Incompatibility of connexin 40 and 43 Hemichannels in gap junctions between mammalian cells is determined by intracellular domains.

Murine connexin 40 (Cx40) and connexin 43 (Cx43) do not form functional heterotypic gap junction channels. This property may contribute to the preferential propagation of action potentials in murine conductive myocardium (expressing Cx40) which is surrounded by working myocardium, expressing Cx43. When mouse Cx40 and Cx43 were individually expressed in cocultured human HeLa cells, no punctate immunofluorescent signals were detected on apposed plasma membranes between different transfectants, using antibodies specific for each connexin, suggesting that Cx40 and Cx43 hemichannels do not dock to each other. We wanted to identify domains in these connexin proteins which are responsible for the incompatibility. Thus, we expressed in HeLa cells several chimeric gene constructs in which different extracellular and intracellular domains of Cx43 had been spliced into the corresponding regions of Cx40. We found that exchange of both extracellular loops (E1 and E2) in this system (Cx40*43E1,2) was required for formation of homotypic and heterotypic conductive channels, although the electrical properties differed from those of Cx40 or Cx43 channels. Thus, the extracellular domains of Cx43 can be directed to form functional homo- and heterotypic channels. Another chimeric construct in which both extracellular domains and the central cytoplasmic loop (E1, E2, and C2) of Cx43 were spliced into Cx40 (Cx40*43E1,2,C2) led to heterotypic coupling only with Cx43 and not with Cx40 transfectants. Thus, the central cytoplasmic loop of Cx43 contributed to selectivity. A third construct, in which only the C-terminal domain (C3) of Cx43 was spliced into Cx40, i.e., Cx40*43C3, showed neither homotypic nor heterotypic coupling with Cx40 and Cx43 transfectants, suggesting that the C-terminal region of Cx43 determined incompatibility.

Connexin35 mediates electrical transmission at mixed synapses on Mauthner cells.

Auditory afferents terminating as "large myelinated club endings" on goldfish Mauthner cells are identifiable "mixed" (electrical and chemical) synaptic terminals that offer the unique opportunity to correlate physiological properties with biochemical composition and specific ultrastructural features of individual synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we demonstrate that gap junctions at these synapses contain connexin35 (Cx35). This connexin is the fish ortholog of the neuron-specific human and mouse connexin36 that is reported to be widely distributed in mammalian brain and to be responsible for electrical coupling between many types of neurons. Similarly, connexin35 was found at gap junctions between neurons in other brain regions, suggesting that connexin35-mediated electrical transmission is common in goldfish brain. Conductance of gap junction channels at large myelinated club endings is known to be dynamically modulated by the activity of their colocalized glutamatergic synapses. We show evidence by confocal microscopy for the presence of the NR1 subunit of the NMDA glutamate receptor subtype, proposed to be a key regulatory element, at these large endings. Furthermore, we also show evidence by FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor is present at postsynaptic densities closely associated with gap junction plaques containing Cx35 at mixed synapses across the goldfish hindbrain. Given the widespread distribution of electrical synapses and glutamate receptors, our results suggest that the plastic properties observed at these identifiable junctions may apply to other electrical synapses, including those in mammalian brain.

Functional expression of the new gap junction gene connexin47 transcribed in mouse brain and spinal cord neurons.

A new mouse gap junction gene that codes for a protein of 46,551 Da has been identified and designated connexin47 (Cx47). It mapped as a single-copy gene to mouse chromosome 11. In human HeLa cells and Xenopus oocytes, expression of mouse Cx47 or a fusion protein of Cx47 and enhanced green fluorescent protein induced intercellular channels that displayed strong sensitivity to transjunctional voltage. Tracer injections in Cx47-transfected HeLa cells revealed intercellular diffusion of neurobiotin, Lucifer yellow, and 4',6-diamidino-2-phenylindole. Recordings of single channels yielded a unitary conductance of 55 pS main state and 8 pS substate. Cx47 mRNA expression was high in spinal cord and brain but was not found in retina, liver, heart, and lung. A low level of Cx47 expression was detected in ovaries. In situ hybridizations demonstrated high expression in alpha motor neurons of the spinal cord, pyramidal cells of the cortex and hippocampus, granular and molecular layers of the dentate gyrus, and Purkinje cells of the cerebellum as well as several nuclei of the brainstem. This expression pattern is distinct from, although partially overlapping with, that of the neuronally expressed connexin36 gene. Thus, electrical synapses in adult mammalian brain are likely to consist of different connexin proteins depending on the neuronal subtype.

