Omori et al., 1996) cannot explain the observed phenotypic effects of CMTX mutations, since only ... more Omori et al., 1996) cannot explain the observed phenotypic effects of CMTX mutations, since only one X chro-E. Brady Trexler, Vytas K. Verselis, mosome is active in somatic cells. The phenotype could and Thaddeus A. Bargiello* arise if CMTX mutations behaved epistatically by sup-Department of Neuroscience pressing the function of other connexins that may be Albert Einstein College of Medicine normally coexpressed in Schwann cells, but the expres-Bronx, New York 10461 sion of other connexins subject to trans-dominant effects has not yet been demonstrated in myelinating Schwann cells. A recent study of 27 CMTX families has Summary suggested that nonsense mutations of Cx32 may produce more severe forms of the disease than missense The relationship between the loss of connexin 32 funcmutations (Ionasescu et al., 1996). However, as mistion and clinical manifestations of X-linked Charcotsense mutations can result in functional channels with Marie-Tooth (CMTX) disease is unknown. Here, we altered properties as well as nonfunctional channels, report that eight of nine CMTX mutations investigated biophysical characterizations of channels formed by form channels with measurable electrical conducmissense mutations are required to determine the motance. Single-channel studies of two mutations demlecular basis of the loss of Cx32 function and to establish onstrate reduced junctional permeability caused by a if changes in function correlate with the severity of the decrease in either pore size (S26L) or open channel disease. probability (M34T) that favors residency in a low-con-In peripheral nerve, Cx32 is found in myelinating ductance substate. Permeation of second messen-Schwann cells, where it is believed to form reflexive gap gers such as cAMP through reflexive gap junctions junctions (Scherer et al., 1995) that shorten the commubetween adjacent cytoplasmic loops of myelinating nication pathway between the periaxonal cytoplasm and Schwann cells is likely to be reduced or absent in these the peripheral cytoplasm in which the cell nucleus is channels. We propose that CMTX mutations impair located. It is likely that reduced communication through this pathway perturbs some aspect of normal glial-neuthe transduction of signals arising from normal glialronal interactions that are critical to the maintenance of neuronal interactions and thereby cause demyelinmyelin sheaths and axons (Doyle and Colman, 1993). ation and axonal degeneration. The characterization of CMTX mutations that only partially alter channel function should help to establish the * To whom correspondence should be addressed.
CHAPTER 14 Molecular Determinants of Voltage Gating of Gap Junctions Formed by Connexin32 and 26 ... more CHAPTER 14 Molecular Determinants of Voltage Gating of Gap Junctions Formed by Connexin32 and 26 Thaddeus A. Bargiello, Seunghoon Oh, Yi Ri, Priscilla E. Purnick, and Vytas K. Verselis Department of Neuroscience, Albert Einstein College of Medicine, Bronx, ...
Channels formed by the connexin family of proteins display multiple forms of voltage dependence t... more Channels formed by the connexin family of proteins display multiple forms of voltage dependence that have different sensitivities and time courses. Intercellular (junctional) channels are sensitive to two orientations of applied voltage: inside-out or transmembrane ...
The period (per) locus, which controls biological rhythms in Drosophila, was originally defined b... more The period (per) locus, which controls biological rhythms in Drosophila, was originally defined by three chemically induced mutations. Flies carrying the pero mutation were arrhythmic, whereas pers and perl mutants had circadian behavioural rhythms with 19-hour and 29-hour periodicities, respectively. Wild-type flies have 24-hour rhythms. Here we compare the per locus DNA sequences of the three mutants with the parental wild-type. The pers and perl mutations lead to amino-acid substitutions, whereas pero introduces an early translation stop (amber). The results indicate that the protein product of per controls biological rhythms. We also report that the abundance of this protein may set the pace of the Drosophila clock. Although circadian rhythms are restored when arrhythmic (per-) Drosophila are transformed with per locus DNA, flies receiving identical transforming DNA segments can produce rhythms with periods that differ by more than 12 hours. Transcription studies reveal a tenfol...
