Gap junction dynamics: reversible effects of divalent cations

Reversible changes in gap junction structure similar to those previously seen to parallel electrical uncoupling (9, 33, 34) are produced by treating with Ca++ or Mg++ gap junctions isolated in EDTA from calf lens fibers. The changes, characterized primarily by a switch from disordered to crystalline particle packings, occur at a [Ca++] of 5 x 10(-7) M or higher and a [Mg++] of 1 x 10(-3) M or higher and can be reversed by exposing the junctions to Ca++- and Mg++-free EGTA solutions. Similar changes are obtained in junctions of rat stomach epithelia incubated at 37 degrees C in well-oxygenated Tyrode''s solutions containing a Ca++ ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187). Deep etching experiments on isolated lens junctions show that the true cytoplasmic surface of the junctions (PS face) is mostly bare, suggesting that the particles may not be connected to cytoskeletal elements. A hypothesis is proposed suggesting a mechanism of particle aggregation and channel narrowing based on neutralization of negative charges by divalent cations or H+.     

Essential dynamics of reversible peptide folding: memory-free conformational dynamics governed by internal hydrogen bonds

A principal component analysis has been applied on equilibrium simulations of a beta-heptapeptide that shows reversible folding in a methanol solution. The analysis shows that the configurational space contains only three dense sub-states. These states of relatively low free energy correspond to the native left-handed helix, a partly helical intermediate, and a hairpin-like structure. The collection of unfolded conformations form a relatively diffuse cloud with little substructure. Internal hydrogen-bonding energies were found to correlate well with the degree of folding. The native helical structure folds from the N terminus; the transition from the major folding intermediate to the native helical structure involves the formation of the two most C-terminal backbone hydrogen bonds. A four-state Markov model was found to describe transition frequencies between the conformational states within error limits, indicating that memory-effects are negligible beyond the nanosecond time-scale. The dominant native state fluctuations were found to be very similar to unfolding motions, suggesting that unfolding pathways can be inferred from fluctuations in the native state. The low-dimensional essential subspace, describing 69% of the collective atomic fluctuations, was found to converge at time-scales of the order of one nanosecond at all temperatures investigated, whereas folding/unfolding takes place at significantly longer time-scales, even above the melting temperature.     

Muscular fatigue: effects of hydrogen ions and inorganic phosphate

During muscular fatigue two metabolites, hydrogen ions (H+) and inorganic phosphate (Pi), increase in concentration. The effect of increase in [H+] has been modeled mathematically for a system containing creatine kinase (EC 2.7.3.2), adenylate kinase (EC 2.7.4.3), and the appropriate concentrations of their substrates. Assuming that no other equilibrium reactions are involved, the result of acidification should be a useful increase in the ratio [ATP]/[ADP]. It is also shown by a reanalysis of earlier 31P NMR studies that the observed combination of increased [H+] and increased [Pi] leads to an increase in the monobasic phosphate concentration [Pi-] that is inversely proportional to the force of contraction. This suggests that Pi- may be a direct inhibitor of the actomyosin ATPase system.     

Gap junction channel gating

Over the last two decades, the view of gap junction (GJ) channel gating has changed from one with GJs having a single transjunctional voltage-sensitive (V(j)-sensitive) gating mechanism to one with each hemichannel of a formed GJ channel, as well as unapposed hemichannels, containing two, molecularly distinct gating mechanisms. These mechanisms are termed fast gating and slow or 'loop' gating. It appears that the fast gating mechanism is solely sensitive to V(j) and induces fast gating transitions between the open state and a particular substate, termed the residual conductance state. The slow gating mechanism is also sensitive to V(j), but there is evidence that this gate may mediate gating by transmembrane voltage (V(m)), intracellular Ca(2+) and pH, chemical uncouplers and GJ channel opening during de novo channel formation. A distinguishing feature of the slow gate is that the gating transitions appear to be slow, consisting of a series of transient substates en route to opening and closing. Published reports suggest that both sensorial and gating elements of the fast gating mechanism are formed by transmembrane and cytoplamic components of connexins among which the N terminus is most essential and which determines gating polarity. We propose that the gating element of the slow gating mechanism is located closer to the central region of the channel pore and serves as a 'common' gate linked to several sensing elements that are responsive to different factors and located in different regions of the channel.     

