Abstract
Large-conductance, voltage- and Ca2+-activated K+ channels (MaxiK, BK) are broadly expressed ion channels typically observed as a plasma membrane protein in various cell types. In murine astrocyte primary cultures, which are more indicative of in-vivo reactive astrocytes rather than resting astrocytes, our previous results using high-resolution confocal microscopy have revealed the novel finding that MaxiK pore-forming α subunit (MaxiKα) is distributed intracellularly, colocalized along the microtubule network. This MaxiKα association with microtubules was further confirmed by in vitro His-tag pulldown assays, co-immunoprecipitation assays from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine (20mM) or thapsigargin (1μM), resulted in increased MaxiKα labeling at the plasma membrane. More notably U46619, a stable 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. We now show using whole-cell patch clamp recordings that U46619 stimulated cells develop a ∼3-fold increase in current amplitude. This data indicates that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. These changes in MaxiKα plasma membrane distribution are effectively blocked by preincubating astrocytes with a cell permeable Ca2+-chelator, BAPTA-AM, or by microtubule disruption prior to stimulation. While microtubules are largely absent in mature astrocytes, our immunohistochemistry results in brain slices show that cortical astrocytes in the developing newborn mouse brain (P1) have a robust expression of microtubules that significantly colocalize with MaxiKα. The results of this study provide the novel insight that suggests 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 reactive astrocytes. Supported by NIH.
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