Proceedings of the National Academy of Sciences of the United States of America, Jan 3, 2009
Connexin 32 (Cx32), a gap junction protein, is found within the para-nodal region and Schmidt-Lan... more Connexin 32 (Cx32), a gap junction protein, is found within the para-nodal region and Schmidt-Lanterman incisures of myelinating Schwann cells (SCs). In developing and regenerating peripheral nerves, pro-myelinating SCs express Cx32 mRNA and protein in conjunction with the expression of myelin specific genes. Neuregulin-1 (Nrg1), a member of the neuregulin family of growth factors, controls SC proliferation and differentiation depending on the cellular environment and the particular stage of SC maturation. Primary cultures of purified SCs from newborn mouse sciatic nerve were used to characterize both the role of Nrg1 in the expression of Cx32 and, conversely, the role of Cx32 in SC responsiveness to Nrg1. Glial growth factor 2, an isoform of Nrg1, up-regulated Cx32 in both proliferating and non-proliferating SCs. However, SCs from Cx32-KO mice exhibited a significantly smaller mitogenic response to glial growth factor 2. Electrical coupling between Cx32-KO SCs did not differ from t...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 19, 2003
X-linked Charcot-Marie-Tooth disease is an inherited peripheral neuropathy arising in patients wi... more X-linked Charcot-Marie-Tooth disease is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding connexin 32 (Cx32). Cx32 is expressed at the paranodes and Schmidt-Lantermann incisures of myelinating Schwann cells in which it is believed to form a reflexive pathway between the abaxonal and adaxonal cytoplasmic domains. Patients with the Val181Ala (V181A) mutation have a severe peripheral neuropathy. Experiments using a nude mouse xenograft system show that Schwann cells expressing only this mutant form of Cx32 are profoundly impaired in their ability to support the earliest stages of regeneration of myelinated fibers. Coupling between paired Xenopus oocytes expressing V181A is reduced compared with the coupling between oocytes expressing wild-type human Cx32 (32WT), and protein levels assayed by Western blot are substantially lower. Immunocytochemisty shows that Neuro2a cells expressing the V181A mutant have very few gap junction plaques compared wi...
Voltage is an important parameter that regulates the conductance of both intercellular and plasma... more Voltage is an important parameter that regulates the conductance of both intercellular and plasma membrane channels (undocked hemichannels) formed by the 21 members of the mammalian connexin gene family. Connexin channels display two forms of voltage-dependence, rectification of ionic currents and voltage-dependent gating. Ionic rectification results either from asymmetries in the distribution of fixed charges due to heterotypic pairing of different hemichannels, or by channel block, arising from differences in the concentrations of divalent cations on opposite sides of the junctional plaque. This rectification likely underpins the electrical rectification observed in some electrical synapses. Both intercellular and undocked hemichannels also display two distinct forms of voltage-dependent gating, termed Vj (fast)-gating and loop (slow)-gating. This review summarizes our current understanding of the molecular determinants and mechanisms underlying these conformational changes derived from experimental, molecular-genetic, structural, and computational approaches.
Previous studies have shown that charge substitutions in the amino terminus of a chimeric connexi... more Previous studies have shown that charge substitutions in the amino terminus of a chimeric connexin, Cx32 * 43E1, which forms unapposed hemichannels in Xenopus oocytes, can result in a threefold difference in unitary conductance and alter the direction and amount of open channel current rectification. Here, we determine the charge selectivity of Cx32 * 43E1 unapposed hemichannels containing negative and/or positive charge substitutions at the 2nd, 5th and 8th positions in the N-terminus. Unlike Cx32 intercellular channels, which are weakly anion selective, the Cx32 * 43E1 unapposed hemichannel is moderately cation selective. Cation selectivity is maximal when the extracellular surface of the channel is exposed to low ionic strength solutions implicating a region of negative charge in the first extracellular loop of Cx43 (Cx43E1) in influencing charge selectivity analogous to that reported. Negative charge substitutions at the 2nd, 5th and 8th positions in the intracellular N-terminus substantially increase the unitary conductance and cation selectivity of the chimeric hemichannel. Positive charge substitutions at the 5th position decrease unitary conductance and produce a non-selective channel while the presence of a positive charge at the 5th position and negative charge at the 2nd results in a channel with conductance similar to the parental channel but with greater preference for cations. We demonstrate that a cysteine substitution of the 8th residue in the N-terminus can be modified by a methanthiosulphonate reagent (MTSEA-biotin-X) indicating that this residue lines the aqueous pore at the intracellular entrance of the channel. The results indicate that charge selectivity of the Cx32 * 43E1 hemichannel can be determined by the combined actions of charges dispersed over the permeation pathway rather than by a defined region that acts as a charge selectivity filter.
