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 NS_NAD). 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 NS_NAD channel. Similar to the single NS_NAD 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 NS_NAD 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 NS_NAD 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.

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Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Thesis (M.S.P.H.)--University of Michigan, 1939.