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Conformational changes in a pore-forming region underlie voltage-dependent "loop gating" of an unapposed connexin hemichannel.

Tang Q, Dowd TL, Verselis VK, Bargiello TA - J. Gen. Physiol. (2009)

Bottom Line: Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism.Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state.We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.

View Article: PubMed Central - PubMed

Affiliation: Dominic P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA-biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd2+ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd2+ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.

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Western blots of WT (Cx32*Cx43E1) and mutant (A43C) membrane-inserted hemichannels. + and − symbols at the bottom of each panel denote treatment of the sample with or without either 50 mM DTT or 10 mM TPEN before SDS-electrophoresis through 5–40% gradient polyacrylamide gels. The position of pre-stained molecular weight standards (Thermo Fisher Scientific) are presented as bars on the right side of the figure. The band with molecular weight ∼50 kD corresponds to a connexin dimer, whereas the monomer has an electrophoretic mobility comparable to a molecular weight standard of ∼27 kD. Only treatment with DTT reduces the dimer to the monomeric connexin form.
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fig4: Western blots of WT (Cx32*Cx43E1) and mutant (A43C) membrane-inserted hemichannels. + and − symbols at the bottom of each panel denote treatment of the sample with or without either 50 mM DTT or 10 mM TPEN before SDS-electrophoresis through 5–40% gradient polyacrylamide gels. The position of pre-stained molecular weight standards (Thermo Fisher Scientific) are presented as bars on the right side of the figure. The band with molecular weight ∼50 kD corresponds to a connexin dimer, whereas the monomer has an electrophoretic mobility comparable to a molecular weight standard of ∼27 kD. Only treatment with DTT reduces the dimer to the monomeric connexin form.

Mentions: Although electrophysiological studies indicate that low levels of A43C currents observed 1–2 d after injection likely occur as a consequence of metal coordination, Western blots of membrane-inserted A43C channels cultured in ND96 media containing 5 mM Ca2+, a concentration that strongly favors connexin hemichannel closure and appears to increase the survival time of injected oocytes presumably by reducing connexin membrane currents, demonstrate that disulfide bonds can form between neighboring A43C residues (Fig. 4). As shown in Fig. 4 A, ∼75–80% of connexin immunoreactivity is localized to a band whose molecular weight corresponds to that expected for a connexin dimer (54 kD), whereas the remainder corresponds to the lower molecular weight monomer (27 kD). Treatment of the sample with 50 mM DTT shifts all immunoreactivity to the monomeric form. Notably, no molecular weight forms larger than dimers are observed in Western blots of homomeric A43C channels. Similar results were obtained in 3 of 30 Western blot experiments. In most cases (27 of 30 blots), the intensity of the dimer is less than that of the monomer, accounting for ∼10–20% of the total immunoreactivity (Fig. 4 B). In this experiment, the dimer was reduced to the monomeric form after treatment with 50 mM DTT but was insensitive to treatment with 10 mM TPEN. Fig. 4 C illustrates a Western blot of parental Cx32*Cx43E1 membrane-inserted unapposed hemichannels. Only the connexin monomer is detected, and there is no change in mobility after DTT treatment. The results of the biochemical experiments indicate that disulfide bond formation may contribute in part to the reduced levels of A43C current, although in most cases it appears that binding of endogenous divalent metal ions, such as Cd2+, have a larger effect.


Conformational changes in a pore-forming region underlie voltage-dependent "loop gating" of an unapposed connexin hemichannel.

Tang Q, Dowd TL, Verselis VK, Bargiello TA - J. Gen. Physiol. (2009)

Western blots of WT (Cx32*Cx43E1) and mutant (A43C) membrane-inserted hemichannels. + and − symbols at the bottom of each panel denote treatment of the sample with or without either 50 mM DTT or 10 mM TPEN before SDS-electrophoresis through 5–40% gradient polyacrylamide gels. The position of pre-stained molecular weight standards (Thermo Fisher Scientific) are presented as bars on the right side of the figure. The band with molecular weight ∼50 kD corresponds to a connexin dimer, whereas the monomer has an electrophoretic mobility comparable to a molecular weight standard of ∼27 kD. Only treatment with DTT reduces the dimer to the monomeric connexin form.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2713147&req=5

fig4: Western blots of WT (Cx32*Cx43E1) and mutant (A43C) membrane-inserted hemichannels. + and − symbols at the bottom of each panel denote treatment of the sample with or without either 50 mM DTT or 10 mM TPEN before SDS-electrophoresis through 5–40% gradient polyacrylamide gels. The position of pre-stained molecular weight standards (Thermo Fisher Scientific) are presented as bars on the right side of the figure. The band with molecular weight ∼50 kD corresponds to a connexin dimer, whereas the monomer has an electrophoretic mobility comparable to a molecular weight standard of ∼27 kD. Only treatment with DTT reduces the dimer to the monomeric connexin form.
Mentions: Although electrophysiological studies indicate that low levels of A43C currents observed 1–2 d after injection likely occur as a consequence of metal coordination, Western blots of membrane-inserted A43C channels cultured in ND96 media containing 5 mM Ca2+, a concentration that strongly favors connexin hemichannel closure and appears to increase the survival time of injected oocytes presumably by reducing connexin membrane currents, demonstrate that disulfide bonds can form between neighboring A43C residues (Fig. 4). As shown in Fig. 4 A, ∼75–80% of connexin immunoreactivity is localized to a band whose molecular weight corresponds to that expected for a connexin dimer (54 kD), whereas the remainder corresponds to the lower molecular weight monomer (27 kD). Treatment of the sample with 50 mM DTT shifts all immunoreactivity to the monomeric form. Notably, no molecular weight forms larger than dimers are observed in Western blots of homomeric A43C channels. Similar results were obtained in 3 of 30 Western blot experiments. In most cases (27 of 30 blots), the intensity of the dimer is less than that of the monomer, accounting for ∼10–20% of the total immunoreactivity (Fig. 4 B). In this experiment, the dimer was reduced to the monomeric form after treatment with 50 mM DTT but was insensitive to treatment with 10 mM TPEN. Fig. 4 C illustrates a Western blot of parental Cx32*Cx43E1 membrane-inserted unapposed hemichannels. Only the connexin monomer is detected, and there is no change in mobility after DTT treatment. The results of the biochemical experiments indicate that disulfide bond formation may contribute in part to the reduced levels of A43C current, although in most cases it appears that binding of endogenous divalent metal ions, such as Cd2+, have a larger effect.

Bottom Line: Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism.Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state.We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.

View Article: PubMed Central - PubMed

Affiliation: Dominic P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA-biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd2+ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd2+ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.

Show MeSH
Related in: MedlinePlus