Proceedings of the National Academy of Sciences, 1996
Gap junction channels are formed by members of the connexin gene family and mediate direct interc... more Gap junction channels are formed by members of the connexin gene family and mediate direct intercellular communication through linked hemichannels (connexons) from each of two adjacent cells. While for most connexins, the hemichannels appear to require an apposing hemichannel to open, macroscopic currents obtained fromXenopus oocytes expressing rat Cx46 suggested that some hemichannels can be readily opened by membrane depolarization [
Proceedings of the National Academy of Sciences, 1991
Gap junctions are formed by a family of homologous proteins termed connexis. Their chnes are dode... more Gap junctions are formed by a family of homologous proteins termed connexis. Their chnes are dodecaners, and homomeric forms differ In their erts with respect to control by vdtage and other gating stimuli. We report here the properties of coupling from expr n of connexin complementary RNAs (cRNAs; sense to mRNA, antisense to cDNA) in Xenopus oocyte pairs in which endogenous coupling was blocked by Injection ofDNA oligonudeotides antisense to the mRNA of Cx38, the principal endogenous connexin. We found that a connexin recently sequenced from rat liver, Cx26, formed functional gap junons whose conductance exhibited voltage dependence with unua characteristics suggestive of two gating s. Junctional conductance (gj) was ieased to a smali degree by depolarization and decreased by hyperpolarization of either cell in a coupled jalr, indicating dependence on the potential between the inside and outside of the cells (V« .). These chag were fast compared with the resolution of their measuremeit (ca. 10 ms). On a slower tmesce, large transJunctional potent (Vi) Of either sign caused a more substantial decrease In conductance similr to that previously reported for several other gap Junctions. Homotypic Junctions formed of another connexn, Cx32, exhibited a similar slow dependence on Vj but no dependence on V". In contrast, heterotypic jun s between an oocyte expressing Cx26 and one expressing Cx32 were electrically asymmetric; they exhibited a greater fast change in gj, whichdepended, however, on Vj, suchthatgj increasedwith 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 ofthe opposite sgn. These data indcate that properties of the hemich contributed by the two connexins in the heterotypic case were chaned frwo their properties in homotypic junt The fast change in gj may involve a mechaism an us to that at fast fig electrical synapses. Experiments in which oocytes e g Cx32 were pared with oocytes empresslng both Cx26 and Cx32 demonstrated that asymmetric junctions would form between oocytes exp g both connexins, thereby confirming their potential relevance in viv6, where the same coupled cells are known to express both proteins. Several gap-junction proteins or connexins have been identified in rat tissues by cDNA cloning, Cx32 and Cx26 from liver, Cx43 from heart, and Cx46 from lens (1-4).
Proceedings of the National Academy of Sciences, 2006
The connexins are a family of at least 20 homologous proteins in humans that form aqueous channel... more The connexins are a family of at least 20 homologous proteins in humans that form aqueous channels connecting the interiors of coupled cells and mediating electrical and chemical communication. Mutations in the gene for human connexin 31 (hCx31) are associated with disorders of the skin and auditory system. Alterations in functional properties of Cx31 junctions are likely to play a role in these diseases; nonetheless, little is known about the properties of the wild-type channels. Here we show that hCx31 channels, like other connexin channels, are gated by voltage and close at low pH and when exposed to long-chain alkanols. Singlechannel conductance of the fully open channel is Ϸ85 pS, and it is permeable to Lucifer yellow, Alexa Fluor 350 , ethidium bromide, and DAPI, which have valences of ؊2, ؊1, ؉1, and ؉2, respectively. In contrast to what has been reported for mouse Cx31, hCx31 appears to form functional heterotypic channels with all four connexins tested, Cx26, Cx30, Cx32, and Cx45. These findings provide an important first step in evaluating the pathogenesis of inherited human diseases associated with mutations in the gene for Cx31. electrical coupling ͉ erythrokeratodermia variabilis ͉ gap junction ͉ intercellular communication Conflict of interest statement: No conflicts declared.
Search by Subject Search using Medical Subject Headings (< b> MeSH</b>), a controlled... more Search by Subject Search using Medical Subject Headings (< b> MeSH</b>), a controlled vocabulary for indexing life sciences content.< br/> Note that some records do not have MeSH. These include Patents and the latest PubMed and PubMed Central records.
Gap junctions are intercellular channels formed by the serial, head to head arrangement of two he... more Gap junctions are intercellular channels formed by the serial, head to head arrangement of two hemichannels. Each hemichannel is an oligomer of six protein subunits, which in vertebrates are encoded by the connexin gene family. All intercellular channels formed by connexins are sensitive to the relative difference in the membrane potential between coupled cells, the transjunctional voltage (Vj), and gate by the separate action of their component hemichannels (Harris, A.L., D.C. Spray, and M.V. Bennett. 1981. J. Gen. Physiol. 77:95–117). We reported previously that the polarity of Vj dependence is opposite for hemichannels formed by two closely related connexins, Cx32 and Cx26, when they are paired to form intercellular channels (Verselis, V.K., C.S. Ginter, and T.A. Bargiello. 1994. Nature. 368:348–351). The opposite gating polarity is due to a difference in the charge of the second amino acid. Negative charge substitutions of the neutral asparagine residue present in wild-type Cx32...