Single-channel SCAM identifies pore-lining residues in the first extracellular loop and first transmembrane domains of Cx46 hemichannels.

Gap junction (GJ) channels provide an important pathway for direct intercellular transmission of signaling molecules. Previously we showed that fixed negative charges in the first extracellular loop domain (E1) strongly influence charge selectivity, conductance, and rectification of channels and hemichannels formed of Cx46. Here, using excised patches containing Cx46 hemichannels, we applied the substituted cysteine accessibility method (SCAM) at the single channel level to residues in E1 to determine if they are pore-lining. We demonstrate residues D51, G46, and E43 at the amino end of E1 are accessible to modification in open hemichannels to positively and negatively charged methanethiosulfonate (MTS) reagents added to cytoplasmic or extracellular sides. Positional effects of modification along the length of the pore and opposing effects of oppositely charged modifying reagents on hemichannel conductance and rectification are consistent with placement in the channel pore and indicate a dominant electrostatic influence of the side chains of accessible residues on ion fluxes. Hemichannels modified by MTS-EA+, MTS-ET+, or MTS-ES- were refractory to further modification and effects of substitutions with positively charged residues that electrostatically mimicked those caused by modification with the positively charged MTS reagents were similar, indicating all six subunits were likely modified. The large reductions in conductance caused by MTS-ET+ were visible as stepwise reductions in single-channel current, indicative of reactions occurring at individual subunits. Extension of single-channel SCAM using MTS-ET+ into the first transmembrane domain, TM1, revealed continued accessibility at the extracellular end at A39 and L35. The topologically complementary region in TM3 showed no evidence of reactivity. Structural models show GJ channels in the extracellular gap to have continuous inner and outer walls of protein. If representative of open channels and hemichannels, these data indicate E1 as constituting a significant portion of this inner, pore-forming wall, and TM1 contributing as pore-lining in the extracellular portion of transmembrane span.

Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation.

pH is a potent modulator of gap junction (GJ) mediated cell-cell communication. Mechanisms proposed for closure of GJ channels by acidification include direct actions of H+ on GJ proteins and indirect actions mediated by soluble intermediates. Here we report on the effects of acidification on connexin (Cx)46 cell-cell channels expressed in Neuro-2a cells and Cx46 hemichannels expressed in Xenopus oocytes. Effects of acidification on hemichannels were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the exposed hemichannel face. Both types of Cx46 channel were found to be sensitive to cytoplasmic pH, and two effects were evident. A rapid and reversible closure was reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible loss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at pH 6.4. Hemichannels remained sensitive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was maintained near resting ( approximately 10(-7) M) levels. Thus, Cx46 hemichannel pH gating does not depend on cytoplasmic intermediates or a rise in [Ca2+]. Rapid application of low pH to the cytoplasmic face of open hemichannels resulted in a minimum latency to closure near zero, indicating that Cx46 hemichannels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible from the cytoplasmic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+ permeation through the pore to reach an internal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is located directly on Cx46 near the pore entrance on the cytoplasmic side.

Effect of transmural vagal stimulation on electrotonic current spread in the rabbit sinoatrial node.

