Expression, Regulation and Functional Roles of the Intermediate Conductance Ca2+-dependent K+ channel, KCa3.1 (KCNN4/IK/SK4) in the MLS-9 Microglial Cell Line and Primary Rat Alternatively Activated by Interleukin IL-4
Bibliographic record
Abstract
`Alternative' activation of microglia by interleukin (IL)-4 up-regulates anti-inflammatory molecules and aids with repair processes. Blockers of the Ca2+-activated K+ channel, KCa3.1 (KCNN4/IK1/SK4) improve the outcome in several rodent models of CNS damage but nothing was known about this channel and its roles in the alternative activated state. Therefore, I first characterized the KCa3.1 current in primary rat microglia (unstimulated and IL-4 treated), and in the MLS-9 rat microglial cell line. KCa3.1 current expression was low in untreated microglia; fewer than 30% displayed a small-amplitude current, and it required a positive gating modulator, riluzole or NS309. In contrast, KCa3.1 current was reliably activated in every MLS-9 cell tested, making this cell line useful for studying channel activation and regulation. In MLS-9 cells, I discovered a selective coupling of KCa3.1 to UTP-mediated P2Y2 receptor activation. Following P2Y2-mediated Ca2+ store depletion, KCa3.1 was activated by Ca2+ entry through Orai1/CRAC channels, and it then promoted Ca2+ influx and refilling of Ca2+ stores; however, the Ca2+ required for channel activation was surprisingly high. In alternative-activated primary microglia (IL-4 treated), I discovered that both KCNN4 mRNA and KCa3.1 current were greatly up-regulated, and it then contributed to several functional outcomes. The signaling pathway involved binding of IL-4 to the Type I receptor, activation of the Ras/MEK/ERK pathway, and AP-1 mediated induction of the KCNN4 gene. I discovered that, once KCa3.1 current was induced in alternative activated microglia, UTP activated it, and both the UTP-mediated Ca2+ influx and migration required KCa3.1 current. Next, I found that KCa3.1 is post-translationally regulated by cGMP protein kinase and by PI3K via a signaling cascade involving intracellular ROS, which activates CaMKII and increases the KCa3.1 current. A similar process was seen in MLS-9 cells. Subsequently, KCa3.1 was involved in a positive feedback loop that potentiated ROS production. Overall, these discoveries demonstrate that KCa3.1 is up-regulated in alternative activated rat microglia, and then contributes to several fundamental processes. This work will contribute to our understanding of microglia after CNS damage or disease and to the potential for targeting KCa3.1 to improve the outcomes.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".