Large-conductance, voltage- and calcium-sensitive potassium channels (MaxiK) in astrocytes: A novel intracellular interaction with the cytoskeleton

Large-conductance, voltage- and Ca2+-activated K + channels (MaxiK, BK) are broadly expressed ion channels minimally assembled by a tetrameric conformation of the pore-forming alpha-subunits (MaxiKalpha) and typically observed as plasma membrane proteins in various cell types. MaxiK channels have been well characterized in neuronal function in the brain; however the role of MaxiK in glia is currently not well understood. Previous electrophysiological studies have measured MaxiK currents in cultured astrocytes confirming the presence of this K+ channel; however, the subcellular distribution and function of astrocytic MaxiK have yet to be demonstrated in this cellular system.;In murine astrocyte primary cultures, this work shows that MaxiKalpha is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiKalpha was visualized by three independent labeling approaches: (1) MaxiKalpha-specific antibodies, (2) expressed EGFP-labeled MaxiKalpha, and (3) fluorophore-conjugated iberiotoxin, a specific MaxiK pore-blocker. This MaxiKalpha 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 MaxiKalpha 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 and other pathophysiological conditions, also elicits a similar increase in MaxiKalpha surface labeling.;Whole-cell patch clamp recordings of U46619 stimulated cells develop a ∼3-fold increase in current density 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 MaxiKalpha. 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 MaxiKalpha stores to the plasma membrane of developing murine astrocytes.