OBJECTIVE: The effect of vagal stimulation on the decay of electrotonic potential caused by intracellular current injection and on input resistance was measured in the sinoatrial node of isolated rabbit right atria. METHODS: Studies were performed on New Zealand White rabbits weighing approximately 2-3 kg. Vagal stimulation was achieved by transmural stimulation of intramural nerve fibres in the presence of propranolol. A K+ perfused suction electrode was used to inject hyperpolarising current pulses; input resistance was measured by means of a double barrel microelectrode. RESULTS: Vagal stimulation which caused a 14-20% increase of cycle length diminished electronic potential significantly by a decrease of membrane resistance. The input resistance of the sinoatrial node was not affected. Space constant values calculated by using either a one or a two dimensional model of electrotonic current spread were decreased on average by 13% and 14% respectively. CONCLUSIONS: The results from this study show that vagal stimulation which gave rise to a moderate negative chronotropic effect and marked changes in action potential configuration of nodal fibres affects the electrotonic interaction within the sinoatrial node. This may have consequences for the electrical activity and synchronisation of the sinoatrial nodal fibres.

Pore-lining residues identified by single channel SCAM studies in Cx46 hemichannels.

The substituted cysteine accessibility method was applied to single Cx46 hemichannels to identify residues that participate in lining the aqueous pore of channels formed of connexins. Criteria for assignment to the pore included reactivity to sulfydryl-specific methanethiosulfonate (MTS) reagents from both sides of an open hemichannel and observable effects on open channel properties. We demonstrate reactivity to MTS reagents over a stretch of seventeen amino acids, D51 through L35, that constitute segments of E1 and TM1. Qualitatively, the nature of the effects caused by the Cys substitutions alone and their modification with MTS reagents of either charge indicate side chain valence is most influential in determining single channel properties with D51 and L35 defining the extracellular and intracellular limits, respectively, of the identified pore-lining region. A number of Cys substitutions beyond L35 in TM1 caused severe alterations in hemichannel function and precluded assignment to the pore. Although all six subunits can be modified by smaller MTS reagents, modifications appear limited to fewer subunits with larger reagents.

Short-range functional interaction between connexin35 and neighboring chemical synapses.

Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication--chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre- and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35- and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.

Cryosurgical ablation of right parietal and septal accessory atrioventricular connections without the use of extracorporeal circulation. A new surgical technique.

A new technique for definitive surgical treatment of the preexcitation syndrome without the use of extracorporeal circulation has been elaborated and clinically used in 25 patients with Wolff-Parkinson-White syndrome. Seventeen patients had right parietal accessory atrioventricular connections and eight had septal connections. In patients with right-sided connections the fat pad in the coronary sulcus was dissected with an ultrasonic scalpel. Ablation of accessory atrioventricular connections was obtained by applying local cryothermia. The evidence of preexcitation and arrhythmias disappeared in all patients. All patients recovered without surgical complications and there were no deaths. The described technique offers considerable advantages over previous surgical methods in selected patients with Wolff-Parkinson-White syndrome.

Connexin hemichannels and cell-cell channels: comparison of properties.

Connexin46 (Cx46) forms functional hemichannels in the absence of contact by an apposed hemichannel and we have used these hemichannels to study gating and permeation at the single channel level with high time resolution. Using both cell-attached and -excised patch configurations, we find that single Cx46 hemichannels exhibit some properties expected of half of a gap junction channel, as well as novel properties. Cx46 hemichannels have a large unitary conductance (approximately 300 pS) and a relatively large pore as inferred from permeability to TEA. Both monovalent cations and anions can permeate, but cations are substantially more permeable. The open channel conductance shows marked inward rectification in symmetric salts. We find that the conductance and permeability properties of Cx46 cell-cell channels can be explained by the series addition of two hemichannels. These data suggest that the pore structures of unapposed hemichannels and cell-cell channels are conserved. Also like cell-cell channels, unapposed Cx46 hemichannels are closed by elevated levels of H+ or Ca2+ ions on the cytoplasmic face. Closure occurs in excised patches indicating that the actions of these agents do not require a soluble cytoplasmic factor. Fast (<0.5 ms) application of H+ to either side of the open hemichannel causes an immediate small reduction in unitary conductance followed by complete closure with latencies that are dependent on H+ concentration and side of application; sensitivity is much greater to H+ on the cytoplasmic side. Closure by cytoplasmic H+ does not require that the hemichannel be open. Thus, H+ ions readily permeate Cx46 hemichannels, but at high enough concentration close them by acting at a cytoplasmic site(s) that causes a conformational change resulting in complete closure. Extracellular H+ may permeate to act on the cytoplasmic site or act on a lower affinity extracellular site. Thus, the unapposed hemichannel is a valuable tool in addressing fundamental questions concerning the operation of gap junction channels that are difficult to answer by existing methods. The ability of Cx46, and perhaps other connexins, to form functional unapposed hemichannels that are opened by moderate depolarization may represent an unexplored role of connexins as mediators of transport across the plasma membrane.

