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Ca2+- and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: from microtubules to the plasma membrane.

ABSTRACT: Large-conductance, voltage- and Ca2+-activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming alpha-subunits (MaxiKalpha) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show 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, also elicits a similar increase in MaxiKalpha surface labeling. Whole-cell patch clamp recordings of U46619-stimulated cells develop a approximately 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 MaxiK. 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 MaxiK stores to the plasma membrane of developing murine astrocytes.

SUBMITTER: Ou JW

PROVIDER: S-EPMC2713352 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Ca2+- and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: from microtubules to the plasma membrane.

Ou J W JW Kumar Y Y Alioua A A Sailer C C Stefani E E Toro L L

Glia 20090901 12

Large-conductance, voltage- and Ca2+-activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming alpha-subunits (MaxiKalpha) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show 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 ...[more]

PMID: 19170178

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Project description:A major contributing factor to the high mortality rate associated with acute myeloid leukemia and multiple myeloma is the development of resistance to chemotherapy. We have shown that the combination of tipifarnib, a nonpeptidomimetic farnesyltransferase inhibitor (FTI), with bortezomib, a proteosome inhibitor, promotes synergistic death and overcomes de novo drug resistance in acute myeloid leukemia cell lines. Experiments were undertaken to identify the molecular mechanisms by which tipifarnib produces cell death in acute myeloid leukemia and multiple myeloma cell lines (U937 and 8226, respectively). Tipifarnib, but not other FTIs tested [N-[4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl]methionine methyl ester trifluoroacetate salt (FTI-277) and 2'-methyl-5-((((1-trityl-1H-imidazol-4-yl)methyl)amino)methyl)-[1,1'-biphenyl]-2-carboxylic acid (FTI-2153), promotes elevations in intracellular free-calcium concentrations ([Ca(2+)](i)) in both cell lines. These elevations in [Ca(2+)](i) were accompanied by highly dynamic plasmalemmal blebbing and frequently resulted in membrane lysis. The tipifarnib-induced elevations in [Ca(2+)](i) were not blocked by thapsigargin or ruthenium red, but were inhibited by application of Ca(2+)-free extracellular solution and by the Ca(2+) channel blockers Gd(3+) and La(3+). Conversely, 2-aminoethoxydiphenyl borate (2-APB) potentiated the tipifarnib-evoked [Ca(2+)](i) overload. Preventing Ca(2+) influx diminished tipifarnib-evoked cell death, whereas 2-APB potentiated this effect, demonstrating a link between tipifarnib-induced Ca(2+) influx and apoptosis. These data suggest that tipifarnib exerts its effects by acting on a membrane channel with pharmacological properties consistent with store-operated channels containing the Orai3 subunit. It is noteworthy that Orai3 transcripts were found to be expressed at lower levels in tipifarnib-resistant 8226/R5 cells. Our results indicate tipifarnib causes cell death via a novel mechanism involving activation of a plasma membrane Ca(2+) channel and intracellular Ca(2+) overload.

Dataset Information

Ca2+- and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: from microtubules to the plasma membrane.

Publications

Ca2+- and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: from microtubules to the plasma membrane.

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