Effects of Ca2+ and Zn2+ on trifluoperazine-S100 proteins interactions: induced circular dichroism and fluorescence spectra

Interactions of trifluoperazine (TFP) with S100 proteins, EF-hand type Ca2+-binding proteins, in the presence of Ca2+ and Zn2+ were studied with induced circular dichroism (CD) and fluorescence spectra. The positive CD bands of TFP were induced at around 265 nm by adding either S100a or S100a0 protein in the presence of Ca2+. No CD band of TFP was, however, induced by adding S100b protein in the presence of Ca2+. Addition of Zn2+ to the TFP/S100 protein solutions did not induce any CD band at all. The fluorescence intensity of 2-p-toluidinylnaphthalene 6-sulfonate (TNS) bound to S100a or S100a0 protein decreased by adding TFP in the presence of Ca2+, while that bound to S100b protein decreased by adding TFP in the presence of Zn2+, indicating that TFP binds to S100a protein and S100a0 protein in a Ca2+-dependent manner and to S100b protein in a Zn2+-dependent manner. From these results together with other experimental findings it was suggested that (1) TFP binds to S100a protein and S100a0 protein in the presence of Ca2+, with half-saturation points of 18 and 3 microM, respectively, (2) TFP binds to S100b protein only in the presence of Zn2+, (3) alpha-subunit of S100 protein binds to TFP specifically in a Ca2+-dependent manner and beta-subunit in a Zn2+-dependent manner.     

Structural and ion-binding properties of an S100b protein mixed disulfide: comparison with the reappraised native S100b protein properties

S100b protein, chemically modified by thioethanol groups (linked via disulfide bonds to two out of four Cys per dimer) was largely similar to reduced native S100b protein in its overall structure and differed only by small modifications extending, however, to the whole protein structure. Studies combining direct Ca2+ binding and associated conformational changes revealed that this chemical modification markedly increased the Ca2(+)-binding affinities (especially in the presence of physiological concentrations of K+ and Mg2+) and introduced a strong positive cooperativity. Different binding models are discussed and it emerges that in both proteins the Ca2(+)-binding sites are not equivalent and probably interact. Like the reduced protein, chemically modified S100b protein binds four Zn2+ ions in two classes of sites (of high and low affinities). Whereas the overall Zn2+ affinity was only slightly decreased, the binding sequence was probably reversed by the introduction of thioethanol groups. Moreover, in the presence of zinc, the Ca2+ affinities were higher and even identical, in both proteins.     

S-100b protein regulates aggregation and fusion of cardiolipin vesicles

We have recently shown that S-100b protein interacts with the polar surface of cardiolipin vesicles [6]. This interaction produces changes in the secondary structure of S-100b as well as changes in the structural organization of cardiolipin vesicles. We report here on the effects of S-100b on cardiolipin vesicles as investigated by turbidity, terbium-dipicolinate fluorescence and freeze-fracture. Experiments were carried out in the absence and in the presence of Ca2+. In the absence of Ca2+ (0.1 mM EDTA), S-100b favors the aggregation and fusion of vesicles to some extent. Under these conditions, electron microscope analyses reveal the presence of fused vesicles along with particles similar to those observed in protein reconstituted systems or to lipid particles observed during fusional processes. In the presence of Ca2+, S-100b counteracts the Ca2(+)-dependent tendency of vesicles to aggregate and fuse. Under these conditions, bilayer phases along with hexagonal phases can be observed by electron microscopy. The latter effects of S-100b are not due to chelation of Ca2+ because of the relative concentrations of S-100b and Ca2+ under our experimental conditions and since much larger concentrations of EDTA are required to produce the S-100b effects. We propose that the dimeric nature of S-100b plays a major role in these events. In the absence of Ca2+, the S-100b molecules probably cross-link adjacent vesicles, one subunit contacting one vesicle and the other subunit contacting another vesicle through electrostatic bonds. In the presence of Ca2+, due to the large changes occurring in the conformation of the protein (which loses about 52% of its alpha-helical content), S-100b associates strongly with the polar surface of individual vesicles, thus generating some kind of physical barrier to aggregation and fusion of vesicles.     

