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Recent work introduced two distinct classes of lipids as potent regulators of Kir channels: long-chain acyl-CoA (LC-CoA) esters (e.g., oleoyl-CoA) and phosphatidylinositol phosphates (e.g., PIP2). PIP2 is known to activate all members of the Kir family whereas LC-CoA esters appear to be a specific activators of KATP channels. Here we studied the effect of LC-CoA esters on different Kir channels heterologously expressed in Xenopus laevis oocytes. In agreement with previous reports, none of the Kir channels tested (Kir1.1, Kir2.1, Kir3.4, Kir4.1, and Kir7.1) were activated by oleoyl-CoA. Quite the contrary, we found LC-CoA esters to be potent inhibitors of all Kir channels (except Kir6.2). PIP2 reversed the inhibitory effect of oleoyl-CoA, suggesting a displacement of PIP2 by oleoly-CoA from a common binding site as the mechanism behind the oleoyl-CoA inhibition. This notion is supported by mutant channels with reduced PIP2 affinity and biochemical studies assaying the binding of PIP2 and oleoyl-CoA on N[H.sub.2]- and COOH-terminal fragments of Kir2.1 and Kir6.2. In addition, we provide evidence that PIP2 and LC-CoA regulate the activity of Kir channels via strucmral changes of in the selectivity filter of the channel's pore.

Adenosine triphosphate (ATP)-sensitive potassium ([K.sub.ATP]) channels couple electrical activity to cellular metabolism through their inhibition by intracellular ATP. ATP inhibition of [K.sub.ATP] 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 ([PIP.sub.2]) and phosphatidylinositol-4-phosphate (PIP) controlled ATP inhibition of cloned [K.sub.ATP] channels ([K.sub.ir] 6.2 and SUR1). These phospholipids acted on the [K.sub.ir] 6.2 subunit and shifted ATP sensitivity by several orders of magnitude. Receptor-mediated activation of phospholipase C resulted in inhibition of [K.sub.ATP]-mediated currents. These results represent a mechanism for control of excitability through phospholipids., Modulation of [K.sub.ATP] channels by activation of metabotropic receptors and cell metabolism is an important pathway for regulation of cell excitability (l). A common feature of these regulatory effects is [...]

Long-chain fatty acids acyl coenzyme A esters (LC-CoA) are obligate intermediates of fatty acid metabolism and have been shown to activate KATP channels but to inhibit most other Kir channels (e.g. Kir2.1) by direct channel binding. The activation of KATP channels by elevated levels of LC-CoA may be involved in the pathophysiology of type 2 diabetes, the hypothalamic sensing of circulating fatty acids and the regulation of cardiac KATP channels. However, LC-CoA are effectively buffered in the cytoplasm and it is currently not clear whether their free concentration can reach levels sufficient to affect Kir channels in vivo. Here, we report that extracellular oleic acid complexed with albumin at an unbound concentration of 81 ± 1 nm strongly activated KATP channels and inhibited Kir2.1 channels in Chinese hamster ovary (CHO) cells as well as endogenous Kir currents in human embryonic kidney (HEK293) cells. These effects were only seen in the presence of a high concentration of glucose (25 mm), a condition known to promote the accumulation of LC-CoA by inhibiting their mitochondrial uptake via carnitine-palmitoyl-transferase-1 (CPT1). Accordingly, pharmacological inhibition of CPT1 by etomoxir restored the effects of oleic acid under low glucose conditions. Finally, triacsin C, an inhibitor of the acyl-CoA synthetase, which is necessary for LC-CoA formation, abolished the effects of extracellular oleic acid on the various Kir channels. These results establish the direct regulation of Kir channels by the cytoplasmic accumulation of LC-CoA, which might be of physiological and pathophysiological relevance in a variety of tissues. [ABSTRACT FROM AUTHOR]