The fully open state of heterotypic gap junction channels formed by pairing cells expressing conn... more The fully open state of heterotypic gap junction channels formed by pairing cells expressing connexin 32 (Cx32) with those expressing connexin 26 (Cx26) rectifies in a way that cannot be predicted from the current–voltage (I–V) relation of either homotypic channel. Using a molecular genetic analysis, we demonstrate that charged amino acids positioned in the amino terminus (M1 and D2) and first extracellular loop (E42) are major determinants of the current–voltage relation of the fully open state of homotypic and heterotypic channels formed by Cx26 and Cx32. The observed I–V relations of wild-type and mutant channels were closely approximated by those obtained with the electrodiffusive model of Chen and Eisenberg (Chen, D., and R. Eisenberg. 1993.Biophys. J. 64:1405–1421), which solves the Poisson-Nernst-Plank equations in one dimension using charge distribution models inferred from the molecular analyses. The rectification of the Cx32/Cx26 heterotypic channel results from the asymme...
Background: Arg 75 in connexins is highly conserved. Results: Disease-causing mutations at this p... more Background: Arg 75 in connexins is highly conserved. Results: Disease-causing mutations at this position cause loss of function; for Cx32, loss of a positive charge appears to be critical. Conclusion: Positive charge at position 75 is required for normal Cx32 function. Significance: Better understanding of the effects of mutations of this position in Cx32 may have relevance to pathogenesis of several human diseases.
The substituted cysteine accessibility method was applied to single Cx46 hemichannels to identify... more 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.
CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy ari... more CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32). In this communication, we describe the expression levels and biophysical parameters of seven mutant forms of Cx32 associated with CMTX, when expressed in paired Xenopus oocytes. Paired oocytes expressing the R15Q and H94Q mutants show junctional conductances not statistically different from that determined for Cx32WT, though both show a trend toward reduced levels. The S85C and G12S mutants induce reduced levels of junctional conductance. Three other mutants (R15W, H94Y and V139M) induce no conductance above baseline when expressed in paired oocytes. Analysis of the conductance voltage relations for these mutants shows that the reduced levels of conductance are entirely (H94Y and V139M) or partly (S85C and R15W) explicable by a reduced open probability of the mutant hemichannels. The R15Q and H94Q mutations also show alterations in the conductance voltage relations that would be expected to minimally (H94Q) or moderately (R15Q) reduce the available gap junction communication pathway. The reduction in G12S induced conductance cannot be explained by alterations in hemichannel open probability and are more likely due to reduced junction formation. These results demonstrate that many CMTX mutations lead to loss of function of Cx32. For these mutations, the loss of function model is likely to explain the pathogenesis of CMTX.
Intercellular channels formed by connexins (gap junctions) are sensitive to the application of tr... more Intercellular channels formed by connexins (gap junctions) are sensitive to the application of transjunctional voltage (V j ), to which they gate by the separate actions of their serially arranged hemichannels (
Loop-gating is one of two voltage-dependent mechanisms that regulate the open probability of conn... more Loop-gating is one of two voltage-dependent mechanisms that regulate the open probability of connexin channels. The loop-gate permeability barrier is formed by a segment of the first extracellular loop (E1) (the parahelix) and appears to be accompanied by straightening of the bend angle between E1 and the first transmembrane domain (TM1). Here, all-atom molecular dynamics simulations are used to identify and characterize interacting van der Waals and electrostatic networks that stabilize the parahelices and TM1/E1 bend angles of the open Cx26 hemichannel. Dynamic fluctuations in an electrostatic network in each subunit are directly linked to the stability of parahelix structure and TM1/E1 bend angle in adjacent subunits. The electrostatic network includes charged residues that are pore-lining and thus positioned to be voltage sensors. We propose that the transition to the closed state is initiated by voltage-driven disruption of the networks that stabilize the open-state parahelix configuration, allowing the parahelix to protrude into the channel pore to form the loop-gate barrier. Straightening of the TM1/E1 bend appears to be a consequence of the reorganization of the interacting networks that accompany the conformational change of the parahelix. The electrostatic network extends across subunit boundaries, suggesting a concerted gating mechanism.