Reinvestigation of the sulfhydryl reactivity in bovine brain S100b (beta beta) protein and the microtubule-associated tau proteins. Ca2+ stimulates disulfide cross-linking between the S100b beta-subunit and the microtubule-associated tau(2) protein

Zn2+ and Ca2+ affect the conformation of bovine brain S100b (beta beta) protein and the exposure of its Cys-84 beta. Zn2+ binding to high-affinity sites of native S100b protected the sulfhydryl groups against the thiol-specific reagent 5,5'-dithiobis(2-nitrobenzoate) and antagonized the Ca2+-stimulated reactivity of Cys-84 beta toward the reagent. Spectroscopic studies on the fluorescence properties of labeled S100b with the fluorescent probes bimane and acrylodan at Cys-84 beta confirmed the antagonistic effect of Ca2+ and Zn2+ with respect to the conformational properties of the protein. Measurements of fluorescence dynamics on bimane-labeled S100b indicated that the slow monomer-dimer equilibrium that characterizes the apoprotein at micromolar concentrations was shifted to the monomer form in the presence of Zn2+, a fact that could explain the previously reported Zn2+-dependent increase of S100b protein affinity for calcium. The difference in the effects of Ca2+ and Zn2+ on the reactivity of Cys-84 beta in S100b was confirmed when we observed that Ca2+ and Zn2+ have opposite actions on the formation of disulfide bridges between Cys-84 beta of the S100b beta-subunit and sulfhydryl groups on the microtubule-associated tau(2) protein. Ca2+ stimulated the covalent complex formation whereas Zn2+ inhibited it. We suggest that Zn2+ may have a modulatory function on Cys-84 beta reactivity in the S100b beta-subunit in vivo. Two types of divalent complexes between tau(2) and beta-subunit were formed in the presence of Ca2+, an equimolar complex tau(2)-beta 1 and a complex of one molecule of tau(2) with two beta-subunits, tau(2)-beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen binding by hemocyanin from Levantina hierosolima. I. Exclusion of subunit interactions as a basis for cooperativity.

The effect of oxygen on the distribution of hemocyanin from Levantina hierosolima among the three sedimenting species 20, 60, and 100 S was determined under two sets of experimental conditions: (a) at pH 7.63 in the absence of Ca2+, where oxygen binding in noncooperative; (b) at pH 8.20 in the presence of 2 x 10-3 M Ca2+, where oxygen binding is cooperative. A comparison of the results in the two cases eliminates the possibility that equilibrium between species with different oxygen affinities is responsible for the cooperative behavior. Cooperative oxygen binding was demonstrated for the 20S subunits at pH 8.80 and 1 x 10-3 M Ca2+. Under these conditions, the concentration of calcium is sufficient to affect the oxygen affinity, but the concentration of calcium plus proton is not sufficient to bring about association. The findings exclude interactions among 20S subunits as a basis for cooperativity in hemocyanin.     

19F-n.m.r. study of trifluoperazine-S100 protein interaction: effects of Ca2+ and Zn2+

19F-n.m.r. spectra were measured to investigate the effects of Ca2+ and Zn2+ on the interaction of trifluoperazine (TFP) with three S100 proteins. It was found that TFP binds to S100a and S100ao proteins irrespective of the presence of Ca2+ and Zn2+, while in the presence of Ca2+ the apparent affinity of TFP to the proteins was greater than that in its absence or in the presence of Zn2+. In contrast, the binding affinity of TRP to S100b protein in the presence and absence of metal ions was lower than to S100a and S100ao proteins. These results suggested that TFP binds to each S100 protein in two ways: one is Ca2(+)- or Zn2(+)-dependent specific manner and another is Ca2(+)- or Zn2(+)-independent non-specific manner.     