[Passive electrical properties of the atrioventricular region of the rabbit heart]. / Passivnye élektricheskie svoistva atrioventrikuliarnoi oblasti serdtsa krolika.

Passive electrical properties of AV node region of rabbit's heart were studied. Electrotonic anisotropism (a9) was characteristic of the entire AV node. a9 was the largest in the region of entry from the crista terminalis and in the lower node. The input resistance, RBX, of AV node was 1.3 to two-fold that measured in atrial trabeculae. Analysis of the results yielded the following specific resistance values for midnodal region: 1 Ohm--cm2 for contacts, 1200 Ohms-cm for intracellular space and 1300 Ohms-cm2 for electrogenic membrane. The nature of intercellular interaction and AV delay is discussed on the basis of the results obtained.

[Model of a passive 3-dimensional anisotropic syncytium as a continuous medium]. / Model' passivnogo trekhmernogo anizotropnogo sintsitiia kak nepreryvnoi sredy

A model is being proposed for analysis passive electrical properties of the syncitium with anisotropy. In terms of the model the potential is expected to decay as approximately e-r/r with an increase of the distance, r, from a polarising electrode. Input resistance of the three-dimensional syncitium loosely depends upon the resistance of electrogenic membrane, and thus should be mainly accounted for by the resistances of the myoplasm and intercellular contacts.

[Electrical connections between myocardial fibers and genesis of the electrocardiogram]. / Elektricheskaia sviaz' mezhdu voloknami miokarda i genez élektrokardiogrammy

The ECG genesis was analysed in the light of electric anisotropism of myocardium. The radial dipole P-r of interfiber currents for man's heart is estimated to be 50 mV with cm-2. These currents could be essential in ECG generation.

[The effect of acetylcholine on passive electrical properties and intercellular electrical communications in the sinoatrial node of the rabbit heart]. / Vliianie atsetilkholina na passivnye élektricheskie svoistva i mezhkletochnuiu élektricheskuiu sviaz' v sinoatrialnom uzle serdtsa krolika.

The effect of acetylcholine (ACH) on passive electric properties and intercellular coupling was studied using microelectrode technique in experiments on isolated preparations of the sinoatrial area of the rabbit's heart. The space constant of electrotonic decay (lambda delta) and input resistance (RBX) were measured. ACH was found to reduce lambda delta and increase RBX. Analysis of the experimental data, using the model of thin layer 3-dimensional syncytium, revealed that ACH of 10(-7) M, 10(-6) M and 10(-5) M increased the resistance of intracellular syncytium by factor of 1.25, 1.36 and 1.43, respectively, and decreased the resistance of the electrogenic membrane by factor of 1.13, 1.12 and 1.23, respectively. It is concluded that ACH increases the resistance of intercellular contacts in the sinoatrial node.

[Dicrete zones of electrical connection between Purkinje terminals and muscle fibers in dog ventricles]. / Diskretnye zony élektricheskoi sviazi terminalei Purkin'e s myshechnymi voloknami v zheludochke sobaki

Activity of Purkinje terminals (P) and neighbouring muscle (M) fibres (P -- M-pairs) in various regions of dog's right ventricle was recorded. It has been shown that transmission of stimulation from P-to M-fibres is abserved not over all endocardial surface, rather in descrete sites -- zones of P--M-interaction. The zones ellips like with the axes 300X100 micron, they are located relatively far from one another -- at the distance of 800-2000 microns. The total area of the zones equals 5% of endocardial surface. P-M delay is less within the zone of connection of P--M-fibres (4 msec) and greater outside the zone (up to 10 msec).

