter. Here we investigated whether Gd(3+) block interferes with Ca(2+)-dependent inactivation, which requires Ca(2+) entry through the same site. Using brief pulses to 200 mV that relieve Gd(3+) block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd(3+) block of the Ca(2+) influx that enhances inactivation. However, we also found that the extent of Gd(3+) block did not change when inactivation was reduced by abolition of Ca(2+)/calmodulin interaction, showing that Gd(3+) does not block the inactivated channel. Thus, Gd(3+) block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd(3+) site or reduces its affinity, or that Ca(2+) occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca(2+)-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117-11123), and that Ca(2+)-inactivated channels conduct Na(+) when Ca(2+) is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709-717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca(2+) so that Ca(2+) ion blocks the pore. A minimal model, in which the inactivation "gate" is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca(2+).

Connexin channel permeability to cytoplasmic molecules.

Connexin channels are known to be permeable to a variety of cytoplasmic molecules. The first observation of second messenger junctional permeability, made approximately 30 years ago, sparked broad interest in gap junction channels as mediators of intercellular molecular signaling. Since then, much has been learned about the diversity of connexin channels with regard to isoform diversity, tissue and developmental distribution, modes of channel regulation, assembly, expression, biochemical modification and permeability, all of which appear to be dynamically regulated. This information has expanded the potential roles of connexin channels in development, physiology and disease, and made their elucidation much more complex--30 years ago such an orchestra of junctional dynamics was unanticipated. Only recently, however, have investigators been able to directly address, in this more complex framework, the key issue: what specific biological molecules, second messengers and others, are able to permeate the various types of connexin channels, and how well? An important related issue, given the ever-growing list of connexin-related pathologies, is how these permeabilities are altered by disease-causing connexin mutations. Together, many studies show that a variety of cytoplasmic molecules can permeate the different types of connexin channels. A few studies reveal differences in permeation by different molecules through a particular type of connexin channel, and differences in permeation by a particular molecule through different types of connexin channels. This article describes and evaluates the various methods used to obtain these data, presents an annotated compilation of the results, and discusses the findings in the context of what can be inferred about mechanism of selectivity and potential relevance to signaling. The data strongly suggest that highly specific interactions take place between connexin pores and specific biological molecular permeants, and that those interactions determine which cytoplasmic molecules can permeate and how well. At this time, the nature of those interactions is unclear. One hopes that with more detailed permeability and structural information, the specific molecular mechanisms of the selectivity can be elucidated.

Electrical coupling and its channels.

As the physiology of synapses began to be explored in the 1950s, it became clear that electrical communication between neurons could not always be explained by chemical transmission. Instead, careful studies pointed to a direct intercellular pathway of current flow and to the anatomical structure that was (eventually) called the gap junction. The mechanism of intercellular current flow was simple compared with chemical transmission, but the consequences of electrical signaling in excitable tissues were not. With the recognition that channels were a means of passive ion movement across membranes, the character and behavior of gap junction channels came under scrutiny. It became evident that these gated channels mediated intercellular transfer of small molecules as well as atomic ions, thereby mediating chemical, as well as electrical, signaling. Members of the responsible protein family in vertebrates-connexins-were cloned and their channels studied by many of the increasingly biophysical techniques that were being applied to other channels. As described here, much of the evolution of the field, from electrical coupling to channel structure-function, has appeared in the pages of the Journal of General Physiology.

Isoelectric points and post-translational modifications of connexin26 and connexin32.

The isoelectric points of the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) were determined by isoelectric focusing in free fluids. The isoelectric points were significantly more acidic than predicted from amino acid sequences and different from each other, allowing homomeric channels to be resolved separately. The isoelectric points of the homomeric channels bracketed the isoelectric points of heteromeric Cx26/Cx32 channels. For heteromeric channels, Cx26 and Cx32 were found in overlapping, pH-focused fractions, indicating quaternary structure was retained. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify post-translational modifications of Cx26 and Cx32 cytoplasmic domains, including the first reported post-translational modifications of Cx26. Suspected modifications were hydroxylation and/or phosphorylation near the amino terminus of both connexins, gamma-carboxyglutamate residues in the cytoplasmic loop of both connexins, phosphorylation in the carboxyl-terminal domain of Cx32, and palmitoylation at the carboxyl-terminus of Cx32. These modifications contribute to the measured acidic isoelectric points of Cx26 and Cx32, whereas their low molecular masses would not appreciably change connexin SDS-PAGE mobility. Most of these modifications have not previously been identified for connexins and may be instrumental in guiding and understanding novel aspects of channel trafficking and molecular mechanisms of channel regulation.

Genomic instability induced in distant progeny of bystander cells depends on the connexins expressed in the irradiated cells.

PURPOSE: To examine the time window during which intercellular signaling though gap junctions mediates non-targeted (bystander) effects induced by moderate doses of ionizing radiation; and to investigate the impact of gap junction communication on genomic instability in distant progeny of bystander cells. MATERIALS AND METHODS: A layered cell culture system was developed to investigate the propagation of harmful effects from irradiated normal or tumor cells that express specific connexins to contiguous bystander normal human fibroblasts. Irradiated cells were exposed to moderate mean absorbed doses from 3.7 MeV α particle, 1000 MeV/u iron ions, 600 MeV/u silicon ions, or 137Cs γ rays. Following 5 h of co-culture, pure populations of bystander cells, unexposed to secondary radiation, were isolated and DNA damage and oxidative stress was assessed in them and in their distant progeny (20-25 population doublings). RESULTS: Increased frequency of micronucleus formation and enhanced oxidative changes were observed in bystander cells co-cultured with confluent cells exposed to either sparsely ionizing (137Cs γ rays) or densely ionizing (α particles, energetic iron or silicon ions) radiations. The irradiated cells propagated signals leading to biological changes in bystander cells within 1 h of irradiation, and the effect required cellular coupling by gap junctions. Notably, the distant progeny of isolated bystander cells also exhibited increased levels of spontaneous micronuclei. This effect was dependent on the type of junctional channels that coupled the irradiated donor cells with the bystander cells. Previous work showed that gap junctions composed of connexin26 (Cx26) or connexin43 (Cx43) mediate toxic bystander effects within 5 h of co-culture, whereas gap junctions composed of connexin32 (Cx32) mediate protective effects. In contrast, the long-term progeny of bystander cells expressing Cx26 or Cx43 did not display elevated DNA damage, whereas those coupled by Cx32 had enhanced DNA damage. CONCLUSIONS: In response to moderate doses from either sparsely or densely ionizing radiations, toxic and protective effects are rapidly communicated to bystander cells through gap junctions. We infer that bystander cells damaged by the initial co-culture (expressing Cx26 or Cx43) die or undergo proliferative arrest, but that the bystander cells that were initially protected (expressing Cx32) express DNA damage upon sequential passaging. Together, the results inform the roles that intercellular communication play under stress conditions, and aid assessment of the health risks of exposure to ionizing radiation. Identification of the communicated molecules may enhance the efficacy of radiotherapy and help attenuate its debilitating side-effects.