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The CO2 sensitivity of transjunctional voltage (Vj) gating was studied by dual voltage clamp in oocytes expressing mouse Cx40 or its COOH terminus (CT)-truncated mutant (Cx40-TR).Vj sensitivity, determined by a standardVj protocol (20 mVVj steps, 120 mV maximal), decreased significantly with exposure to 30% CO2. The Boltzmann values of control versus CO2-treated oocytes were:V0?=?36.3 and 48.7 mV,n?=?5.4 and 3.7, andGj min?=?0.21 and 0.31, respectively. CO2 also affected the kinetics ofVj-dependent inactivation of junctional current (Ij); the time constants of two-term exponentialIj decay, measured atVj?=?60 mV, increased significantly with CO2 application. Similar results were obtained with Cx40-TR, suggesting that CT does not play a role in this phenomenon. The sensitivity of Cx40 channels to 100% CO2 was also unaffected by CT truncation. There is evidence that CO2 decreases theVj sensitivity of Cx26, Cx50 and Cx37 as well, whereas it increases that of Cx45 and Cx32 channels. Since Cx40, Cx26, Cx50 and Cx37 gate at the positive side ofVj, whereas Cx45 and Cx32 gate at negativeVj, it is likely thatVj behavior with respect to CO2-induced acidification varies depending on gating polarity, possibly involving the function of the postulatedVj sensor (NH2-terminus). [ABSTRACT FROM AUTHOR]

The major intrinsic protein (MIP26) of bovine lens membranes, purified by HPLC, was incorporated into liposomes and planar bilayers. Permeability of MIP26 channels was studied in liposomes by a spectrophotometric osmotic-swelling assay, and channel electrical properties were monitored in planar bilayers following liposome fusion. Particle formation in liposomes was determined by freeze fracture. MIP26 channels were permeable to KCl and sucrose. In planar bilayers, channel-conductance transitions were observed only after addition of liposomes to both chambers and with voltages greater than +/- 20 mV. Channel open probability decreased progressively as voltage increased, and an open probability of 50% was at 60-80 mV, indicating that the channels are voltage dependent. Histograms of single-channel current amplitudes at 80 mV showed a Gaussian distribution that peaked at 10 pA (approximately 120 pS), after subtraction of 1 pA baseline current. Frequency distributions of open and closed times at 80 mV were single exponential functions with time constants of 0.13 and 1.9 sec, respectively. Open time constants ranged from 0.1 to 0.3 sec, and closed time constants ranged from 1 to 7 sec. Cs+ did not decrease conductance, but reduced mean open time from 0.2 to 0.038 sec and mean closed time from 1.5 to 0.38 sec. The increase in channel flickering with Cs+ occurred in bursts. TEA affected neither conductance nor kinetics. Channel events were also observed in Na+ solutions (zero K+). These data indicate that MIP26 channels are not K(+)-selective channels. Channel characteristics such as: permeability to molecules larger than small ions, conductance greater than 100 pS, long open and closed time constants, etc., are similar to those of gap junction channels. [ABSTRACT FROM AUTHOR]

This 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-1-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. [ABSTRACT FROM AUTHOR]

Electrical uncoupling of crayfish septate axons with acidification has been shown to cause a substantial increase in [Ca2+]i which closely matches in percent the increase in junctional resistance. To determine the origin of [Ca2+]i increase, septate axons have been exposed either to drugs that influence Ca2+ release from internal stores, caffeine and ryanodine, or to treatments that affect Ca2+ entry. A large increase in junctional resistance and [Ca2+]i maxima above controls resulted from addition of caffeine (10-30 mM) to acetate solutions, while a substantial decrease in both parameters was observed when exposure to acetate-caffeine was preceded by caffeine pretreatment. In contrast, ryanodine (1-10 microM) always caused a significant decrease in junctional resistance and [Ca2+]i maxima when applied either together with acetate or both before and with acetate. Calcium channel blockers such as La3+, Cd2+ and nisoldipine had no effect, while an increase in the [Ca2+] of acetate solutions either decreased junctional resistance and [Ca2+]i maxima or had no effect. The data suggest that cytoplasmic acidification causes an increase in [Ca2+]i by releasing Ca2+ from caffeine and ryanodine-sensitive Ca2+ stores. The increase in [Ca2+]i results in a decrease in gap junction conductance. [ABSTRACT FROM AUTHOR]

Neutral-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, and [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 acidification of similar magnitude. With acetate, [Ca2+]i increased by approximately one order of magnitude from basal values of 0.1-0.3 microM. 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 approximately 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. [ABSTRACT FROM AUTHOR]