Postsynaptic effects of nickel ions at the insect neuromuscular junction

The effect of extracellular nickel on the excitatory postsynaptic response at the insect neuromuscular junction was studied in the segmental muscle of the larval mealworm Tenebrio molitor. The response to L-glutamate applied iontophoretically (glutamate potential, GP) was potentiated in the presence of Ni2+ though the excitatory postsynaptic potential (EPSP) was reduced. It seems unlikely that Ni2+ acts at the same binding site as L-glutamate does since the value of the limiting slope of double logarithmic plots for the action of glutamate was increased in the presence of Ni2+. The potentiation of GP in the presence of Ni2+ cannot be ascribed to competition between Ni2+ and Ca2+ since GP amplitude did not show any dependence on the concentration of Ca2+. Nickel ions did not alter the reversal potential of excitatory postsynaptic current (EPSC) and glutamate current (GC) under the voltage clamp condition, whereas the amplitude of GC was potentiated in the presence of Ni2+. The time constant of the decay of EPSC showed a weak voltage dependency: the more depolarized the membrane, the more prolonged the time constant. In the presence of 1 mM Ni2+ the amplitude of miniature EPSCs (MEPSCs) increased and the half decay time was prolonged significantly. These results suggest that Ni2+ interacts with charged groups near the glutamate receptor-channel complex so that the kinetics of the channel are altered.     

Protein phosphorylation and hydrogen ions modulate calcium-induced closure of gap junction channels.

The regulation of the cell-to-cell pathway formed by gap junctions seems to involve the interaction of the junctional channels with either calcium or hydrogen ions, as well as protein phosphorylation and calmodulin. These mechanisms of junctional regulation have been considered to act independently on specific sites of the gap junction protein; however, the possibility that they may be interrelated has not been adequately explored mainly due to the difficulties involved in simultaneous measurement of intracellular cations and protein phosphorylation. To further understanding of mechanisms regulating gap junctions, we have internally perfused coupled lateral axons from crayfish with solutions containing different calcium and hydrogen concentrations under conditions favoring phosphorylation, while monitoring the junctional conductance. We found that calcium ions regulate cell communication probably through a direct interaction with the channel protein. Phosphorylation and low pH do not alter junctional conductance themselves, but appear only to modulate the effects of calcium, possibly by altering the affinity of the channel for calcium. We propose that a combination of free intracellular calcium and protein phosphorylation form an important physiological mechanism regulating intercellular communication.     

Gap junction structures: Analysis of the x-ray diffraction data

Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W.C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate to the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profile and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of the X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low resolution three- dimensional representations of the structures of gap junctions.     

Gap junction permeability: selectivity for anionic and cationic probes

Gap junction channels formed by different connexins exhibit specific permeability to a variety of larger solutes including second messengers, polypeptides, and small interfering RNAs. Here, we report the permeability of homotypic connexin26 (Cx26), Cx40, Cx43, and Cx45 gap junction channels stably expressed in HeLa cells to solutes with different size and net charge. Channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe. All four connexins allowed passage of both cationic and anionic probes, but the transfer rates were connexin dependent. The negatively charged probes [Lucifer yellow (LY; median axial diameter 9.9 ?, charge -2), carboxyfluorescein (CF; 8.2 ?; -2), and Alexa Fluor350 (AF350, 5.4 ?; -1)] exhibited the following permeability order: Cx43 > Cx45 > Cx26 > Cx40. In contrast, for the positively charged species permeability, the orders were as follows: Cx26 ≈ Cx43 ≈ Cx40 ≈ Cx45 for N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl) amino] ethanaminium (NBD-m-TMA; 5.5 ?, +1) and Cx26 ≥ Cx43 ≈ Cx40 > Cx45 for ethidium bromide (10.3 ?, +1). Comparison of probe permeability relative to K(+) revealed that Cx43 and Cx45 exhibited similar permeability for NBD-m-TMA and AF350, indicating weak charge selectivity. However, lesser transfer of CF and LY through Cx45 relative to Cx43 channels suggests stronger size-dependent discrimination of solute. The permeability of NBD-m-TMA for Cx40 and Cx26 channels was approximately three times higher than to anionic AF350 despite the fact that both have similar minor diameters, suggesting charge selectivity. In conclusion, these results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge, suggesting that channel selectivity may be a key factor in cell signaling.     

