ATP-sensitive K؉ channels (K ATP channels) of pancreatic -cells play key roles in glucose-stimulated insulin secretion by linking metabolic signals to cell excitability. Membrane phosphoinositides, in particular phosphatidylinositol 4,5-bisphosphates (PIP 2 ), stimulate K ATP channels and decrease channel sensitivity to ATP inhibition; as such, they have been postulated as critical regulators of K ATP channels and hence of insulin secretion in -cells. Here, we tested this hypothesis by manipulating the interactions between K ATP channels and membrane phospholipids in a -cell line, INS-1, and assessing how the manipulations affect membrane excitability and insulin secretion. We demonstrate that disruption of channel interactions with PIP 2 by overexpressing PIP 2 -insensitive channel subunits leads to membrane depolarization and elevated basal level insulin secretion at low glucose concentrations. By contrast, facilitation of channel interactions with PIP 2 by upregulating PIP 2 levels via overexpression of a lipid kinase, phosphatidylinositol 4-phosphate 5 kinase, decreases the ATP sensitivity of endogenous K ATP channels by ϳ26-fold and renders INS-1 cells hyperpolarized, unable to secrete insulin properly in the face of high glucose. Our results establish an important role of the interaction between membrane phosphoinositides and K ATP channels in regulating insulin secretion. Diabetes 54:2852-2858, 2005 P ancreatic -cells secrete insulin in response to glucose stimulus. The ATP-sensitive K ϩ (K ATP ) channel, a complex of four inwardly rectifying K ϩ channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits, is a key component in this stimulus-secretion coupling process (1-3). The hallmark features of K ATP channels are their sensitivities to intracellular nucleotides ATP and ADP, the derivatives of glucose metabolism (1,2). ATP inhibits channel activity, whereas ADP, in complex with Mg 2ϩ , stimulates channel activity. It is now generally accepted that the physiological activity of K ATP channels is regulated primarily by the relative concentrations of ATP and ADP (1,4,5). As plasma glucose increases, ATP concentration increases and ADP concentration decreases, resulting in K ATP channel closure, membrane depolarization, Ca 2ϩ influx, and insulin release. Conversely, when glucose decreases, the concentration ratio of ATP to ADP decreases, leading to K ATP channel opening, membrane hyperpolarization, and termination of insulin secretion. The importance of ATP and ADP in regulating K ATP channels in vivo has been confirmed by the finding that mutations that reduce channel sensitivity to ATP or MgADP are causative in permanent neonatal diabetes or congenital hyperinsulinism, respectively (5-9).The discovery that membrane phosphoinositides, in particular the most abundant phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (10), stimulate K ATP channel activity and antagonize the inhibitory effect of ATP in isolated membrane patches (11,12) has led to the proposal that in addition ...