Oxidative stress activates a novel non-selective cation channel in insulin-secreting cells
Single channel recordings from CRI-G1 insulin-secreting cells were used to characterize a novel ion channel. The presence of both Ca2+ and β-NAD+ at the cytoplasmic aspect of the membrane are required for channel activity. This is the first ion channel described which requires internal β-NAD+ for activity (thus termed NSNAD). The channel was found to be permeable to all monovalent (Na+, K+ and Cs+) and divalent cations tested (Ca2+, Mg2+, Ba2+, and Mn2+). The slope conductance is relatively large (70 - 90pS) compared to other non-selective cation channels and also has extremely slow kinetics (open and closed times in the range of seconds). Whole-cell voltage clamp experiments illustrate that internal β-NAD+ activates a cation current consistent with activation of the NSNAD channel. Similar to the single NSNAD channel, the β-NAD+-activated current was sensitive to the internal concentrations of both Ca2+ and β-NAD+. The non-selective nature of this cation current was confirmed by replacement of the internal K+ with Cs+ which did not diminish the β-NAD+-activated current. Additionally, replacement of external cations with the impermeant NMDG abolished the β-NAD+-activated current. The diabetogenic agent alloxan was found to irreversibly depolarize CRI-GI cells by opening a non-selective cation channel with characteristics similar to the NSNAD channel. The channel activated by alloxan is characterized by a slope conductance of approximately 70 pS and very slow (seconds) kinetics. Channel activity is lost upon excision of the patch, but can be re-activated by the application of internal β-NAD+. The mechanism of alloxan-induced depolarization and channel activation appears to be through the production of reactive oxygen species (ROS). This data indicates that oxidative stress generated by both alloxan and H2O2 causes the activation of the NSNAD channel which results in irreversible collapse of the membrane potential and massive Ca+ influx leading to eventual cell death. This may represent a component of the destruction of pancreatic β-cells during type I diabetes and possibly other pathologies in which oxidative stress is implicated.