Omori et al., 1996) cannot explain the observed phenotypic effects of CMTX mutations, since only ... more Omori et al., 1996) cannot explain the observed phenotypic effects of CMTX mutations, since only one X chro-E. Brady Trexler, Vytas K. Verselis, mosome is active in somatic cells. The phenotype could and Thaddeus A. Bargiello* arise if CMTX mutations behaved epistatically by sup-Department of Neuroscience pressing the function of other connexins that may be Albert Einstein College of Medicine normally coexpressed in Schwann cells, but the expres-Bronx, New York 10461 sion of other connexins subject to trans-dominant effects has not yet been demonstrated in myelinating Schwann cells. A recent study of 27 CMTX families has Summary suggested that nonsense mutations of Cx32 may produce more severe forms of the disease than missense The relationship between the loss of connexin 32 funcmutations (Ionasescu et al., 1996). However, as mistion and clinical manifestations of X-linked Charcotsense mutations can result in functional channels with Marie-Tooth (CMTX) disease is unknown. Here, we altered properties as well as nonfunctional channels, report that eight of nine CMTX mutations investigated biophysical characterizations of channels formed by form channels with measurable electrical conducmissense mutations are required to determine the motance. Single-channel studies of two mutations demlecular basis of the loss of Cx32 function and to establish onstrate reduced junctional permeability caused by a if changes in function correlate with the severity of the decrease in either pore size (S26L) or open channel disease. probability (M34T) that favors residency in a low-con-In peripheral nerve, Cx32 is found in myelinating ductance substate. Permeation of second messen-Schwann cells, where it is believed to form reflexive gap gers such as cAMP through reflexive gap junctions junctions (Scherer et al., 1995) that shorten the commubetween adjacent cytoplasmic loops of myelinating nication pathway between the periaxonal cytoplasm and Schwann cells is likely to be reduced or absent in these the peripheral cytoplasm in which the cell nucleus is channels. We propose that CMTX mutations impair located. It is likely that reduced communication through this pathway perturbs some aspect of normal glial-neuthe transduction of signals arising from normal glialronal interactions that are critical to the maintenance of neuronal interactions and thereby cause demyelinmyelin sheaths and axons (Doyle and Colman, 1993). ation and axonal degeneration. The characterization of CMTX mutations that only partially alter channel function should help to establish the * To whom correspondence should be addressed.
CHAPTER 14 Molecular Determinants of Voltage Gating of Gap Junctions Formed by Connexin32 and 26 ... more CHAPTER 14 Molecular Determinants of Voltage Gating of Gap Junctions Formed by Connexin32 and 26 Thaddeus A. Bargiello, Seunghoon Oh, Yi Ri, Priscilla E. Purnick, and Vytas K. Verselis Department of Neuroscience, Albert Einstein College of Medicine, Bronx, ...
Channels formed by the connexin family of proteins display multiple forms of voltage dependence t... more Channels formed by the connexin family of proteins display multiple forms of voltage dependence that have different sensitivities and time courses. Intercellular (junctional) channels are sensitive to two orientations of applied voltage: inside-out or transmembrane ...
The period (per) locus, which controls biological rhythms in Drosophila, was originally defined b... more The period (per) locus, which controls biological rhythms in Drosophila, was originally defined by three chemically induced mutations. Flies carrying the pero mutation were arrhythmic, whereas pers and perl mutants had circadian behavioural rhythms with 19-hour and 29-hour periodicities, respectively. Wild-type flies have 24-hour rhythms. Here we compare the per locus DNA sequences of the three mutants with the parental wild-type. The pers and perl mutations lead to amino-acid substitutions, whereas pero introduces an early translation stop (amber). The results indicate that the protein product of per controls biological rhythms. We also report that the abundance of this protein may set the pace of the Drosophila clock. Although circadian rhythms are restored when arrhythmic (per-) Drosophila are transformed with per locus DNA, flies receiving identical transforming DNA segments can produce rhythms with periods that differ by more than 12 hours. Transcription studies reveal a tenfol...
