Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple electrical activity to cellular metabolism through their inhibition by intracellular ATP. ATP inhibition of KATP channels varies among tissues and is affected by the metabolic and regulatory state of individual cells, suggesting involvement of endogenous factors. It is reported here that phosphatidylinositol-4, 5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) controlled ATP inhibition of cloned KATP channels (Kir6.2 and SUR1). These phospholipids acted on the Kir6.2 subunit and shifted ATP sensitivity by several orders of magnitude. Receptor-mediated activation of phospholipase C resulted in inhibition of KATP-mediated currents. These results represent a mechanism for control of excitability through phospholipids.

Phosphatidylinositol polyphosphates (PIPs) are potent modulators of Kir channels. Previous studies have implicated basic residues in the C terminus of Kir6.2 channels as interaction sites for the PIPs. Here we examined the role of the N terminus and identified an arginine (Arg-54) as a major determinant for PIP 2 modulation of ATP sensitivity in K ATP channels. Mutation of Arg-54 to the neutral glutamine (R54Q) and, in particular, to the negatively charged glutamate (R54E) impaired PIP 2 modulation of ATP inhibition, while mutation to lysine (R54K) had no effect. These data suggest that electrostatic interactions between PIP 2 and Arg-54 are an essential step for the modulation of ATP sensitivity. This N-terminal PIP 2 site is highly conserved in Kir channels with the exception of the pH-gated channels Kir1.1, Kir4.1, and Kir5.1 that contain a neutral residue at the corresponding positions. Introduction of an arginine at this position in Kir1.1 channels rendered the N-terminal PIP 2 site functional largely increasing the PIP 2 affinity. Moreover, Kir1.1 channels lose the ability to respond to physiological changes of the intracellular pH. These results explain the need of a silent N-terminal PIP 2 site in pH-gated channels and highlight the N terminus as an important region for PIP 2 modulation of Kir channel gating.Kir channels are a superfamily of eukaryotic channel proteins that are expressed in many tissues and responsible for important physiological processes such as cell excitability, insulin secretion, K ϩ homeostasis, vascular tone, and regulation of the heart rate. Four subunits assemble to a channel. Each subunit contains two transmembrane segments with cytoplasmic N-and C-terminal domains and a connecting loop forming the pore (1). Some members of the Kir channel family are endowed with gating mechanisms such as ATP gating (K ATP channels) (2) and pH gating (Kir1.1 and Kir4.1 channels) (3). These gating mechanisms are central for the diverse functions of Kir channels in physiology and the understanding of the related pathophysiology. Kir1, Kir4, and Kir5 channels, that are predominantly expressed in epithelia, are exquisitely sensitive to changes in intracellular pH in the physiological range (3-5). This pH sensitivity is mediated by the protonation of a lysine in the N terminus (Lys-80 in Kir1.1) that induces closure of the channel's pore by an allosteric mechanism (pH gating) (3, 6). Even small changes in the pH sensitivity can cause severe kidney defects such as the Bartter syndrome (3), highlighting the physiological importance of proper pH gating in Kir1.1 channels. Kir6 channels display a very ubiquitous expression pattern and, in coassembly with the sulfonylurea receptor (SUR), 1 represent the ATP-sensitive K ϩ channels (K ATP channels) (7). Intracellular ATP closes K ATP channels by binding to the Kir6.2 subunits (ATP gating), whereas the SURs act as regulatory subunits endowing the channel with sensitivity to MgADP and pharmacological compounds. The ATP/ADP dependence of K ATP channels couple...