This 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 of Xenopus 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 X 10(-8) - 1 X 10(-7) M) strongly inhibits uncoupling. The inhibition starts after 30, 50 and 60 min of treatment with 1 X 10(-7), 7 X 10(-8) and 5 X 10(-8) M 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. [ABSTRACT FROM AUTHOR]

Electron microscopy has revealed marked and typical modifications of collagen in the part of the artery affected by Buerger's disease. Thickening of the fibrils due to dissociation of the subfilaments and their fragmentation and disintegration into an amorphous substance are described. The close similarity between these alterations of collagen fibrils in the arteries and the ones previously observed in the collagen of the ganglia has led us to put forward the hypothesis that there is a systematic development in this disease based on a particular lesion of collagen. [ABSTRACT FROM AUTHOR]

The chemical gating of gap junction channels is mediated by cytosolic calcium (Ca 2+ i ) at concentrations ([Ca 2+ ] i ) ranging from high nanomolar (nM) to low micromolar (µM) range. Since the proteins of gap junctions, connexins/innexins, lack high-affinity Ca 2+ -binding sites, most likely gating is mediated by a Ca 2+ -binding protein, calmodulin (CaM) being the best candidate. Indeed, the role of Ca 2+ -CaM in gating is well supported by studies that have tested CaM blockers, CaM expression inhibition, testing of CaM mutants, co-localization of CaM and connexins, existence of CaM-binding sites in connexins/innexins, and expression of connexins (Cx) mutants, among others. Based on these data, since 2000, we have published a Ca 2+ -CaM-cork gating model. Despite convincing evidence for the Ca 2+ -CaM role in gating, a recent study has proposed an alternative gating model that would involve a direct electrostatic Ca 2+ -connexin interaction. However, this study, which tested the effect of unphysiologically high [Ca 2+ ] i on the structure of isolated junctions, reported that neither changes in the channel's pore diameter nor connexin conformational changes are present, in spite of exposure of isolated gap junctions to [Ca 2+ ] i as high at the 20 mM. In conclusion, data generated in the past four decades by multiple experimental approaches have clearly demonstrated the direct role of Ca 2+ -CaM in gap junction channel gating.

Gap junction channels are regulated by gates sensitive to cytosolic acidification and trans-junctional voltage (Vj). We propose that the chemical gate is a calmodulin (CaM) lobe. The fast-Vj gate is made primarily by the connexin's NH 2 -terminus domain (NT). The chemical gate closes the channel slowly and completely, while the fast-Vj gate closes the channel rapidly but incompletely. The chemical gate closes with increased cytosolic calcium concentration [Ca 2+ ] i and with Vj gradients at Vj's negative side. In contrast, the fast-Vj gate closes at the positive or negative side of Vj depending on the connexin (Cx) type. Cxs with positively charged NT close at Vj's negative side, while those with negatively charged NT close at Vj's positive side. Cytosolic acidification alters in opposite ways the sensitivity of the fast-Vj gate: it increases the Vj sensitivity of negative gaters and decreases that of positive gaters. While the fast-Vj gate closes and opens instantaneously, the chemical gate often shows fluctuations, likely to reflect the shifting of the gate (CaM's N-lobe) in and out of the channel's pore., Competing Interests: The author declares no conflicts of interest.

This review describes and discusses unusual axonal structural details and evidence for unmasking sulfhydryl groups (-SH) in axoplasmic membranes resulting from electrical stimulation or asphyxia. Crayfish axons contain fenestrated septa (FS) that, in phase contrast, micrographs appear as repeated striations. In the electron microscope, each septum is made of two cross-sectioned membranes containing ~55 nm pores, each occupied by a microtubule. Thin filaments, which we believe are made of kinesin, bridge the microtubule to the edge of the pore. FS are believed to play a role in axoplasmic flow. The axons also display areas in which axon and sheath glial cell plasma membranes are sharply curved and project into the axoplasm. In freeze-fractures, the protoplasmic leaflet (P-face) of the projections appears as elongated indentations containing parallel chains of particles. The sheath glial cell plasma membrane also contains particles, but they are irregularly aggregated. The axons also display areas where axonal and glial plasma membranes fuse, creating intercellular pores. In axons fixed during electrical stimulation, the plasma membrane, the outer membrane of mitochondria, membranes of other cytoplasmic organelles, and gap junctions increase in electron opacity and thickness, resulting from unmasking of sulfhydryl groups (-SH). Similar changes occur in asphyxiated nerve cords.