Pip2-Channel Interaction as a Critical Element in Regulation of SK Channel Activity

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP2), a minor acidic phospholipid found in the inner leaflet of the plasma membrane, plays a major role in cellular signaling. While it is well documented that PIP2 can regulate the activity of many ion channels through direct interactions, the affinity of PIP2 is so high for some channel proteins that its physiological role as a modulator has been questioned. Here we demonstrate that PIP2 is an essential cofactor for activation of small-conductance K+ channels (SK2) by Ca2+-bound calmodulin (CaM), with the PIP2-binding site located at the interface of the CaM-SK2 complex near the CaM N-lobe. Because of the high sensitivity of PIP2 for the channel complex, global PIP2 hydrolysis produces only a small inhibitory effect on SK2 channel activity. Phosphorylation of CaM, when complexed with SK, at T79 by Casein Kinase 2 (CK2), mimicked by the T79D mutation in mammalian cells, is known to inhibit Ca2+-dependent activation of SK channels by an unknown mechanism of action. We show that the phosphomimetic T79D mutation can effectively reduce the PIP2 sensitivity of SK2 channel by 12-fold and confer susceptibility to inhibition by PIP2 hydrolysis. We propose the converged signaling of CaM phosphorylation and PIP2 hydrolysis as the underlying mechanism for inhibition of SK channels in neurons induced by neurotransmitters, which contributes to the regulation of synaptic plasticity and thus learning and memory. Additionally we observed that changes of protein conformation in close proximity to PIP2 binding site by a small molecule, NS309, can effectively enhance the interaction between the protein and PIP2, and potentiate channel activity. These results demonstrate the PIP2-channel interaction as a critical element in both physiological and pharmacological regulation of SK channel activity.