Camillo Peracchia

Lens cell-to-cell channel protein: I. Self-assembly into liposomes and permeability regulation by calmodulin.

SummaryLens fibers are coupled by communicating junctions which contain a 28-kDalton protein (MIP26) believed to be the main component of the cell-to-cell channel. To study the permeability properties and regulation of these channels, anin vitro system has been developed in which MIP26 isolated from calf lens is incorporated into liposomes and the resulting channels are studied spectrophotometrically by a swelling assay. Liposome vesicles were prepared using a sonication/resuspension method. Incorporation efficiency was monitored by freeze-fracture. Vesicles were resuspended in 6% Dextran T-10. Assay buffer was identical, except for isotonic substitution of sucrose for T-10. MIP26-incorporated (but not control) vesicles swell under isotonic conditions indicating sucrose entry (via channels) followed by water to maintain osmotic balance. In the absence of calmodulin, calcium ion has no effect on channel permeability. On the contrary, vesicles prepared with equimolar amounts of MIP26 and CaM do not swell in the presence of calcium ion, indicating that the channels can be closed. Addition of EGTA to these vesicles reinitiates swelling—evidence that the channel gating mechanism is reversible. Magnesium ion has no effect on either type of vesicle.

Two distinct gating mechanisms in gap junction channels: CO2-sensitive and voltage-sensitive

The chemical gating of single-gap junction channels was studied by the dual whole-cell voltage-clamp method in HeLa cells transfected with connexin43 (HeLa43) and in fibroblasts from sciatic nerves. Junctional current (Ij), single-channel conductance, and Ij kinetics were studied in cell pairs during CO2 uncoupling and recoupling at small transjunctional voltages (Vj < 35 mV: Vj gating absent) and at high Vj (Vj > 40 mV: Vj gating strongly activated). In the absence of Vj gating, CO2 exclusively caused Ij slow transitions from open to closed channel states (mean transition time: approximately 10 ms), corresponding to a single-channel conductance of approximately 120 pS. At Vj > 40 mV, Vj gating induced fast Ij flickering between open, gamma j(main state), and residual, gamma j(residual), states (transition time: approximately 2 ms). The ratio gamma j(main state)/gamma j(residual) was approximately 4-5. No obvious correlation between Ij fast flickering and CO2 treatment was noticed. At high Vj, in addition to slow Ij transitions between open and closed states, CO2 induced slow transitions between residual and closed states. During recoupling, each channel reopened by a slow transition (mean transition time: approximately 10 ms) from closed to open state (rarely from closed to residual state). Fast Ij flickering between open and residual states followed. The data are in agreement with the hypothesis that gap junction channels possess two gating mechanisms, and indicate that CO2 induces channel gating exclusively by the slow gating mechanism.

INHIBITION OF CALMODULIN EXPRESSION PREVENTS LOW-PH-INDUCED GAP JUNCTION UNCOUPLING IN XENOPUS OOCYTES

The relationship among intracellular pH (pHi), −log10 intracellular Ca2+ concentration (pCai) and gap junctional conductance, the participation of Ca2+ stores, and the role of calmodulin in channel regulation have been studied inXenopus oocytes, expressing the native connexin (Cx38), exposed to external solutions bubbled with 100% CO2. The time courses of pHi [measured with 2′,7′-bis(2-carboxyethyl)-5,6-carboxylluorscein (BCECF)], (pCai) (measured with the membrane-associated fura-C18) and junctional conductance (measured with a double voltage-clamp protocol) were compared. The data obtained confirm previous evidence for a closer relationship of junctional conductance with (pCai) than with pHi. Evidence for an inhibitory effect of intracellularly injected ruthenium red or 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) on CO2-induced uncoupling, coupled to negative results with Ca2+-free external solutions, point to a low-pHi-induced Ca2+ release from internal stores, likely to be primarily mitochondria. The hypothesis proposing a participation of calmodulin in channel gating was tested by studying the effects of calmodulin expression inhibition by intracellular injection of oligonucleotides antisense to the two calmodulin mRNAs expressed in the oocytes. Calmodulin mRNA was permanently eliminated in 5 h. The oocytes injected with the antisense nucleotides progressively lost the capacity to uncouple with CO2 within 72 h. The effect of CO2 on junctional conductance was reduced by ≈60% in 24 h, by ≈76% in 48 h and by ≈93% in 72 h. Oocytes that had lost gating sensitivity to CO2. partially recovered gating competency following calmodulin injection. The data suggest that lowered pHi uncouples gap junctions by a Ca2+-calmodulin-mediated mechanism.

The paranodal axo-glial junction in the central nervous system studied with thin sections and freeze-fracture

The lateral belts of the myelin sheath wind helically around the paranodal region of the axon. The lateral belt coil leaves an imprint on the axon and thus confers a conspicuous, indented configuration to the freeze-fracture faces of the axolemma. The contact area between the axolemma and the lateral belt membrane is the site of an extensive and unusual cell junction (axo-glial junction). In thin sections the junctional membranes are undulated, the peaks in one membrane mirroring the peaks in the other. The transverse bands (intercellular septa) are in register with the undulations. The intercellular space measures about 30 A. In freeze-fracture replicas, the undulations are evident as alternating ridges and grooves which run strictly parallel and are oriented at an angle with respect to the helical path of the lateral belt. Both junctional membranes contain parallel rows of intramembrane particles which coincide with the ridges and grooves and, therefore, with the intercellular septa. The center-to-center distance between septa or, equivalently, between adjacent rows of particles measures approximately 250 A. Although the axo-glial junction possesses structurally symmetrical features, there exist important differences between the two junctional membranes. The intramembrane particles of the glial and the axonal membrane differ in cleaving properties. Furthermore, in some of the fibres the E face of the junctional axolemma displays a crystalline array which is not present in the fracture faces of the glial membrane. The axo-glial junction is limited to the paranodal region, although the inner belt of the myelin sheath may form occasional junctional spots with the internodal region proper of the axolemma. The classification and the presumptive functions of the paranodal axo-glial junction are briefly discussed.

Is calmodulin involved in the regulation of gap junction permeability

The cell-to-cell channels of gap junctions mediate the direct exchange of ions and small metabolites between neighboring cells. A number of studies have shown that these channels close when the intracellular free calcium or hydrogen concentration increases, the result being cell-to-cell uncoupling. Since most of the calcium-activated biological phenomena are mediated by calmodulin (CaM), an obvious question is whether or not CaM is involved in the mechanism of cell coupling regulation. Data from the present study, showing the inhibitory effects of a calmodulin blocker on electrical uncoupling in Xenopus embryo cells, suggest a possible CaM participation in the uncoupling mechanism.

Increase in gap junction resistance with acidification in crayfish septate axons is closely related to changes in intracellular calcium but not hydrogen ion concentration

SummaryNeutral-carrier pH-and Ca-sensitive microelectrodes were used to investigate the relationship between junctional electrical resistance and either pHi or [Ca2+]i in crayfish septate axons uncoupled by acidification. For measuring [Ca2+]i a new neutral carrier sensor sensitive to picomolar [Ca2+] and virtually insensitive to other ions was used. Uncoupling was induced by superfusing the axons with Na-acetate solutions (pH 6.3). With acetate, the time course of changes in junctional resistance differed markedly from that of pHi or [H+]i peaked 40–90 sec before junctional resistance. The difference in shape and peak time between pHi and junctional resistance curves caused significant hysteresis in the pHi versus junctional resistance relationship. In addition, junctional resistance maxima reached with slow acidification rates were 3–4 times greater than those with fast acidifications of similar magnitude. With acetate, [Ca2+]i, increased by approximately one order of magnitude from basal values of 0.1–0.3 μm. The curves describing the time course of changes in [Ca2+]i and junctional resistance matched well with each other in shape, peak time and magnitude. Both junctional resistance and [Ca2+]i recovered following a single exponential decay with a time constant of ∼2 min. Different rates of acidification caused increases in [Ca2+]i and junctional resistance comparable in magnitude. The data indicate that the increase in junctional resistance induced by acidification is more closely related to [Ca2+]i than to [H+]i.

Effects of the anesthetics heptanol, halothane and isoflurane on gap junction conductance in crayfish septate axons: A calcium- and hydrogen-independent phenomenon potentiated by caffeine and theophylline, and inhibited by 4-aminopyridine

SummaryThis study has monitored junctional and nonjunctional resistance. [Ca2+]i and [H−]i, and the effects of various drugs in crayfish septate axons exposed to neutral anesthetics. The uncoupling efficiency of heptanol and halothane is significantly potentiated by caffeine and theophylline. The modest uncoupling effects of isoflurane, described here for the first time, are also enhanced by caffeine. Heptanol causes a decrease in [Ca2+]i and [H+]i both in the presence and absence of either caffeine or theophylline. A similar but transient effect on [Ca2+]i is observed with halothane. 4-Aminopyridine strongly inhibits the uncoupling effects of heptanol. The observed decrease in [Ca2−]i with heptanol and halothane and negative results obtained with different [Ca2+]o, Ca2+-channel blockers (nisoldipine and Cd2+) and ryanodine speak against a Ca2+ participation. Negative results obtained with 3-isobutyl-l-methylxanthine, forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester and H7, superfused in the presence and absence of caffeine and/or heptanol. indicate that neither the heptanol effects nor their potentiation by caffeine are mediated by cyclic nucleotides, adenosine receptors and kinase C. The data suggest a direct effect of anesthetics. possibly involving both polar and hydrophobic interactions with channel proteins. Xanthines and 4-aminopyridine may participate by influencing polar interactions. The potentiating effect of xanthines on cell-to-cell uncoupling by anesthetics may provide some clues on the nature of cardiac arrhythmias in patients treated with theophylline during halothane anesthesia.

