Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage.
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Base Sequence↗
Biomedical subjects
M V Bennett
Publications and source records attributed to M V Bennett.
Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32.
Heterotypic gap junctions formed by pairing Xenopus oocytes expressing hemichannels formed of Cx32 with those expressing hemichannels formed of Cx26 displayed novel transjunctional voltage (Vj) dependence not predicted by the behavior of these connexins in homotypic configurations. Rectification of initial and steady-state currents was observed. Relative positivity and negativity on the Cx26 side of the junction resulted in increased and decreased initial conductance (gj0), respectively. Only relative positivity on the Cx26 decreased steady-state conductance (gj infinity). This behavior suggested that interactions between hemichannels influences gap junction gating. The role of the first extracellular loop (E1) in these interactions was examined by pairing Cx32 and Cx26 with a chimeric connexin in which Cx32 E1 was replaced with Cx26 E1 (Cx32*26E1). Both junctions rectified with gj0/Vj relations that were less steep than that observed for Cx32/Cx26. Decreases in gj infinity occurred for either polarity Vj in the Cx32/Cx32*26E1 junction. Mutation of two amino acids in Cx26 E1 increased the steepness of both the gj0/Vj and gj infinity/Vj relations. These data demonstrate that fast rectification can arise from mismatched E1 domains and that E1 may contribute to the voltage sensing mechanisms underlying both fast and slow Vj-dependent processes.
Amino Acid Sequence↗
Biophysics of gap junctions.
Gap junction channels, now known to be formed of connexins, connect the interiors of apposed cells. These channels can be opened and closed by various physiological stimuli and experimental treatments. They are permeable to ions and neutral molecules up to a size of about 1 kDa or 1.5 nm diameter, including second messengers and metabolites. The processes of gating and of permeation are the subject of this review. Voltage is a readily applied stimulus, and transjunctional voltages, or those between cytoplasm and exterior, affect most junctions. Single channel transitions between open and closed states are rapid and presumably involve a charge movement as occurs with channels of electrically excitable channels of nerve and muscle. Identification of gating domains and charges by domain replacement and site-directed mutagenesis is being pursued. Raising cytoplasmic H+ or Ca2+ concentrations rapidly reduces junctional conductance, and this action is generally reversible, at least in part. A number of lipophilic alcohols, fatty acids and volatile anesthetics have similar actions. Phosphorylation also modulates junctional conductance, and in several cases, sites of phosphorylation are known. These gating processes appear similar to those induced by voltage. Permeability measurement indicates that the channel is aqueous and that permeation is by diffusion with only minor interactions with the channel wall. Differences among junctions are known, but further characterization of connexin and cell specificity is required.
Animals↗
Pinealocytes in rats: connexin identification and increase in coupling caused by norepinephrine.
Dye coupling was observed between pinealocytes in acutely dissected pineal glands of adult rats. Pinealocytes maintained in culture were also electrically coupled. Connexins 26 and 43 and their respective mRNAs were present but neither connexin32 nor its mRNA were detected. Pinealocytes expressed only connexin26 whereas connexin43 was confined to astrocytes. In 5-day-old cultures of pinealocytes the incidence of dye coupling and level of immunodetectable connexin26 were low, and both were increased by norepinephrine (NE). The increase in incidence of coupling was maximal at around 6 h after treatment and was prevented by inhibitors of protein or mRNA synthesis. NE-induced metabolic and electrical synchronization mediated by gap junctions may favor melatonin secretion.
Animals↗
Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage.