Proceedings of the National Academy of Sciences of the United States of America, Jan 3, 2009
Connexin 32 (Cx32), a gap junction protein, is found within the para-nodal region and Schmidt-Lan... more Connexin 32 (Cx32), a gap junction protein, is found within the para-nodal region and Schmidt-Lanterman incisures of myelinating Schwann cells (SCs). In developing and regenerating peripheral nerves, pro-myelinating SCs express Cx32 mRNA and protein in conjunction with the expression of myelin specific genes. Neuregulin-1 (Nrg1), a member of the neuregulin family of growth factors, controls SC proliferation and differentiation depending on the cellular environment and the particular stage of SC maturation. Primary cultures of purified SCs from newborn mouse sciatic nerve were used to characterize both the role of Nrg1 in the expression of Cx32 and, conversely, the role of Cx32 in SC responsiveness to Nrg1. Glial growth factor 2, an isoform of Nrg1, up-regulated Cx32 in both proliferating and non-proliferating SCs. However, SCs from Cx32-KO mice exhibited a significantly smaller mitogenic response to glial growth factor 2. Electrical coupling between Cx32-KO SCs did not differ from t...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 19, 2003
X-linked Charcot-Marie-Tooth disease is an inherited peripheral neuropathy arising in patients wi... more X-linked Charcot-Marie-Tooth disease is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding connexin 32 (Cx32). Cx32 is expressed at the paranodes and Schmidt-Lantermann incisures of myelinating Schwann cells in which it is believed to form a reflexive pathway between the abaxonal and adaxonal cytoplasmic domains. Patients with the Val181Ala (V181A) mutation have a severe peripheral neuropathy. Experiments using a nude mouse xenograft system show that Schwann cells expressing only this mutant form of Cx32 are profoundly impaired in their ability to support the earliest stages of regeneration of myelinated fibers. Coupling between paired Xenopus oocytes expressing V181A is reduced compared with the coupling between oocytes expressing wild-type human Cx32 (32WT), and protein levels assayed by Western blot are substantially lower. Immunocytochemisty shows that Neuro2a cells expressing the V181A mutant have very few gap junction plaques compared wi...
Voltage is an important parameter that regulates the conductance of both intercellular and plasma... more Voltage is an important parameter that regulates the conductance of both intercellular and plasma membrane channels (undocked hemichannels) formed by the 21 members of the mammalian connexin gene family. Connexin channels display two forms of voltage-dependence, rectification of ionic currents and voltage-dependent gating. Ionic rectification results either from asymmetries in the distribution of fixed charges due to heterotypic pairing of different hemichannels, or by channel block, arising from differences in the concentrations of divalent cations on opposite sides of the junctional plaque. This rectification likely underpins the electrical rectification observed in some electrical synapses. Both intercellular and undocked hemichannels also display two distinct forms of voltage-dependent gating, termed Vj (fast)-gating and loop (slow)-gating. This review summarizes our current understanding of the molecular determinants and mechanisms underlying these conformational changes derived from experimental, molecular-genetic, structural, and computational approaches.
Previous studies have shown that charge substitutions in the amino terminus of a chimeric connexi... more Previous studies have shown that charge substitutions in the amino terminus of a chimeric connexin, Cx32 * 43E1, which forms unapposed hemichannels in Xenopus oocytes, can result in a threefold difference in unitary conductance and alter the direction and amount of open channel current rectification. Here, we determine the charge selectivity of Cx32 * 43E1 unapposed hemichannels containing negative and/or positive charge substitutions at the 2nd, 5th and 8th positions in the N-terminus. Unlike Cx32 intercellular channels, which are weakly anion selective, the Cx32 * 43E1 unapposed hemichannel is moderately cation selective. Cation selectivity is maximal when the extracellular surface of the channel is exposed to low ionic strength solutions implicating a region of negative charge in the first extracellular loop of Cx43 (Cx43E1) in influencing charge selectivity analogous to that reported. Negative charge substitutions at the 2nd, 5th and 8th positions in the intracellular N-terminus substantially increase the unitary conductance and cation selectivity of the chimeric hemichannel. Positive charge substitutions at the 5th position decrease unitary conductance and produce a non-selective channel while the presence of a positive charge at the 5th position and negative charge at the 2nd results in a channel with conductance similar to the parental channel but with greater preference for cations. We demonstrate that a cysteine substitution of the 8th residue in the N-terminus can be modified by a methanthiosulphonate reagent (MTSEA-biotin-X) indicating that this residue lines the aqueous pore at the intracellular entrance of the channel. The results indicate that charge selectivity of the Cx32 * 43E1 hemichannel can be determined by the combined actions of charges dispersed over the permeation pathway rather than by a defined region that acts as a charge selectivity filter.