[Intercellular coupling in the sinus node of the rabbit heart]. / Mezhkletochnaia sviaz' v sinusnom uzle serdtsa krolika

Passive electrical properties of SA node region of rabbit's heart were studied. Electrotonic anisotropism was characteristic of the entire SA node; anisotropism decreased moving from crista terminalis to interatrial septum. The input resistance, RBX, was the largest in the region of true pacemakers and decreased moving to the perinodal region. Analysis of the results yielded specific resistance values for contacts and electrogenic membrane of 3 Ohms-cm3 and 10(4) Ohms-cm2 respectively.

[Anisotropy of stationary rates and delays in extrasystolic waves in the dog heart]. / Anizotropiia statsionarnykh skorostei i zaderzhek ékstrasistolicheskikh voln v serdtse sobaki

Excitation propagation in the ventricle and auricle bands of the dog's myocardium has been studied. The anisotropy of stationary valocities and delays of the initiation of extrasistole exciation has been shown: along the fibres the velocity is higher and the delay smaller than across the fibres. The levels of anisotropy of velocities and delays are almost equal in each structure. The strongest anisotropy of delays and velocities (-4,5) is observed in the ventricle muscle layer; the weakest (1,7)-in a specialized layer. The anisotropy of velocities and delays is the greater, the greater is the electrotone anisotropy. A new mechanism of extrasistole initiation (reentry) in the system homogenous by the membrane properties, but with anisotropy of intercellular bonds is considered.

[Electrophysiological aspects of the interrelationship between the normal and hypoxic portions of the myocardium]. / Elektrofiziologischeskie aspekty vzaimosviazi mezhdu normal'nym i gipoksicheskim uchastkom miokarda.

The results of studying the electrophysiological parameters on the boundary between the normal and the hypoxic area of the myocardium are discussed. The experiments were conducted on isolated preparation of the rabbit atrium with the use of microelectrodes for recording the transmembrane action potentials. The existence of the "maintenance" effect is shown, which is manifested by the better survival of the hypoxic area located nearer to the transitional zone as compared to the survival of a more distant area. The "maintenance" effect is due to the electrotonic interrelationship and to the intercellular diffuse processes of the ions and metabolites. With the continuation of the effect of hypoxia, the transitional zone becomes narrower, one of the causes of which is the disturbed intercellular relationship.

[Automatism of the sino-atrial node and sinoatrial conduction of excitation in hypoxia]. / Avtomatiia sinusovogo uzla i sinoatrial'noe provedenie vozbuzhdeniia v usloviiakh gipoksii.

The effect of experimental hypoxia on the automatic activity of the sinus node and sinoauricular transmission of excitation was studied in 17 isolated preparations of rabbit hearts. A greater frequency of the shifts of sinoauricular delay was noted under hypoxia. Local electrophoretic applications of adrenalin demonstrated that the shifts of the delay occur because of the shifts of pacemakers. The mean frequency of the automatic activity recorded in the atria falls to zero between 6 and 20 min of hypoxia. Hypoxia causes the development of sinoauricular blocks and atrial arrhythmias in the form of individual extrasystoles, high-frequency discharges, or alternations of series of impulses with "mute" periods. The increased tendency towards arrhythmias in the sinoauricular zone is, according to the authors, due to the growing heterogeneity of the duration of action potentials that increases more than three-fold by the 10th minute of hypoxia. The possible reflection of the excitation wave ("echo") from the sinus node zone with an inhibited excitability was demonstrated.

[Characteristics of the operations on the heart conduction system in the pre-excitation syndrome]. / Osobennosti operatsii na provodiashchikh putiakh serdtsa pri sindrome predvozbuzhdeniia.

Observation of 13 patients with the septal location of additional conducting pathways is given, 2 of them had James' fibres, 1 had a combination of James' and Mahaim bundles, in the remainder the Kent's bundle was in the anterior position. Removal of conduction through Kent's and His' bundles is described in detail. The syndrome of pre-excitation was successfully removed in 10 out of 12 patients, 1 had relapses later and 2 died. A detailed analysis of all 35 operated on patients with the pre-excitation syndrome is given. Good results were obtained in 88.57%.