Charge Shielding of PIP(2) by Cations Regulates Enzyme Activity of Phospholipase C

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP(2) binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP(2), a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP(2)-PLC interaction. This charge shielding of PIP(2) was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP(2) analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP(2) via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP(2) was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP(2) hydrolysis by PLC as triggered by M(1) muscarinic receptors with a potency order of Mg(2+) < spermine(4+) < neomycin(6+). Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg(2+) and polyamines modulate the activity of PLCs by controlling the amount of free PIP(2) available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically.