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J Gen Physiol
2015 Sep 01;1463:245-54. doi: 10.1085/jgp.201511375.
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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
.
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Cysteine-scanning mutagenesis combined with thiol reagent modification is a powerful method with which to define the pore-lining elements of channels and the changes in structure that accompany channel gating. Using the Xenopus laevis oocyte expression system and two-electrode voltage clamp, we performed cysteine-scanning mutagenesis of several pore-lining residues of connexin 26 (Cx26) hemichannels, followed by chemical modification using a methanethiosulfonate (MTS) reagent, to help identify the position of the gate. Unexpectedly, we observed that the effect of MTS modification on the currents was reversed within minutes of washout. Such a reversal should not occur unless reducing agents, which can break the disulfide thiol-MTS linkage, have access to the site of modification. Given the permeability to large metabolites of connexin channels, we tested whether cytosolic glutathione (GSH), the primary cell reducing agent, was reaching the modified sites through the connexin pore. 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. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay, we confirmed that homomeric Cx26 and Cx32 and heteromeric Cx26/Cx32 are permeable to GSH and other endogenous reductants. These results show that, for wide pores, accessibility of cytosolic reductants can lead to reversal of MTS-based thiol modifications. This potential for reversibility of thiol modification applies to on-cell accessibility studies of connexin channels and other channels that are permeable to large molecules, such as pannexin, CALHM, and VRAC.
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Figure 1. 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.
Figure 2. Reversible chemical modification by MTS at cysteine residues lining the Cx26 pore. (A) MTSES modification at D50C. (Left) Current traces from an oocyte expressing Cx26 D50C hemichannels in response to a depolarizing pulse from â80 to 0 mV. Black trace is the current before modification. Orange trace is a representative trace in the presence of MTSES after reaching the maximal effect of the modification. Blue trace is the current after wash off of MTSES. (Right) Time course of the maximal tail currents in the absence, the presence, and after wash off of MTSES. Solid blue line represents single-exponential fit for recovery from modification. (B and C) The corresponding data for MTSES modification at positions D46C and G45C, respectively. Experimental procedure as described in A. All traces were obtained in the presence of 0.25 mM of extracellular Ca2+.
Figure 3. Decreased levels of cellular rGSH decelerate or diminish reversibility of MTS modification. BSO and TBHO2, which decrease cytoplasmic rGSH levels, decelerate and/or diminish reversibility of the MTSES modification. (A) A representative experiment for MTSES modification at position D50C. (Left) Dots show the time course of maximal tail current for an oocyte pretreated with 2 mM TBHO2, which was then exposed to MTSES. Wash off of MTSES was in the presence of TBHO2. (Right) Same experiment, but with 24â48-h preexposure to BSO. The tail currents were measured in the presence of 0.25 mM of extracellular Ca2+ after a depolarizing pulse from â80 to 0 mV. (B and C) Representative experiments for MTS modification at positions D46C and G45C, respectively, as described in A.
Figure 4. BSO or TBHO2 treatment significantly decreases recovery after MTSES modification. Percentage of recovery from MTSES modification after BSO (gray bars) or TBHO2 (black bars) treatment in oocytes expressing D50C, D46C, or G45C. White bars correspond to recovery from MTSES modification without any treatment. The values were normalized at steady state or after 300 s of MTSES wash off. Error bars represent the mean ± SEM of at least eight independent measurements.
Figure 5. Connexin hemichannels are permeable to cytosolic reductants. (A; left) Scheme of TSF method. (Right) Chemical structure of 3-PA and endogenous cellular reductants. (B) Percentage of liposomes permeable to cytosolic reducing agents for liposomes containing either homomeric or heteromeric hemichannels formed by Cx32, Cx26, and Cx32/26. Orange bars, GSH; blue bars, NADH; green bars, NADPH. Homomeric and heteromeric hemichannels were fully permeable through all tested reducers. 3-PA (black bars), a trisaccharide derivative known to be permeable through Cx32 channels but not Cx26-containing channels, was used as control. Error bars represent the mean ± SEM of at least three independent measurements.
Figure 6. Irreversible maleimide modification at cysteine residues lining the pore. (A) Comparison of the chemical structures of MTSES and maleimide ES. (B) Time course of the peak tail current in the absence (black), the presence (red), or after wash off (blue) of maleimide ES for modification of D50C hemichannels expressed in Xenopus oocytes. (C) Maleimide ES modification at positions G45C, as described in A.
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