Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels.

Suh BC, Inoue T, Meyer T, Hille B.

Science. 2006 Dec 1;314(5804):1454-7. PMID: 16990515

Objective:   One of the major signaling pathways near the cell membrane involves the activation of phospholipase-C to cleave phosphotidylinositol 4,5-bisphosphate (PIP2) in to diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3).  It should, however, be noted that PIP2 does not serve only as a reserve to be cleaved as needed, but also has the important function of stabilizing the “open” state of KCNQ potassium channels. One challenge researchers have faced is in examining the effects of PIP2 depletion on KCNQ channels without activating the downstream effects that result from activating the entire cascade.  In this paper, a novel method of PIP2 depletion was developed that did not result in production of DAG or IP3.

Methods:    NIH3T3 fibroblast cells were transfected with KCNQ channel subunits and synthetic fusion constructs which would allow for the relocation of a cytosolic PIP2-(5)-phosphatase to the membrane in response to the application of a rapamycin analog, iRap.  The phosphatase was fused to a CFP reporter.  The cells also were made to express a membrane-localized YFP-bound PIP2 reporter that would relocate to the cytosol after cleavage of the 5-phosphate.  Cells were monitored for changes in fluorescence in response to drug exposure and currents were recorded by whole-cell voltage clamp.

Results:

·         When exposed to iRap, cytosolic phosphatase-CFP successfully relocated to the membrane while the YFP-PIP2sensor relocated to the cytosol.  When a phosphatase-dead or phosphatase-missing construct was used, it relocated to the membrane but the PIP2 sensor did not relocate to the cytosol.

·         When using a drug to activate PLC, they could see YFP-PIP2sensor relocation without the CFP-phosphatase relocation.  This relocation was reversible when the drug was washed out.  Under iRap and PLC-activation conditions, potassium currents were inhibited by PIP2 depletion.

·         Under conditions of PLC inhibition, an upstream PLC activator had no effect while iRap did, demonstrating that iRap did not have crossover effects on the PLC pathway.  This was confirmed by use of a DAG indicator and a calcium sensor (indicative of IP3 production).

·         Another construct was used in which iRap activated a kinase to regenerate PIP2 from other phosphatidyl inositols.  This caused PLC activation to have a smaller effect on KCNQ currents.

Conclusions:

·         KCNQ channels require a constant supply of PIP2 to maintain the stability of their “open” conformation.

·         New fusion constructs allow depletion of PIP2 without generation of DAG or IP3

·         New constructs also allow the regeneration of PIP2 faster than a cell could normally do it.

 

-DH