Gap junctional communication during neuromuscular junction formation

We have tested whether gap junctions form between nerve and muscle during their initial contact, before establishing the chemical synapse. Embryonic Xenopus stage 18-20 myotomes and neural tubes were permeabilized with DMSO to load appropriate reagents, dissociated, and cocultured. When myotomes, loaded with Lucifer yellow, were cocultured with unlabeled neural tube cells, 23% of the neurons contained dye after 24 hr. Affinity-purified gap junction antibodies loaded into myocytes or neurons reduced neuronal labeling significantly to 5%. [3H]uridine nucleotide transfer was observed in both directions between myocytes and neurons. Again gap junction antibodies substantially reduced recipient label. In all cases preimmune IgGs did not reduce transfer. When acetylcholine receptor clustering was examined in cultures containing gap junction antibodies, no difference in the number of neuronally induced AChR clusters was observed. This suggests that the cluster-inducing signal between nerve and muscle does not pass through gap junctions.     

Gap junction ultrastructure in three states of conductance

Summary Junctional conductance between the epidermal cells of the beetle Tenebrio molitor is raised after exposure to the hormone 20-hydroxyecdysone and lowered reversibly by exposure to chlorpromazine. Gap Junctional particle size, density and arrangement associated with these conductance changes were studied. We found no significant difference in particle density in gap junctions of control (2456±471 particles/m², mean ±S.D.) and hormone-treated epidermis (2490±315); however, a significant increase in packing density occurred in chlorpromazine-uncoupled epidermis (3133±665). The particles are randomly arranged in all three states of conductance. Particle size measurements show that the E-face gap junctional particles are heterogeneous with a mean diameter ±S.D. of 15.2±2.0 nm. No significant difference in particle size between controls and experimentals was detected. Although glutaraldehyde irreversibly uncoupled these cells, the absence of glutaraldehyde fixation but presence of glycerol induced marked alterations in the appearance of the gap junctions such that quantification was no longer possible. From this particle-packing data and our previous thin-section data, we estimate that there are 90000 gap junctional particles per cell (within junctional plaques). The conductance of a single gap junctional channel (assuming one population) changes from 94 pS to 213 pS after hormone treatment.     

Gap junction assembly: multiple connexin fluorophores identify complex trafficking pathways

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

Gap junction channels: distinct voltage-sensitive and -insensitive conductance states.

All mammalian gap junction channels are sensitive to the voltage difference imposed across the junctional membrane, and parameters of voltage sensitivity have been shown to vary according to the gap junction protein that is expressed. For connexin43, the major gap junction protein in the cardiovascular system, in the uterus, and between glial cells in brain, voltage clamp studies have shown that transjunctional voltages (Vj) exceeding +/- 50 mV reduce junctional conductance (gj). However, substantial gj remains at even very large Vj values; this residual voltage-insensitive conductance has been termed gmin. We have explored the mechanism underlying gmin using several cell types in which connexin43 is endogenously expressed as well as in communication-deficient hepatoma cells transfected with cDNA encoding human connexin43. For pairs of transfectants exhibiting series resistance-corrected maximal gj (gmax) values ranging from < 2 to > 90 nS, the ratio gmin/gmax was found to be relatively constant (about 0.4-0.5), indicating that the channels responsible for the voltage-sensitive and -insensitive components of gj are not independent. Single channel studies further revealed that different channel sizes comprise the voltage-sensitive and -insensitive components, and that the open times of the larger, more voltage-sensitive conductance events declined to values near zero at large voltages, despite the high gmin. We conclude that the voltage-insensitive component of gj is ascribable to a voltage-insensitive substate of connexin43 channels rather than to the presence of multiple types of channels in the junctional membrane. These studies thus demonstrate that for certain gap junction channels, closure in response to specific stimuli may be graded, rather than all-or-none.     

Gap junction channel gating modulated through protein phosphorylation

As a ubiquitous post-translation modification process, protein phosphorylation has proven to be a key mechanism in regulating the function of several membrane proteins, including transporters and channels. Connexins, pannexins, and innexins are protein families that form gap junction channels essential for intercellular communication. Connexins have been intensely studied, and most of their isoforms are known to be phosphorylated by protein kinases that lead to modifications in tyrosine, serine, and threonine residues, which have been reported to affect, in one way or another, intercellular communication. Despite the abundant reports on changes in intercellular communication due to the activation or inactivation of numerous kinases, the molecular mechanisms by which phosphorylation alters channel gating properties have not been elucidated completely. Hence, this chapter will cover some of the current, relevant research that attempt to explain how phosphorylation triggers and/or modulates gap junction channel gating.     