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 μM). We provide evidence that CoA and diadenosine polyphosphates (e.g. Ap4A) are ligands of the inhibitory ATP‐binding site on Kir6.2. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol phosphates (PIPs, e.g. PIP[sub2]) and long-chain acyl-CoA esters (e.g. oleoyl-CoA) are potent activators of K[subATP] channels that are thought to link K[subATP] 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) K[subATP] channels in a way very similar to PIP[sub2]. Mutations (R54Q, R176A) in the C- and N-terminus of Kir6.2 that greatly reduced the PIP[sub2] modulation of ATP sensitivity likewise reduced the modulation by oleoy1-CoA, indicating that the two lipids interact with the same site. Polyvalent cations reduced the effect of oleoyl-CoA and PIP[sub2] on the ATP sensitivity with similar potency suggesting that electrostatic interactions are of similar importance. However, experiments with differently charged inhibitory adenosine phosphates (ATP[sup4-], ADP&sup3-; and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP&sup2-;)) and diadenosine tetraphosphate (Ap[sub4]A[sup5-]) ruled out a mechanism where oleoyl-CoA or PIP[sub2] attenuate ATP inhibition by reducing ATP binding through electrostatic repulsion. Surprisingly, CoA (the head group of oleoyl-CoA) did not activate but inhibited K[subATP] channels (IC[sub50] 265 ± 33 µ M). We provide evidence that CoA and diadenosine polyphosphates (e.g. Ap[sub4]A) are ligands of the inhibitory ATP-binding site on Kir6.2. [ABSTRACT FROM AUTHOR]

The KATP channel is a heterooctamer composed of two different subunits, four inwardly rectifying K+ channel subunits, either Kir 6.1 or Kir 6.2, and four sulfonylurea receptors (SUR), which belong to the family of ABC transporters. This unusual molecular architecture is related to the complex gating behaviour of these channels. Intracellular ATP inhibits KATP channels by binding to the Kir 6.x subunits, whereas Mg-ADP increases channel activity by a hydrolysis reaction at the SUR. This ATP/ADP dependence allows KATP channels to link metabolism to excitability, which is important for many physiological functions, such as insulin secretion and cell protection during periods of ischemic stress. Recent work has uncovered a new class of regulatory molecules for KATP channel gating. Membrane phospholipids such as phosphoinositol 4,5-bisphosphate and phosphatidylinositiol 4-monophosphate were found to interact with KATP channels resulting in increased open probability and markedly reduced ATP sensitivity. The membrane concentration of these phospholipids is regulated by a set of enzymes comprising phospholipases, phospholipid phosphatases and phospholipid kinases providing a possible mechanism for control of cell excitability through signal transduction pathways that modulate activity of these enzymes. This review discusses the mechanisms and molecular determinants that underlie gating of KATP channel by nucleotides and phospholipids and their physiological implications. [ABSTRACT FROM AUTHOR]

Examines the structural and functional properties of the inward-rectifier potassium channels. Types of cells responsible for setting the resting membrane potential; Cause of the inward-rectification of potassium channels; Block of the channel pore by intracellular polyamines and magnesium.

Inward-rectifier potassium channels (Kir channels) stabilize the resting membrane potential and set a threshold for excitation in many types of cell. This function arises from voltage-dependent rectification of these channels due to blockage by intracellular polyamines. In all Kir channels studied to date, the voltage-dependence of rectification is either strong or weak. Here we show that in cardiac as well as in cloned KATP channels (Kir6.2 + sulfonylurea receptor) polyamine-mediated rectification is not fixed but changes with intracellular pH in the physiological range: inward-rectification is prominent at basic pH, while at acidic pH rectification is very weak. The pH-dependence of polyamine block is specific for KATP as shown in experiments with other Kir channels. Systematic mutagenesis revealed a titratable C-terminal histidine residue (H216) in Kir6.2 to be the structural determinant, and electrostatic interaction between this residue and polyamines was shown to be the molecular mechanism underlying pH-dependent rectification. This pH-dependent block of KATP channels may represent a novel and direct link between excitation and intracellular pH. [ABSTRACT FROM AUTHOR]

Fast N?type inactivation of voltage?dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and occurs by a 'ball?and?chain'?type mechanism. In this mechanism an N?terminal protein domain (inactivation gate) occludes the pore from the cytoplasmic side. In Kv3.4 channels, inactivation is not fixed but is dynamically regulated by protein phosphorylation. Phosphorylation of several identified serine residues on the inactivation gate leads to reduction or removal of fast inactivation. Here, we investigate the structure?function basis of this phospho?regulation with nuclear magnetic resonance (NMR) spectroscopy and patch?clamp recordings using synthetic inactivation domains (ID). The dephosphorylated ID exhibited compact structure and displayed high?affinity binding to its receptor. Phosphorylation of serine residues in the N? or C?terminal half of the ID resulted in a loss of overall structural stability. However, depending on the residue(s) phosphorylated, distinct structural elements remained stable. These structural changes correlate with the distinct changes in binding and unbinding kinetics underlying the reduced inactivation potency of phosphorylated IDs. [ABSTRACT FROM AUTHOR]