Gap junctions are formed by a family of homologous proteins termed connexins. Their channels are dodecamers, and homomeric forms differ in their properties with respect to control by voltage and other gating stimuli. We report here the properties of coupling from expression of connexin complementary RNAs (cRNAs; sense to mRNA, antisense to cDNA) in Xenopus oocyte pairs in which endogenous coupling was blocked by injection of DNA oligonucleotides antisense to the mRNA of Cx38, the principal endogenous connexin. We found that a connexin recently sequenced from rat liver, Cx26, formed functional gap junctions whose conductance exhibited voltage dependence with unusual characteristics suggestive of two gating mechanisms. Junctional conductance (gj) was increased to a small degree by depolarization and decreased by hyperpolarization of either cell in a coupled pair, indicating dependence on the potential between the inside and outside of the cells (Vi-o). These changes were fast compared with the resolution of their measurement (ca. 10 ms). On a slower timescale, large transjunctional potentials (Vj) of either sign caused a more substantial decrease in conductance similar to that previously reported for several other gap junctions. Homotypic junctions formed of another connexin, Cx32, exhibited a similar slow dependence on Vj but no dependence on Vi-o. In contrast, heterotypic junctions between an oocyte expressing Cx26 and one expressing Cx32 were electrically asymmetric; they exhibited a greater fast change in gj, which depended, however, on Vj, such that gj increased with relative positivity on the Cx26 side and decreased with relative negativity on the Cx26 side. There was also a large slow decrease in gj in response to Vj for relative positivity on the Cx26 side but not for Vj of the opposite sign. These data indicate that properties of the hemichannels contributed by the two connexins in the heterotypic case were changed from their properties in homotypic junctions. The fast change in gj may involve a mechanism analogous to that at fast rectifying electrical synapses. Experiments in which oocytes expressing Cx32 were paired with oocytes expressing both Cx26 and Cx32 demonstrated that asymmetric junctions would form between oocytes expressing both connexins, thereby confirming their potential relevance in vivo, where the same coupled cells are known to express both proteins.
Animals↗
Differential expression of three glutamate receptor genes in developing rat brain: an in situ hybridization study.
Non-N-methyl-D-aspartate glutamate receptors (GluRs) are encoded by a gene family, known members of which are designated GluR-1, -2, -3, -4, and -5. The present study examined the developmental pattern of GluR-1, -2, and -3 gene expression in rat brain. In situ hybridization revealed different spatial patterns throughout the brain for the cognate mRNAs at all ages examined, as well as different temporal patterns during development. In the adult all three mRNAs were expressed prominently in the pyramidal and granule layers of the hippocampus and in the Purkinje cell layer of the cerebellum, where detailed differences were apparent at the cellular level. In neocortex, GluR-2 mRNA exhibited prominent lamination and regional differences, which were less marked for GluR-1 and -3 mRNAs. In caudate-putamen GluR-2 mRNA was at high levels, but GluR-1 and -3 mRNAs were not. At early ages transcripts were transiently elevated relative to adult levels. GluR-1 mRNA reached peak expression in cortex at postnatal day 14 (P14) (225% of adult), in striatum at P4 (255% of adult), in hippocampus at P14 (195% of adult), and in cerebellum at P21 (150% of adult). GluR-3 exhibited more modest peaks in neocortex and hippocampus. In contrast, GluR-2 mRNA was at near adult levels throughout the first days of postnatal life and exhibited a peak only in cerebellum at P14 (168% of adult). The finding of differential developmental regulation of the GluR-1, -2, and -3 genes indicates that the receptors they encode may have different influences on synaptic plasticity, neuronal survival, and susceptibility to excitatory amino acid toxicity.
Aging↗
Interaction of Mg2+ and phencyclidine in use-dependent block of NMDA channels.
The interaction between Mg2+ and phencyclidine (PCP) in blocking open N-methyl-D-aspartate (NMDA) channels was investigated in Xenopus oocytes injected with rat brain mRNA. These receptors exhibit the pharmacological and physiological properties of the neuronal receptors, and the oocyte is readily amenable to electrical recording and application of well-controlled chemical stimuli. We found that Mg2+ at physiological concentrations greatly impeded the ability of PCP to block the NMDA channel. The interaction between Mg2+ and PCP was competitive; 0.5 mM Mg2+ caused a four-fold decrease in the potency of PCP in blocking open NMDA channels. Moreover, Mg2+ speeded the recovery from PCP block in the presence of agonist, suggesting that Mg2+ reduced reblock of NMDA channels by PCP that had escaped from open channels. Our observations suggest that the presence of Mg2+ in the channel tends to prevent PCP entry and block. Since depolarization is likely to reduce channel occupancy by Mg2+ more than that by PCP, neural activity may have an important influence on the actions of PCP and related drugs.