Effects of pH, temperature and Ca2+ content on the conformation of alpha-lactalbumin in a medium modelling physiological conditions

Data obtained by the intrinsic protein fluorescence technique showed that, in addition to Ca2+ and Mg2+ ions, bovine alpha-lactalbumin also binds physiologically significant Na+ and K+ ions, the nucleotides ATP, ADP, UTP, UDP and UDP-galactose. The release of the bound Ca2+ ions from the protein in a medium modelling physiological conditions (containing Mg2+, Na+, K+, ATP and ADP in physiological concentrations) induced a transition of the protein from the native state of the Ca2+-loaded form to a state which is a mixture of native and and thermally changed states of the apo- and metal bound forms. Any variations in temperature result in changes in the populations of these states. This may be associated with some Ca2+ and temperature dependent regulation of the protein function. Variations of pH within the physiological limits had little influence on the conformation of both Ca2+-loaded and Ca2+-free alpha-lactalbumin.     

Oligomerization state of S100B at nanomolar concentration determined by large-zone analytical gel filtration chromatography.

S100B is a Ca(2+)-binding protein known to be a non-covalently associated dimer, S100B(beta beta), at high concentrations (0.2-3.0 mM) under reducing conditions. The solution structure of apo-S100B (beta beta) shows that the subunits associate in an antiparallel manner to form a tightly packed hydrophobic core at the dimer interface involving six of eight helices and the C-terminal loop (Drohat AC, Amburgey JC, Abildgaard F, Starich MR, Baldisseri D, Weber DJ. 1996. Solution structure of rat apo-S100B (beta beta) as determined by NMR spectroscopy. Biochemistry 35:11577-11588). The C-terminal loop, however, is also known to participate in the binding of S100B to target proteins, so its participation in the dimer interface raises questions as to the physiological relevance of dimeric S100B (beta beta). Therefore, we investigated the oligomerization state of S100B at low concentrations (1-10,000 nM) using large-zone analytical gel filtration chromatography with 35S-labeled S100B. We found that S100B exists (> 99%) as a non-covalently associated dimer, S100B (beta beta), at 1 nM subunit concentration (500 pM dimer) in the presence or absence of saturating levels of Ca2+, which implies a dissociation constant in the picomolar range or lower. These results demonstrate for the first time that in reducing environments and at physiological concentrations, S100B exists as dimeric S100B (beta beta) in the presence or absence of Ca2+, and that the non-covalent dimer is most likely the form of S100B presented to target proteins.     

Redox sensitive cysteine residues in calbindin D28k are structurally and functionally important

Human calbindin D(28k) is a Ca(2+) binding protein that has been implicated in the protection of cells against apoptosis. In this study, the structural and functional significance of the five cysteine residues present in this protein have been investigated through a series of cysteine-to-serine mutations. The mutants were studied under relevant physiological redox potentials in which conformational changes were monitored using ANS binding. Urea-induced denaturations, as monitored by intrinsic tryptophan fluorescence, were also carried out to compare their relative stability. It was shown that the two N-terminal cysteine residues undergo a redox-driven structural change consistent with disulfide bond formation. The other cysteine residues are not by themselves sufficient at inducing structural change, but they accentuate the disulfide-dependent conformational change in a redox-dependent manner. Mass spectrometry data show that the three C-terminal cysteine residues can be modified by glutathione. Furthermore, under oxidizing conditions, the data display additional species consistent with the conversion of cysteine thiols to sulfenic acids and disulfides to disulfide-S-monoxides. The biological function of calbindin D(28k) appears to be tied to the redox state of the cysteine residues. The two N-terminal cysteine residues are required for activation of myo-inositol monophosphatase, and enzyme activation is enhanced under conditions in which these residues are oxidized. Last, oxidized calbindin D(28k) binds Ca(2+) with lower affinity than does the reduced protein.     

