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Ca2+‐ and thromboxane‐dependent distribution of MaxiK channels in cultured astrocytes: From microtubules to the plasma membrane

Large‐conductance, voltage‐ and Ca2+‐activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore‐forming α‐subunits (MaxiKα) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiKα is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiKα was visualized by three independent labeling approaches: (1) MaxiKα‐specific antibodies, (2) expressed EGFP‐labeled MaxiKα, and (3) fluorophore‐conjugated iberiotoxin, a specific MaxiK pore‐blocker. This MaxiKα association with microtubules was further confirmed by in vitro His‐tag pulldown, co‐immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiKα labeling at the plasma membrane. More notably, U46619, an analog of thromboxane A2 (TXA2), which triggers Ca2+‐release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiKα surface labeling. Whole‐cell patch clamp recordings of U46619‐stimulated cells develop a ∼3‐fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiKα. The results of this study provide the novel insight that suggests that Ca2+ released from intracellular stores may play a key role in regulating the traffic of intracellular, microtubule‐associated MaxiKα stores to the plasma membrane of developing murine astrocytes. © 2009 Wiley‐Liss, Inc.