Recent work has established membrane phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP(2)) as potent regulators of K(ATP) channels controlling open probability and ATP sensitivity. We here investigated the effects of phospholipids on the pharmacological properties of cardiac type K(ATP) (Kir6.2/SUR2A) channels. In excised membrane patches K(ATP) channels showed considerable variability in sensitivity to glibenclamide and ATP. Application of the phosphatidylinositol phosphates (PIPs) phosphatidylinositiol-4-phosphate, PIP(2), and phosphatidylinositol-3,4,5-trisphosphate reduced sensitivity to ATP and glibenclamide closely resembling the native variability. Insertion of the patch back into the oocyte (patch-cramming) restored high ATP and glibenclamide sensitivity, indicating reversible modulation of K(ATP) channels via endogenous PIPs-degrading enzymes. Thus, the observed variability seemed to result from differences in the membrane phospholipid content. PIP(2) also diminished activation of K(ATP) channels by the K(+) channel openers (KCOs) cromakalim and P1075. The properties mediated by the sulphonylurea receptor (sensitivity to sulfonylureas and KCOs) seemed to be modulated by PIPs via a different mechanism than ATP inhibition mediated by the Kir6.2 subunits. First, polycations abolished the effect of PIP(2) on ATP inhibition consistent with an electrostatic mechanism but only weakly affected glibenclamide inhibition and activation by KCOs. Second, PIP(2) had clearly distinct effects on the concentration-response curves for ATP and glibenclamide. However, PIPs seemed to mediate the different effects via the Kir6.2 subunits because a mutation in Kir6.2 (R176A) attenuated simultaneously the effects of PIP(2) on ATP and glibenclamide inhibition. Finally, experiments with various lipids revealed structural features necessary to modulate K(ATP) channel properties and an artificial lipid (dioleoylglycerol-succinyl-nitriloacetic acid) that mimicked the effects of PIPs on K(ATP) channels.

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Phosphatidylinositol phosphates (PIPs, e.g. PIP2) and long-chain acyl-CoA esters (e.g. oleoyl-CoA) are potent activators of KATP channels that are thought to link KATP channel activity to the cellular metabolism of PIPs and fatty acids. Here we show that the two types of lipid act by the same mechanism: oleoyl-CoA potently reduced the ATP sensitivity of cardiac (Kir6.2/SUR2A) and pancreatic (Kir6.2/SUR1) KATP channels in a way very similar to PIP2. Mutations (R54Q, R176A) in the C- and N-terminus of Kir6.2 that greatly reduced the PIP2 modulation of ATP sensitivity likewise reduced the modulation by oleoyl-CoA, indicating that the two lipids interact with the same site. Polyvalent cations reduced the effect of oleoyl-CoA and PIP2 on the ATP sensitivity with similar potency suggesting that electrostatic interactions are of similar importance. However, experiments with differently charged inhibitory adenosine phosphates (ATP4-, ADP3- and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP2-)) and diadenosine tetraphosphate (Ap4A5-) ruled out a mechanism where oleoyl-CoA or PIP2 attenuate ATP inhibition by reducing ATP binding through electrostatic repulsion. Surprisingly, CoA (the head group of oleoyl-CoA) did not activate but inhibited KATP channels (IC50 = 265 +/- 33 muM). We provide evidence that CoA and diadenosine polyphosphates (e.g. Ap4A) are ligands of the inhibitory ATP-binding site on Kir6.2.

A conventional borosilicate glass patch pipette is glued into a plastic jacket, forming the entity of a FlipTip. One or two three-channel modules of recording tip sockets are mounted on a liquid handler platform to take up FlipTips. The tip sockets are connected to preamplifiers (HEKA) and to a suction system. The inner chamber of the tip sockets is filled with intracellular solution (IS), and FlipTips are prefilled with extracellular solution (ES) immediately before use. The FlipTips are then inserted into the recording tip sockets, and suspended cells are taken out of a cell hotel and dispensed into the open back of the FlipTips. Simply by gravity, the cells move down toward the end of the pipette. Continuous gentle suction draws a single cell into the very end of the tip, forming a classical GigaSeal of 1-5GOmega. Stronger suction pulses are then applied to establish the open whole-cell configuration. Under voltage-clamp conditions, the response of an ion channel is measured simultaneously in three to six recording sockets. Alternatively, the perforated patch method is used with Amphoterecine B as the pore-forming agent. Compound delivery is accomplished with a fused silica quartz pipette through the open back of the FlipTip directly onto the cell. All these tasks are performed automatically and completely unattended by the Flyscreen 8500 automated patch-clamp robot. A data throughput of several hundred data points per day can be accomplished with the system.

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