Cosecretion of chaperones and low-molecular-size medium additives increases the yield of recombinant disulfide-bridged proteins

Attempts were made to engineer the periplasm of Escherichia coli to an expression compartment of heterologous proteins in their native conformation. As a first approach the low-molecular-size additive L-arginine and the redox compound glutathione (GSH) were added to the culture medium. Addition of 0.4 M L-arginine and 5 mM reduced GSH increased the yield of a native tissue-type plasminogen activator variant (rPA), consisting of the kringle-2 and the protease domain, and a single-chain antibody fragment (scFv) up to 10- and 37-fold, respectively. A variety of other medium additives also had positive effects on the yield of rPA. In a second set of experiments, the effects of cosecreted ATP-independent molecular chaperones on the yields of native therapeutic proteins were investigated. At optimized conditions, cosecretion of E. coli DnaJ or murine Hsp25 increased the yield of native rPA by a factor of 170 and 125, respectively. Cosecretion of DnaJ also dramatically increased the amount of a second model protein, native proinsulin, in the periplasm. The results of this study are anticipated to initiate a series of new approaches to increase the yields of native, disulfide-bridged, recombinant proteins in the periplasm of E. coli.     

Calcium-binding properties of two high affinity calcium-binding proteins from squid optic lobe

We have studied the calcium-binding properties of two high affinity calcium-binding proteins from squid optic lobes: one, squid calmodulin (SCaM), similar to bovine brain calmodulin (BCaM), the other, squid calcium-binding protein (SCaBP), distinct (Head, J.F., Spielberg, S., and Kaminer, B. (1983) Biochem J. 209, 797-802). Equilibrium dialysis measurements on the squid proteins (and BCaM) were made at 100 mM KCl in the presence and absence of 3 mM Mg2+, and at 400 mM KCl in the presence of 3 mM Mg2+, which more closely resembles the conditions in the squid. SCaM, SCaBP, and BCaM each bind a maximum of 4 Ca2+ ions/molecule of protein under the ionic conditions tested. SCaBP has a higher affinity than SCaM or BCaM for Ca2+ at 100 mM KCl in the absence of Mg2+. However, in the presence of Mg2+, half-maximal binding to SCaBP occurs at a similar pCa value to that observed with calmodulin. Increasing the KCl concentration reduces the affinity of all three proteins for Ca2+. UV absorption measurements showed that the binding of 4 Ca2+ ions/molecule is necessary to complete spectral changes in SCaBP, compared to two for the calmodulins. While Ca2+ causes perturbations in aromatic chromophores in SCaM and SCaBP, Mg2+ causes a significant perturbation only in SCaBP. These Mg2+-induced changes differ qualitatively from those induced by Ca2+.     

Impaired cardiac contractility response to hemodynamic stress in S100A1-deficient mice

Ca(2+) signaling plays a central role in cardiac contractility and adaptation to increased hemodynamic demand. We have generated mice with a targeted deletion of the S100A1 gene coding for the major cardiac isoform of the large multigenic S100 family of EF hand Ca(2+)-binding proteins. S100A1(-/-) mice have normal cardiac function under baseline conditions but have significantly reduced contraction rate and relaxation rate responses to beta-adrenergic stimulation that are associated with a reduced Ca(2+) sensitivity. In S100A1(-/-) mice, basal left-ventricular contractility deteriorated following 3-week pressure overload by thoracic aorta constriction despite a normal adaptive hypertrophy. Surprisingly, heterozygotes also had an impaired response to acute beta-adrenergic stimulation but maintained normal contractility in response to chronic pressure overload that coincided with S100A1 upregulation to wild-type levels. In contrast to other genetic models with impaired cardiac contractility, loss of S100A1 did not lead to cardiac hypertrophy or dilation in aged mice. The data demonstrate that high S100A1 protein levels are essential for the cardiac reserve and adaptation to acute and chronic hemodynamic stress in vivo.     