Animals↗
Gap junctions: new tools, new answers, new questions.
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Amino Acid Sequence↗
Biophysical properties of gap junctions between freshly dispersed pairs of mouse pancreatic beta cells.
Coupling between beta cells through gap junctions has been postulated as a principal mechanism of electrical synchronization of glucose-induced activity throughout the islet of Langerhans. We characterized junctional conductance between isolated pairs of mouse pancreatic beta cells by whole-cell recording with two independent patch-clamp circuits. Most pairs were coupled (67%, n = 155), although the mean junctional conductance (gj) (215 +/- 110 pS) was lower than reported in other tissues. Coupling could be recorded for long periods, up to 40 min. Voltage imposed across the junctional or nonjunctional membranes had no effect on gj. Up to several hours of treatment to increase intracellular cAMP levels did not affect gj. Electrically coupled pairs did not show transfer of the dye Lucifer yellow. Octanol (2 mM) reversibly decreased gj. Lower concentrations of octanol (0.5 mM) and heptanol (0.5 mM) than required to uncouple beta cells decreased voltage-dependent K+ and Ca2+ currents in nonjunctional membranes. Although gj recorded in these experiments would be expected to be provided by current flowing through only a few channels of the unitary conductance previously reported for other gap junctions, no unitary junctional currents were observed even during reversible suppression of gj by octanol. This result suggests either that the single channel conductance of gap junction channels between beta cells is smaller than in other tissues (less than 20 pS) or that the small mean conductance is due to transitions between open and closed states that are too rapid or too slow to be resolved.
Animals↗
A voltage-dependent gap junction in Drosophila melanogaster.
Steady-state and kinetic analyses of gap junctional conductance, gi, in salivary glands of Drosophila melanogaster third instar larvae reveal a strong and complex voltage dependence that can be elicited by two types of voltages. Voltages applied between the cells, i.e., transjunctional voltages, Vj, and those applied between the cytoplasm and the extracellular space, inside-outside voltages, Vi,o, markedly alter gj. Alteration of Vi-o while holding Vj = O,i.e., by equal displacement of the voltages in the cells, causes gj to increase to a maximum on hyperpolarization and to decrease to near zero on depolarization. These conductance changes associated with Vi-o are fit by a model in which there are two independent gates in series, one in each series, one in each membrane, where each gate is equally sensitive to Vi-o and exhibits first order kinetics. Vj's generated by applying voltage steps of either polarity to either cell, substantially reduce gj. These conductance changes exhibit complex kinetics that depend on Vi-o as well as Vj. At more positive Vi-o's, the changes in gj have two phases, an early phase consisting of of a decrease in gj for either polarity of Vj and a later phase consisting of an increase in gj on hyperpolarizing either cell and a decrease on depolarizing either cell. At negative Vi-o's in the plateau region of the gj-Vi-o relation, the later slow increase in gj is absent on hyperpolarizing either cell. Also, the early decrease in gj for either polarity of Vj is faster the more positive the Vi-o. The complex time course elicited by applying voltage steps to one cell can be explained as combined actions of Vi-o and Vj, with the early phase ascribable to Vj, but influenced by Vi-o, and the later phase to the changes in Vi-o associated with the generation of Vj. The substantially different kinetics and sensitivity of changes in gj by Vi-o and Vj suggests that the mechanisms of gating by these two voltages are different. Evidently, these gap-junction channels are capable of two distinct, but interactive forms of voltage dependence.
Animals↗
Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II.
Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. CA2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228-239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.
Amino Acid Sequence↗
Polyamines potentiate responses of N-methyl-D-aspartate receptors expressed in xenopus oocytes.
