Induced PIP2-Mediated Ion Channel Gating at the Plasma Membrane

Research Summary

The iDimerize Inducible Heterodimerization technology is used to understand the role of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by phospholipase C (PLC), in mediating KCNQ ion channel closure. Authors show that induced PIP2 depletion is sufficient for turning off KCNQ channels, without requiring other PLC-associated signaling molecules. Paper: Suh et al. (2010) Neuron, 67(23):224–38.

Ligand Induced PIP2- Mediated Ion Channel Gating at the Plasma Membrane

Advantages of inducible dimerization

Experimental design

Human embryonic kidney tsA-201 cells were created that deplete PIP2 using inositol polyphosphate 5-phosphatase (Inp54p), thus avoiding other signaling molecules generated downstream of PLC. Yeast Inp54p was fused with a DmrA domain and cyan fluorescent protein (CFP). A DmrC domain was anchored at the membrane using a signaling sequence, to allow inducible Inp54p recruitment upon addition of A/C Heterodimerizer ligand to the growth media. A yellow fluorescent protein (YFP)-labeled pleckstrin homology (PH) domain, which naturally binds membrane phosphatidylinositol lipids, was used as a sensor for PIP2 levels.*

Key Results

  1. In the presence of ligand, Inp54p accumulation at the membrane and a simultaneous release of fluorescent sensor were observed, confirming PIP2 depletion at the membrane.

  2. Ligand treatment also caused complete and reversible suppression of KCNQ currents, proving that PIP2 depletion suffices in turning off KCNQ channels.

*

Studies are presented using iDimerize components. For complete experimental details please refer to the original publication. The optimal position and copy number of fusion domains varies per study and must be determined empirically.

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