Interactions between the microtubule-associated tau proteins and S100b regulate tau phosphorylation by the Ca2+/calmodulin-dependent protein kinase II

Binding between the microtubule-associated tau protein and S100b protein was demonstrated by affinity chromatography and cross-linking experiments and was manifested in the effect of S100b on tau protein phosphorylation by protein kinase II. All three expressions of the binding showed that S100b discriminates among the four species of tau, revealing for the first time that the different kinds of tau may differ functionally. Noncovalent interaction between tau and S100b depended on the presence of Ca2+ or Zn2+ and resulted in total inhibition of tau phosphorylation by protein kinase II. In the absence of reducing agent, covalent binding studies between Cys84 beta in the carboxyl-terminal region of the S100b-beta subunit and tau proteins confirmed interactions between the two proteins. It is suggested that the homologous calcium-binding domain that characterizes the carboxyl terminus of S100 and the tubulin subunit may be responsible for the common interaction of both proteins with tau proteins. The physicochemical relationship between S100 subunits and p11, the subunit of a substrate for tyrosine kinase, and their similarity in interaction with protein kinase substrates are discussed.     

Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.     

Purification and properties of a novel nucleotide-hydrolysing enzyme (5''-nucleotidase) from Boophilus microplus.

Conformational changes in the beta-subunit of the bovine brain Ca2+-binding protein S100b (S100-beta) accompanying Ca2+ binding were investigated by analysis of the spectroscopic properties of the single tyrosine residue (Tyr17 beta) and flow-dialysis binding experiments. S100-beta binds Ca2+ sequentially at two sites to change the conformation of the protein. The first Ca2+ ion binds to site II beta, a typical Ca2+-binding site in the C-terminal region, and it does not significantly perturb the proximal environment of Tyr17 beta. After the first site is occupied, another Ca2+ ion binds to the N-terminal Ca2+-binding site, I beta, and strengthens a hydrogen bond between Tyr17 beta and a neighbouring carboxylate acceptor group, which results in a large increase in the Tyr17 beta fluorescence spectrum half-width and a positive absorption and c.d. signal between 290 and 275 nm. Ca2+ binding to the S100b.Zn2+6 complex, studied by flow-dialysis and fluorescence measurements showed that, although Zn2+ ions increase the affinity of S100b protein for Ca2+, the Ca2+-binding sequence was not changed. Tb3+ (terbium ion) binding studies on the S100b.Zn2+6 complex proved that Tb3+ antagonizes only Ca2+ binding site II beta and confirmed the sequential occupation of Ca2+-binding sites on the S100b.Zn2+6 complex.     

Multiple conductance states of the purified calcium release channel complex from skeletal sarcoplasmic reticulum.

The CHAPS-solubilized and purified 30S ryanodine receptor protein complex from skeletal sarcoplasmic reticulum (SR) was incorporated into planar lipid bilayers. The resulting electrical activity displayed similar responses to agents such as Ca2+, ATP, ryanodine, or caffeine as the native Ca2+ release channel, confirming the identification of the 30S complex as the Ca2+ release channel. The purified channel was permeable to monovalent ions such as Na+, with the permeability ratio PCa/PNa approximately 5, and was highly selective for cations over anions. The purified channel also showed at least four distinct conductance levels for both Na+ and Ca2+ conducting ions, with the major subconducting level in NaCl buffers possessing half the conductance value of the main conductance state. These levels may be produced by intrinsic subconductances present within the channel oligomer. Several of these conductances may be cooperatively coupled to produce the characteristic 100 +/- 10 pS unitary Ca2+ conductance of the native channel.     

Binding study of metal ions to S100 protein: 43Ca, 25Mg, 67Zn and 39K n.m.r.

The interactions of the S100 protein (S100) with metal cations such as Ca2+, Mg2+, Zn2+ and K+ were studied by the metal n.m.r. spectroscopy. The line widths of 43Ca, 25Mg, 67Zn and 39K n.m.r. markedly increased by adding all S100s. A broad 43Ca n.m.r. band of Ca(2+)-S100a solution was not affected by Zn2+ and K+, while it was greatly decreased by adding Mg2+. The 43Ca n.m.r. spectra of Ca(2+)-S100a0 and -S100b solutions consisted of two slow-exchangeable signals which corresponded to Ca2+ bound to two environmentally different sites of the S100a0. These two 43Ca n.m.r. signals were not affected by Zn2+ and K+. The line width of broad 25Mg n.m.r. band of the Mg(2+)-S100 solution greatly decreased by adding Ca2+, while it did not change by adding Zn2+ and K+. Further, the addition of Ca2+, Mg2+ and K+ did not affect the line width of the 67Zn n.m.r. of the Zn(2+)-S100 solutions. These findings suggest that: (1) Mg2+ binds to all S100s, and at least one of the Mg2+ binding sites of S100 molecule is the same as the Ca2+ binding site; (2) Zn2+ binds to S100s, although the binding site(s) is/are different from Ca(2+)- or Mg(2+)-binding site(s), and the environment of Zn2+ nuclei will not change even though Ca2+ binds to S100s.     