Communicating junctions and calmodulin: Inhibition of electrical uncoupling inXenopus embryo by calmidazolium

SummaryThis paper reports the inhibitory effects of calmidazolium (CDZ), a calmodulin inhibitor, on electrical uncoupling by CO2. Membrane potential and coupling ratio (V2/V1) are measured in two neighboring cells ofXenopus embryos (16 to 64 cell stage) for periods as long as 5.5 hr. Upon exposure to 100% CO2, control cells consistently uncouple even if the CO2 treatments are repeated every 15 min for 2.5 hr. CDZ (5×10−8−1×10−7m) strongly inhibits uncoupling. The inhibition starts after 30, 50 and 60 min of treatment with 1×10−7, 7×10−8 and 5×10−8m CDZ, respectively, is concentration-dependent and partially reversible. In the absence of CO2, CDZ also improves electrical coupling. CDZ has no significant effect on membrane potential and nonjunctional membrane resistance. These data suggest that calmodulin or a calmodulin-like protein participates in the uncoupling mechanism.

Chimeric evidence for a role of the connexin cytoplasmic loop in gap junction channel gating

Gap junction channels are regulated by gates that close upon exposure to 100% CO2, probably via an increase in intracellular Ca2+ concentration, [Ca2+]i. For defining connexin (Cx) domain(s) involved in gating, we have studied chemical and voltage gating sensitivities of channels made of Cx38, Cx32 or chimeras of the above, expressed inXenopus oocytes. Cx38 channels are more sensitive to CO2 and voltage than those of Cx32. A 3-min exposure to 100% CO2 reduces Cx38 junctional conductance (Gj) to 0% of initial values at a maximum rate of 25%/min, whereas even a 15-min exposure to 100% CO2 reduces Cx32Gj by approximately 50% at the slow rate of 9%/min. Of the various Cx32 mutants and Cx32/38 chimeras constructed, two chimeras (Cx32/38I and Cx32/38N) expressed functional channels. Upon exposure to CO2, channels made of Cx32/38I (Cx32 inner loop replaced with that of Cx38) reproduced precisely the uncoupling behavior of Cx38 channels in uncoupling magnitude and in both uncoupling and recoupling rates, whereas channels made of Cx32/38N (N-terminus replaced) behaved closer to Cx32 than to Cx38 channels. Cx38 channels were more voltage sensitive than those of Cx32, withV0, i.e., the transjunctional voltage at which voltage-sensitive conductance is half maximal = 35.3 and 59.5 mV andn, i.e., equivalent gating charge = 3.3 and 2.1, respectively. Of the two chimeras, Cx32/38I channels were similar to Cx38 channels, withV0 = 40.6 mVGj min i.e., the theoretical minimal normalized junctional conductance = 0.35 andn = 3.0, whereas Cx32/38 N channels displayed very low voltage sensitivity, withV0 = 84.8 mVGj min = 0.5 andn = 1.1. The data suggest that the inner loop plays a major role in pH and voltage gating sensitivity, but whether other domains also participate in the gating mechanism cannot be excluded.

Calmodulin interacts with a C-terminus peptide from the lens membrane protein MIP26

Lens fiber cells are coupled by communicating junctions that comprise over 50% of their appositional surfaces. The main intrinsic protein (MIP26) of lens fibers is a 28.2 kDa protein that forms large gap junction-like channels in reconstituted systems. Previously, we have shown that Ca(++)-activated calmodulin (CaM) regulates the permeability of reconstituted MIP26 channels and changes the conformation of MIP26, as measured by intrinsic fluorescence and circular dichroism spectroscopy. Examination of the MIP26 amino acid sequence has revealed a basic amphiphilic alpha-helical segment (Pep C) on the C-terminus with residue distribution similar to that found in other CaM binding proteins. To test the interaction between the amphiphilic segment and CaM, both a 20-mer peptide and trp-substituted fluorescent analog have been synthesized and purified by HPLC. Evidence from spectrofluorometric titration shows that the Pep C binds with CaM in 1:1 stoichiometry and with a kd of approximately 10 nM. Neither Ca++ nor H+ alone affects the conformation of the Pep C. However, when mixed with CaM the Pep C undergoes both a dramatic blue-shift in tryptophan fluorescence emission, indicative of strong hydrophobic interaction, and an increase in circular dichroism absorption in the alpha-helical region. Additional fluorescence blue-shift and alpha-helical content occur when Ca++ is added to the CaM:Pep C complex.