Glutathione release through connexin hemichannels: Implications for chemical modification of pores permeable to large molecules.
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.
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
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Mentions: An example of the effect of MTSES modification is shown in Fig. 2 A (left), which depicts representative current traces in response to a depolarizing pulse in an oocyte expressing D50C mutant hemichannels. The current traces were obtained at 0.25 mM Ca2+ to enhance hemichannel opening (low Ca2+ facilitates Cx26 hemichannel currents). As expected, the holding and tail currents significantly increased in the presence of 500 µM MTSES (Fig. 2 A, left, orange trace), compared with the currents before MTSES exposure (black trace). Strikingly, within 5 min after wash off of MTSES, the holding and tail currents returned to levels similar to those observed before MTSES exposure (blue trace). The time course of the MTSES effect on the peak tail currents and its (unexpected) reversal after MTSES washout were determined (Fig. 2 A, right). Tail currents increased after the addition of MTSES, but after washout, the currents returned to the original levels, with an approximate time constant of 358 s. Similar effects were seen for MTSES exposure to D46C and G45C channels, with reversal of the MTSES effects having time constants of ∼180 and ∼715 s, respectively (Fig. 2, B and C). No modifications were observed in wild-type Cx26 currents in the presence, or after wash off, of 500 µM MTSES (Fig. S1 A). The reversibility of the effect of MTSES on the currents mediated by cysteine mutants suggested that MTS modification was being chemically reversed, as a second application of MTSES has a similar effect to the first application (Fig. S2). The MTS–thiol linkage can only be broken by reducing agents, raising the intriguing possibility that endogenous cytosolic reducing agents permeate the connexin pore to reverse the MTS modification. A recent study showed slow permeation of GSH through Cx46 hemichannels (Slavi et al., 2014). We examined whether MTSES modification was reversible in Cx46 hemichannels containing a substituted cysteine at pore-lining position 43 (E43C). Although Cx46 E43C channels showed slower MTSES modification than that observed for cysteine mutants of Cx26 hemichannels, there was a sustained recovery from the chemical modification after washing off MTSES (Fig. S3), further supporting slow permeation of a reducing agent through connexin channels.
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.