Glutamate, the major excitatory neurotransmitter in the central nervous system, activates at least three types of channel-forming receptors defined by the selective agonists N-methyl-D-aspartate (NMDA), kainate, and quisqualate [or more selectively by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)]. Activation of the NMDA receptor requires glycine as well as NMDA or glutamate. Recent studies have provided evidence that certain polyamines potentiate the binding by NMDA receptors of glycine and the open channel blocker MK-801. To determine whether polyamines alter channel opening, we examined their effects on rat brain glutamate receptors expressed in Xenopus oocytes. Our results demonstrate that spermine potentiates the response of the NMDA receptor but has no effect on responses to kainate and quisqualate. Furthermore, spermine increases the maximum response to NMDA and glycine and acts, at least in part, by increasing the apparent affinity of the NMDA receptor/channel complex for glycine. The present findings and the fact that polyamines are a natural constituent of brain suggest that polyamines may play a role in the regulation of glutamatergic transmission.
Animals↗
Glycine decreases desensitization of N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes and is required for NMDA responses.
In Xenopus oocytes injected with rat brain mRNA, as in neurons, glycine greatly potentiated responses of the N-methyl-D-aspartate (NMDA) type of excitatory amino acid receptor. Injected oocytes generated a partially desensitizing inward current in response to NMDA with 30 nM added glycine. As the added glycine concentration was increased from 30 nM to 1 microM, the NMDA response was increased and exhibited less desensitization. The relationship between the NMDA peak response and added glycine concentration indicated a single component response with apparent affinity of 0.29 microM and a Hill coefficient of 0.77. The desensitized response was also fit by the Hill relation with a lower affinity but similar coefficient. The time course of desensitization at 500 microM NMDA was exponential with a time constant (350 msec) that was independent of glycine concentration between 0.03 and 0.3 microM. At higher glycine concentration a slower component of decay (tau = 1.4 sec) was observed. This component was enhanced by increasing the extracellular Ca2+. NMDA without added glycine evoked a small transient response. However this response was suppressed completely by prewashing with the glycine antagonist 7-chlorokynurenic acid, suggesting that it may have been due to glycine contamination. The dose-response relation for low concentrations of glycine indicated that the measured level of glycine contamination accounted for these responses. These results indicate that glycine has at least two actions at the NMDA receptor: it enables channel opening by the agonist and decreases desensitization.
Animals↗
Projections of giant fibers, a class of reticular interneurons, in the brain of the silver hatchetfish.
Giant fibers are large reticular interneurons that mediate excitatory input from the Mauthner cells to pectoral-fin motoneurons. The present study revealed extensive giant-fiber projections to central targets other than the pectoral-fin motoneurons. Physiologically identified giant fibers were filled intracellularly with Lucifer Yellow. The presence of numerous dendrites suggests that there may be significant integration of non-Mauthner inputs in the giant-fiber cell body. The axon decussates and then bifurcates to form a descending process, that innervates pectoral-fin motoneurons, and an ascending process with collaterals that terminate in the rostral trigeminal, rostral facial and, in some cases, oculomotor and trochlear motor nuclei. The projections are consistent with a role for the giant fibers as mediators of all the cranial components of the Mauthner-initiated startle response. There are also extensive projections to cells near the Mauthner cell and to the medial reticular formation. The latter projections may participate in longer-latency components of the Mauthner-initiated startle response, or they may constitute part of a general arousal pathway. The morphology of giant fibers suggests that they are homologous to cranial relay neurons in the goldfish and T reticular interneurons in zebrafish larvae. Non-Mauthner inputs to giant fibers may participate in non-Mauthner startle responses and contribute to the variability of Mauthner-mediated startle responses.
Animals↗
Neuronal analysis of pharyngeal peristalsis in the gastropod Navanax in terms of identified motoneurons innervating identified muscle bands. I. Muscle band identifiability.
