Measurement of PIP2 metabolism and its relevance for neuronal function

Many plasma membrane proteins have recently been found to require the phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP2) to function. These PIP2-requiring proteins include potassium channels, TRP channels, and several transporters. PIP2 also modulates exocytosis and cell adhesion. Phosphoinsositides are dynamically varying, and we are only beginning to understand the many signals carried by these changes.

In order to measure reaction rates for the enzymes involved in PIP2 metabolism, we induced quick PIP2 depletion in individual tsA201 cells by two methods: muscarinic receptor activation (to activate phospholipase C), or using voltage steps and a voltage-sensitive phosphoinositide 5-phosphatase (VSP). PIP2 was monitored in living cells by patch clamp of the PIP2-sensitive M-current through KCNQ2/3 potassium channels and by epifluorescence microscopy of fluorescent probes that specifically bind to plasma membrane PIP2.

Phosphoinositides are interconverted by lipid kinases and lipid phosphatases. We found that PIP 5-kinase, which produces PIP2 by phosphorylation of its precursor PI(4)P, is relatively fast with a time constant of 10s. The PI 4-kinase, which produces PI(4)P from PI is 10 fold slower. Kinetic modeling allowed us to also determine the activities of the 4-phosphatase and the 5-phosphatase. From this, we were able estimate the concentration of free PIP2at the plasma membrane, which has been unknown so far. We were also able to estimate the concentration of PI(4)P, which is in a similar range as that of PIP2.

Taken together, our findings suggest that a brief PIP2 depletion can be restored quickly by phosphorylation of PI(4)P, whereas a longer depletion of PIP2 (even if it is less pronounced) also requires the much slower PI 4-kinase to recover. As PIP2-sensitive potassium channels regulate neuronal excitability, these changes in PIP2 have immediate consequences on neuronal excitability.

PIP2 depletion is induced by activation of muscarinic M1 and other metabotropic receptors. It therefore underlies some desired and undesired effects of the many clinically used drugs that target these receptors. In addition, PIP2 depletion has been described in models of Alzheimer's disease, such as exposure to oligomeric amyloid-β peptide (Berman et al., Nature Neuroscience 2008). A better understanding of PIP2 metabolism might therefore suggest new treatment strategies for this disease.

(Supported by the NIH and an HFSP fellowship.)