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M. Srinivas; Regulation of Gap Junction Channels by Oxidation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4093.
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Oxidative stress plays a major role in the formation of cataracts. One of the primary modifications associated with oxidative stress in the lens is the oxidation of sulphur–containing amino acid residues, e.g. cysteine and methionine, in proteins. The effect of oxidation on membrane proteins, especially those involved in the lens microcirculatory system such as gap junction channels remains unknown. In this study, the effects of oxidation on gap junction channels formed by Cx46, which is primarily expressed in lens fiber cells, and on those formed by Cx50, which is expressed in both the lens epithelium and fiber cells, were investigated.
Chloramine–T, a membrane permeable oxidizing agent that selectively oxidizes methionine and cysteine residues, was used. The effect of chloramine–T (1 mM) on N2A neuroblastoma cells that were exogenously transfected with Cx46 or Cx50 cDNA was investigated. In addition, to determine whether cysteine or methionine residues were involved in the action of chloramine–T, the effects of dithiothreitol (DTT), a reducing agent that selectively modifies oxidized cysteine residues (but not oxidized methionine residues) were evaluated.
Extracellular chloramine–T (1 mM) caused a complete decrease in junctional conductance of Cx46 and Cx50 within 1 min of its application (98.3 ± 3%, n=6; and 91.1 ± 4, n=4; for Cx50 and Cx46, respectively). The effects of chloramine–T on coupling were largely irreversible, despite extensive washing of cell pairs with oxidant–free solutions for > 6 min, as expected for an oxidation mechanism. Additional single channel studies on Cx50 expressing cells indicated that chloramine–T primarily affects channel open probability. Extracellular DTT (2 mM), applied immediately after chloramine–T, only partially reversed the effects of the oxidizing agent on Cx50 and Cx46 gap junction channels (recovery of 26 ± 4 % of control; n=3 and 31 ± 5 % of control; n=3, for Cx50 and Cx46, respectively after 4 min of DTT). DTT alone had no effect on Cx46 or Cx50 gap junction channels.
Results suggest that oxidation of methionine residues and, to a lesser extent, cysteine residues, affects coupling of lens gap junction channels. Reduction of Cx46– and Cx50–mediated coupling may be one mechanism by which oxidizing agents cause cataract formation.
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