Proceedings of the National Academy of Sciences, 1996
Gap junction channels are formed by members of the connexin gene family and mediate direct interc... more Gap junction channels are formed by members of the connexin gene family and mediate direct intercellular communication through linked hemichannels (connexons) from each of two adjacent cells. While for most connexins, the hemichannels appear to require an apposing hemichannel to open, macroscopic currents obtained fromXenopus oocytes expressing rat Cx46 suggested that some hemichannels can be readily opened by membrane depolarization [
Proceedings of the National Academy of Sciences, 1991
Gap junctions are formed by a family of homologous proteins termed connexis. Their chnes are dode... more Gap junctions are formed by a family of homologous proteins termed connexis. Their chnes are dodecaners, and homomeric forms differ In their erts with respect to control by vdtage and other gating stimuli. We report here the properties of coupling from expr n of connexin complementary RNAs (cRNAs; sense to mRNA, antisense to cDNA) in Xenopus oocyte pairs in which endogenous coupling was blocked by Injection ofDNA oligonudeotides antisense to the mRNA of Cx38, the principal endogenous connexin. We found that a connexin recently sequenced from rat liver, Cx26, formed functional gap junons whose conductance exhibited voltage dependence with unua characteristics suggestive of two gating s. Junctional conductance (gj) was ieased to a smali degree by depolarization and decreased by hyperpolarization of either cell in a coupled jalr, indicating dependence on the potential between the inside and outside of the cells (V« .). These chag were fast compared with the resolution of their measuremeit (ca. 10 ms). On a slower tmesce, large transJunctional potent (Vi) Of either sign caused a more substantial decrease In conductance similr to that previously reported for several other gap Junctions. Homotypic Junctions formed of another connexn, Cx32, exhibited a similar slow dependence on Vj but no dependence on V". In contrast, heterotypic jun s between an oocyte expressing Cx26 and one expressing Cx32 were electrically asymmetric; they exhibited a greater fast change in gj, whichdepended, however, on Vj, suchthatgj increasedwith 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 ofthe opposite sgn. These data indcate that properties of the hemich contributed by the two connexins in the heterotypic case were chaned frwo their properties in homotypic junt The fast change in gj may involve a mechaism an us to that at fast fig electrical synapses. Experiments in which oocytes e g Cx32 were pared with oocytes empresslng both Cx26 and Cx32 demonstrated that asymmetric junctions would form between oocytes exp g both connexins, thereby confirming their potential relevance in viv6, where the same coupled cells are known to express both proteins. Several gap-junction proteins or connexins have been identified in rat tissues by cDNA cloning, Cx32 and Cx26 from liver, Cx43 from heart, and Cx46 from lens (1-4).
Proceedings of the National Academy of Sciences, 2006
The connexins are a family of at least 20 homologous proteins in humans that form aqueous channel... more The connexins are a family of at least 20 homologous proteins in humans that form aqueous channels connecting the interiors of coupled cells and mediating electrical and chemical communication. Mutations in the gene for human connexin 31 (hCx31) are associated with disorders of the skin and auditory system. Alterations in functional properties of Cx31 junctions are likely to play a role in these diseases; nonetheless, little is known about the properties of the wild-type channels. Here we show that hCx31 channels, like other connexin channels, are gated by voltage and close at low pH and when exposed to long-chain alkanols. Singlechannel conductance of the fully open channel is Ϸ85 pS, and it is permeable to Lucifer yellow, Alexa Fluor 350 , ethidium bromide, and DAPI, which have valences of ؊2, ؊1, ؉1, and ؉2, respectively. In contrast to what has been reported for mouse Cx31, hCx31 appears to form functional heterotypic channels with all four connexins tested, Cx26, Cx30, Cx32, and Cx45. These findings provide an important first step in evaluating the pathogenesis of inherited human diseases associated with mutations in the gene for Cx31. electrical coupling ͉ erythrokeratodermia variabilis ͉ gap junction ͉ intercellular communication Conflict of interest statement: No conflicts declared.
Search by Subject Search using Medical Subject Headings (< b> MeSH</b>), a controlled... more Search by Subject Search using Medical Subject Headings (< b> MeSH</b>), a controlled vocabulary for indexing life sciences content.< br/> Note that some records do not have MeSH. These include Patents and the latest PubMed and PubMed Central records.
Gap junctions are intercellular channels formed by the serial, head to head arrangement of two he... more Gap junctions are intercellular channels formed by the serial, head to head arrangement of two hemichannels. Each hemichannel is an oligomer of six protein subunits, which in vertebrates are encoded by the connexin gene family. All intercellular channels formed by connexins are sensitive to the relative difference in the membrane potential between coupled cells, the transjunctional voltage (Vj), and gate by the separate action of their component hemichannels (Harris, A.L., D.C. Spray, and M.V. Bennett. 1981. J. Gen. Physiol. 77:95–117). We reported previously that the polarity of Vj dependence is opposite for hemichannels formed by two closely related connexins, Cx32 and Cx26, when they are paired to form intercellular channels (Verselis, V.K., C.S. Ginter, and T.A. Bargiello. 1994. Nature. 368:348–351). The opposite gating polarity is due to a difference in the charge of the second amino acid. Negative charge substitutions of the neutral asparagine residue present in wild-type Cx32...
