Affinity For PIP Research Articles

Liposome-Based Methods to Study GTPase Activation by Phosphoinositides.

Phosphoinositides (PIPs) are lipid messengers with different functions according to their localization. After their local production by the action of lipid kinases or phosphatases, PIPs regulate various biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, or gene expression through binding of their phosphorylated inositol head group with different protein domains such as PH, PX, and FYVE. It is well known that PIPs regulate the activity of small GTPases by interacting with and activating Guanyl-nucleotide Exchange Factor (GEF) proteins through specific domains such as the ones mentioned above. However, most of the in vitro assays to assess the activation of GTPases focus on the GTPase only and neglect the fact that co-activators, such as membranes and protein activators, have a significant effect in vivo. Herein, we describe not only the classical protein-lipid overlay and liposome sedimentation methods but also an assay we have developed, which contains three partners: a liposome which composition reproduces the membrane of the target of the GTPase, the recombinant specific DH-(PIP affinity) GEF domain, and the recombinant GTPase to be tested by different PIPs. This assay allows us to clearly quantify the GTPase activation.

Mechanisms for Kir channel inhibition by quinacrine: acute pore block of Kir2.x channels and interference in PIP2 interaction with Kir2.x and Kir6.2 channels

Cardiac inward rectifier potassium currents determine the resting membrane potential and contribute repolarization capacity during phase 3 repolarization. Quinacrine is a cationic amphiphilic drug. In this work, the effects of quinacrine were studied on cardiac Kir channels expressed in HEK 293 cells and on the inward rectifier potassium currents, I(K1) and I(KATP), in cardiac myocytes. We found that quinacrine differentially inhibited Kir channels, Kir6.2 ∼ Kir2.3 > Kir2.1. In addition, we found in cardiac myocytes that quinacrine inhibited I(KATP) > I(K1). We presented evidence that quinacrine displays a double action towards strong inward rectifier Kir2.x channels, i.e., direct pore block and interference in phosphatidylinositol 4,5-bisphosphate, PIP(2)-Kir channel interaction. Pore block is evident in Kir2.1 and 2.3 channels as rapid block; channel block involves residues E224 and E299 facing the cytoplasmic pore of Kir2.1. The interference of the drug with the interaction of Kir2.x and Kir6.2/SUR2A channels and PIP(2) is suggested from four sources of evidence: (1) Slow onset of current block when quinacrine is applied from either the inside or the outside of the channel. (2) Mutation of Kir2.3(I213L) and mutation of Kir6.2(C166S) increase their affinity for PIP(2) and lowers its sensitivity for quinacrine. (3) Mutations of Kir2.1(L222I and K182Q) which decreased its affinity for PIP(2) increased its sensitivity for quinacrine. (4) Co-application of quinacrine with PIP(2) lowers quinacrine-mediated current inhibition. In conclusion, our data demonstrate how an old drug provides insight into a dual a blocking mechanism of Kir carried inward rectifier channels.