Synergistic Ca2+ and Cu2+ requirements of the FGF1-S100A13 interaction measured by quartz crystal microbalance: an initial step in amlexanox-reversible non-classical release of FGF1

It is known that fibroblast growth factor-1 (FGF1) lacking a conventional signal peptide sequence shows non-classical release independent of the endoplasmic reticulum-Golgi system. Recent studies reveal that FGF1 is co-released with S100A13, a Ca2+-binding protein that acts as an extracellular cargo molecule. Although both FGF1 and S100A13 are Cu2+-binding proteins, the role of Cu2+, as well as that of Ca2+, in non-classical release, remains to be clarified. In the present study we examined the requirements of both metal ions for the interaction between these two proteins. The addition of Ca2+ significantly increased the ka value, while decreasing the KD value, for the interaction between Strep-tagII-S100A13 and GST-FGF1; both values were obtained by use of a quartz crystal microbalance, a real-time mass-measuring device. The EC50 of Ca2+ to enhance the interaction was 10.11 microM. Although the addition of Cu2+ alone had no effect, it caused a marked potentiation of the Ca2+-enhanced interaction. The EC50 of Cu2+ for the potentiation was 50.45 nM. On the other hand, the EC50 of Ca2+ and the KD values were decreased from 11.69 to 2.07 microM and 0.75 to 0.38 x 10(-7)M, respectively, by the addition of 200 nM Cu2+. The Cu2+-induced potentiation of this interaction was abolished by amlexanox, which inhibits non-classical release of FGF1. All of these findings suggest that synergistic effects of Ca2+ and Cu2+ play a key role in the interaction between FGF1 and S100A13, which is the initial step in non-classical release of FGF1.     

The gap junction cellular internet: connexin hemichannels enter the signalling limelight

Cxs (connexins), the protein subunits forming gap junction intercellular communication channels, are transported to the plasma membrane after oligomerizing into hexameric assemblies called connexin hemichannels (CxHcs) or connexons, which dock head-to-head with partner hexameric channels positioned on neighbouring cells. The double membrane channel or gap junction generated directly couples the cytoplasms of interacting cells and underpins the integration and co-ordination of cellular metabolism, signalling and functions, such as secretion or contraction in cell assemblies. In contrast, CxHcs prior to forming gap junctions provide a pathway for the release from cells of ATP, glutamate, NAD+ and prostaglandin E2, which act as paracrine messengers. ATP activates purinergic receptors on neighbouring cells and forms the basis of intercellular Ca2+ signal propagation, complementing that occuring more directly via gap junctions. CxHcs open in response to various types of external changes, including mechanical, shear, ionic and ischaemic stress. In addition, CxHcs are influenced by intracellular signals, such as membrane potential, phosphorylation and redox status, which translate external stresses to CxHc responses. Also, recent studies demonstrate that cytoplasmic Ca2+ changes in the physiological range act to trigger CxHc opening, indicating their involvement under normal non-pathological conditions. CxHcs not only respond to cytoplasmic Ca2+, but also determine cytoplasmic Ca2+, as they are large conductance channels, suggesting a prominent role in cellular Ca2+ homoeostasis and signalling. The functions of gap-junction channels and CxHcs have been difficult to separate, but synthetic peptides that mimic short sequences in the Cx subunit are emerging as promising tools to determine the role of CxHcs in physiology and pathology.