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Glutathione release through connexin hemichannels: Implications for chemical modification of pores permeable to large molecules.

Tong X, Lopez W, Ramachandran J, Ayad WA, Liu Y, Lopez-Rodriguez A, Harris AL, Contreras JE - J. Gen. Physiol. (2015)

Bottom Line: Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine decreased the cytosolic GSH concentration in Xenopus oocytes and reduced reversibility of MTS modification, as did acute treatment with tert-butyl hydroperoxide, which oxidizes GSH.Cysteine modification based on thioether linkages (e.g., maleimides) cannot be reversed by reducing agents and did not reverse with washout.These results show that, for wide pores, accessibility of cytosolic reductants can lead to reversal of MTS-based thiol modifications.

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Affiliation: Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School, Rutgers University, Newark, NJ 07103 Department of Pharmacology, Bengbu Medical College, Bengbu, Anhui Province 233000, China.

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Residues selected for chemical modification in the Cx26 hemichannel. (A) Top (left) and side view (right) of the human Cx26 hemichannel crystal structure. Four subunits are shown in the side view to show the pore-lining region. (B) Magnification of residues D50, D46, and G45, all highlighted in red.
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fig1: Residues selected for chemical modification in the Cx26 hemichannel. (A) Top (left) and side view (right) of the human Cx26 hemichannel crystal structure. Four subunits are shown in the side view to show the pore-lining region. (B) Magnification of residues D50, D46, and G45, all highlighted in red.

Mentions: Cysteine substitution followed by modification by MTS is a useful tool to define the roles of pore-lining and charged residues in the gating and conductance of connexin channels (Kronengold et al., 2003a; Tang et al., 2009; Verselis et al., 2009; Lopez et al., 2013; Sánchez et al., 2013). Here, this technique was applied to explore the functional roles of three residues (D50, D46, and G45) that line the extracellular side of the pore and have been shown to be involved in electrostatic stabilization of the open state and/or regulation of the channels by extracellular calcium ion. Previous reports from us and other groups have shown that hemichannel currents significantly increase when cysteines substituted at these positions are exposed to and modified by MTSES, a negatively charged MTS reagent (Sánchez et al., 2010, 2013; Lopez et al., 2013; MTSES modification of D50C and D46C reestablishes the native negative charge at this position that stabilizes the open state). The locations of these residues are depicted in Fig. 1.


Glutathione release through connexin hemichannels: Implications for chemical modification of pores permeable to large molecules.

Tong X, Lopez W, Ramachandran J, Ayad WA, Liu Y, Lopez-Rodriguez A, Harris AL, Contreras JE - J. Gen. Physiol. (2015)

Residues selected for chemical modification in the Cx26 hemichannel. (A) Top (left) and side view (right) of the human Cx26 hemichannel crystal structure. Four subunits are shown in the side view to show the pore-lining region. (B) Magnification of residues D50, D46, and G45, all highlighted in red.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4555470&req=5

fig1: Residues selected for chemical modification in the Cx26 hemichannel. (A) Top (left) and side view (right) of the human Cx26 hemichannel crystal structure. Four subunits are shown in the side view to show the pore-lining region. (B) Magnification of residues D50, D46, and G45, all highlighted in red.
Mentions: Cysteine substitution followed by modification by MTS is a useful tool to define the roles of pore-lining and charged residues in the gating and conductance of connexin channels (Kronengold et al., 2003a; Tang et al., 2009; Verselis et al., 2009; Lopez et al., 2013; Sánchez et al., 2013). Here, this technique was applied to explore the functional roles of three residues (D50, D46, and G45) that line the extracellular side of the pore and have been shown to be involved in electrostatic stabilization of the open state and/or regulation of the channels by extracellular calcium ion. Previous reports from us and other groups have shown that hemichannel currents significantly increase when cysteines substituted at these positions are exposed to and modified by MTSES, a negatively charged MTS reagent (Sánchez et al., 2010, 2013; Lopez et al., 2013; MTSES modification of D50C and D46C reestablishes the native negative charge at this position that stabilizes the open state). The locations of these residues are depicted in Fig. 1.

Bottom Line: Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine decreased the cytosolic GSH concentration in Xenopus oocytes and reduced reversibility of MTS modification, as did acute treatment with tert-butyl hydroperoxide, which oxidizes GSH.Cysteine modification based on thioether linkages (e.g., maleimides) cannot be reversed by reducing agents and did not reverse with washout.These results show that, for wide pores, accessibility of cytosolic reductants can lead to reversal of MTS-based thiol modifications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School, Rutgers University, Newark, NJ 07103 Department of Pharmacology, Bengbu Medical College, Bengbu, Anhui Province 233000, China.

Show MeSH