The fully open state of heterotypic gap junction channels formed by pairing cells expressing conn... more The fully open state of heterotypic gap junction channels formed by pairing cells expressing connexin 32 (Cx32) with those expressing connexin 26 (Cx26) rectifies in a way that cannot be predicted from the current–voltage (I–V) relation of either homotypic channel. Using a molecular genetic analysis, we demonstrate that charged amino acids positioned in the amino terminus (M1 and D2) and first extracellular loop (E42) are major determinants of the current–voltage relation of the fully open state of homotypic and heterotypic channels formed by Cx26 and Cx32. The observed I–V relations of wild-type and mutant channels were closely approximated by those obtained with the electrodiffusive model of Chen and Eisenberg (Chen, D., and R. Eisenberg. 1993.Biophys. J. 64:1405–1421), which solves the Poisson-Nernst-Plank equations in one dimension using charge distribution models inferred from the molecular analyses. The rectification of the Cx32/Cx26 heterotypic channel results from the asymme...
Background: Arg 75 in connexins is highly conserved. Results: Disease-causing mutations at this p... more Background: Arg 75 in connexins is highly conserved. Results: Disease-causing mutations at this position cause loss of function; for Cx32, loss of a positive charge appears to be critical. Conclusion: Positive charge at position 75 is required for normal Cx32 function. Significance: Better understanding of the effects of mutations of this position in Cx32 may have relevance to pathogenesis of several human diseases.
The substituted cysteine accessibility method was applied to single Cx46 hemichannels to identify... more 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.
CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy ari... more CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32). In this communication, we describe the expression levels and biophysical parameters of seven mutant forms of Cx32 associated with CMTX, when expressed in paired Xenopus oocytes. Paired oocytes expressing the R15Q and H94Q mutants show junctional conductances not statistically different from that determined for Cx32WT, though both show a trend toward reduced levels. The S85C and G12S mutants induce reduced levels of junctional conductance. Three other mutants (R15W, H94Y and V139M) induce no conductance above baseline when expressed in paired oocytes. Analysis of the conductance voltage relations for these mutants shows that the reduced levels of conductance are entirely (H94Y and V139M) or partly (S85C and R15W) explicable by a reduced open probability of the mutant hemichannels. The R15Q and H94Q mutations also show alterations in the conductance voltage relations that would be expected to minimally (H94Q) or moderately (R15Q) reduce the available gap junction communication pathway. The reduction in G12S induced conductance cannot be explained by alterations in hemichannel open probability and are more likely due to reduced junction formation. These results demonstrate that many CMTX mutations lead to loss of function of Cx32. For these mutations, the loss of function model is likely to explain the pathogenesis of CMTX.
Intercellular channels formed by connexins (gap junctions) are sensitive to the application of tr... more Intercellular channels formed by connexins (gap junctions) are sensitive to the application of transjunctional voltage (V j ), to which they gate by the separate actions of their serially arranged hemichannels (
Loop-gating is one of two voltage-dependent mechanisms that regulate the open probability of conn... more Loop-gating is one of two voltage-dependent mechanisms that regulate the open probability of connexin channels. The loop-gate permeability barrier is formed by a segment of the first extracellular loop (E1) (the parahelix) and appears to be accompanied by straightening of the bend angle between E1 and the first transmembrane domain (TM1). Here, all-atom molecular dynamics simulations are used to identify and characterize interacting van der Waals and electrostatic networks that stabilize the parahelices and TM1/E1 bend angles of the open Cx26 hemichannel. Dynamic fluctuations in an electrostatic network in each subunit are directly linked to the stability of parahelix structure and TM1/E1 bend angle in adjacent subunits. The electrostatic network includes charged residues that are pore-lining and thus positioned to be voltage sensors. We propose that the transition to the closed state is initiated by voltage-driven disruption of the networks that stabilize the open-state parahelix configuration, allowing the parahelix to protrude into the channel pore to form the loop-gate barrier. Straightening of the TM1/E1 bend appears to be a consequence of the reorganization of the interacting networks that accompany the conformational change of the parahelix. The electrostatic network extends across subunit boundaries, suggesting a concerted gating mechanism.
Uploads
Papers by Ted Bargiello