The neuronal basis of pharyngeal ingestion and swallowing can be conveniently studied in the marine mollusc Navanax inermis because of the small number of neurons involved in the behavior, reproducible identifiability of many individual neurons and simple muscle arrangement. The Navanax pharynx contains 3 approximately orthogonal muscle groups, circumferential, longitudinal and radial, arranged in well defined layers. Pharyngeal peristalsis involves sequential circumferential constriction, apparently with coordinated local pharyngeal expansion produced by radial muscle. Circumferential and longitudinal muscles consist of discrete, well defined bands. The number, position and arrangement of circumferential and longitudinal bands show little interanimal variability; these bands are individually identified by sequential number. Regional morphologic specializations of circumferential bands presumably facilitate peristalsis. Circumferential and longitudinal bands form a two dimensional reference system defining position on the pharyngeal surface. In the accompanying paper circumferential motor fields are described in terms of identified motoneurons innervating identified bands, and radial motor fields are located with respect to the coordinate system of overlying longitudinal and circumferential bands.
Animals↗
Neuronal analysis of pharyngeal peristalsis in the gastropod Navanax in terms of identified motoneurons innervating identified muscle bands. II. Radial and circumferential motor fields.
The neuronal basis of pharyngeal ingestion and peristalsis was studied in the gastropod Navanax inermis. Radially and circumferentially oriented muscles produce expansion and constriction of the pharynx. Motor fields of 11 identified radial motoneurons and 13 identified circumferential motoneurons were determined with respect to circumferential and longitudinal muscle band coordinates by muscle movements, electromyography, antidromic stimulation and axonal anatomy. Activation of these identified motoneurons can account for all the elemental pharyngeal movements observed during feeding. Four motoneurons, each innervating most of radial muscle, can mediate ingestion. Three radial motoneurons with anterior motor fields can mediate anterior expansion during sealing of the pharyngeal lips around prey and during regurgitation. Ten circumferential motoneurons have small arciform motor fields, the distributions of which correspond to the regional specializations in circumferential band organization. Arciform constriction can center eccentric ingested prey within the pharyngeal lumen during peristalsis. Arciform constrictions could combine to form an annular constriction in peristalsis. Small, non-overlapping, circumferential motor fields maximize the number of independent annular units available to produce a fine peristaltic wave. Sphincters have more circumferential motoneurons with smaller motor fields; this innervation permits finer motor control. Radial motoneurons with posterior motor fields can produce expansion caudal to a circumferential constriction during peristalsis. Motor fields of regional radial motoneurons show greater interanimal variability than circumferential motor fields, which is correlated with a less essential role of radial motoneurons in peristalsis. Two circumferential motoneurons with giant posterior pharyngeal motor fields can mediate pharyngeal emptying either in swallowing or in regurgitation.
Action Potentials↗
The electromotor system of the electric eel investigated with horseradish peroxidase as a retrograde tracer.
The electromotor system of the electric eel, Electrophorus electricus, was studied by injection of horseradish peroxidase as a retrograde tracer. The electromotor neurons, which innervate the electrocytes, comprise a midline nucleus, largely dorsal to the spinal canal. Spinal motoneurons lie ventrolaterally. The electromotor and skeletal motor neuron populations correspond to the acetylcholinesterase-negative and -positive cells previously described. The medullary relay neurons were labeled following HRP injection into the spinal cord at a level where electromotor neurons occurred, but not after injection into the cord in the abdominal region rostral to these cells. Other medullary neurons, presumably bulbospinal motor fibers, were labeled after both levels of spinal cord injection. The results suggest that these axosomatic synapses, which are electrically transmitting but morphologically mixed, take up retrograde tracers in a manner similar to chemical synapses and that tracer uptake is at least largely at terminal regions.
Animals↗
Fine structure of the tuberous electroreceptor of the high-frequency electric fish, Sternarchus albifrons (gymnotiformes).
Sternarchus emits low voltage biphasic pulses at about 700-900/s. These signals (and changes in them caused by external objects) are detected by the tuberous or phasic electroreceptors. We used electron microscopy to examine extracellular compartments in the current pathway to the receptor cells, which are delineated by cells joined by tight junctions. Highly specialized accessory cells were found to separate the receptor cells from the extracellular space continuous with the exterior. Except for synaptic specializations complements of intramembrane particles of cell membranes were unremarkable and did not correlate with presumed high and low resistivity. We propose an equivalent electrical circuit that is consistent with the morphological and physiological observations.
Animals↗