Gap junction proteins are key drivers of endocrine function

It has long been known that the main secretory cells of exocrine and endocrine glands are connected by gap junctions, made by a variety of connexin species that ensure their electrical and metabolic coupling. Experiments in culture systems and animal models have since provided increasing evidence that connexin signaling contributes to control the biosynthesis and release of secretory products, as well as to the life and death of secretory cells. More recently, genetic studies have further provided the first lines of evidence that connexins also control the function of human glands, which are central to the pathogenesis of major endocrine diseases. Here, we summarize the recent information gathered on connexin signaling in these systems, since the last reviews on the topic, with particular regard to the pancreatic beta cells which produce insulin, and the renal cells which produce renin. These cells are keys to the development of various forms of diabetes and hypertension, respectively, and combine to account for the exploding, worldwide prevalence of the metabolic syndrome. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Gap junction blockers: a potential approach to attenuate morphine withdrawal symptoms

Background

The exact mechanisms of morphine-induced dependence and withdrawal symptoms remain unclear. In order to identify an agent that can prevent withdrawal syndrome, many studies have been performed. This study was aimed to evaluate the effect of gap junction blockers; carbenoxolone (CBX) or mefloquine (MFQ); on morphine withdrawal symptoms in male rat.Adult male Wistar rats (225 – 275 g) were selected randomly and divided into 10 groups. All groups underwent stereotaxic surgery and in order to induce dependency, morphine was administered subcutaneously) Sc) at an interval of 12 hours for nine continuous days. On the ninth day of the experiment, animals received vehicle or CBX (100, 400, 600 μg/10 μl/rat, icv) or MFQ (50, 100 and 200 μg/10 μl/rat, icv) after the last saline or morphine (Sc) injection. Morphine withdrawal symptoms were precipitated by naloxone hydrochloride 10 min after the treatments. The withdrawal signs including: jumping, rearing, genital grooming, abdomen writhing, wet dog shake and stool weight, were recorded for 60 minutes.

Results

Results showed that CBX and MFQ decreased all withdrawal signs; and the analysis indicated that they could attenuate the total withdrawal scores significantly.

Conclusion

Taking together it is concluded that gap junction blockers prevented naloxone-precipitated withdrawal symptoms.     

Short-term experimental acidification of a Welsh stream: comparing the biological effects of hydrogen ions and aluminium

SUMMARY. 1. A soft-water stream iti upland Wales was dosed with sulphuric acid and aluminiutn sulphate at two successive points to create sitnultaneous episodes of low pH, and low pH with increased aluminiutn. Chemical atid biological responses were measured before, during and after the episode and were compared with a reference zone. 2. The pH fell frotn ～7.0 to 4.28 (±0.18 SD) and 5.02 (±0.10) respectively in the acid and aluminium zones. Corresponding aluminium concentrations during the episode were 0.052 g Al m^?3 (±0.008) and 0.347 g Al nr³ (±0.047), the former not differing significantly from the reference zone. The concentration of cadmium rose to 0.002- 0.011 g Cd m^?3in both treated areas, but the concentrations of other metals were unchanged. 3. In situ toxicity tests were performed with macroinvertebrates and fish. Chironomus riparius. Hydropsyche angustipennis and Dinocras cephalotes suffered no mortality. Ecdyonurus venosus, Baetis rhodani and Gammarus pulex showed up to 25% mortality in both treatment zones and further mortalities occurred after the episode. Brown trout Salmo trutta and salmon Valmo salar s howed 7–10% mortality in the acid zone, but 50–87% in the aluminium zone, where salmon had a significantly shorter LT₅₀than trout. 4. The drift of Simuliidae increased during treatment in both acid and aluminium zones. Drift densities of Dixa puherula, Protonemura meyeri, Ephemeralla ignita and Dicranota sp. increased in the aluminium zone. The most pronounced response was by Baetis rhodani in the aluminium zone where drift density increased by ×8.4 during the episode. 5. Baetis rhodani was the only taxon to show a significant decline in benthic density during the treatment, and then only in the aluminium zone. Drift could account for most of the losses. 6. The depth distribution of invertebrates in the substratum differed between zones following treatment. More individuals were present at the surface of the reference zone (1287 m^?2±747) than at the surface of the other zones (<400 m^?2); however, densities at greater depths were similar. These patterns probably reflected differences prior to the treatments.