Identification of residues contributing to the ATP binding site of Kir6.2

The ATP-sensitive potassium (K(ATP)) channel links cell metabolism to membrane excitability. Intracellular ATP inhibits channel activity by binding to the Kir6.2 subunit of the channel, but the ATP binding site is unknown. Using cysteine-scanning mutagenesis and charged thiol-modifying reagents, we identified two amino acids in Kir6.2 that appear to interact directly with ATP: R50 in the N-terminus, and K185 in the C-terminus. The ATP sensitivity of the R50C and K185C mutant channels was increased by a positively charged thiol reagent (MTSEA), and was reduced by the negatively charged reagent MTSES. Comparison of the inhibitory effects of ATP, ADP and AMP after thiol modification suggests that K185 interacts primarily with the beta-phosphate, and R50 with the gamma-phosphate, of ATP. A molecular model of the C-terminus of Kir6.2 (based on the crystal structure of Kir3.1) was constructed and automated docking was used to identify residues interacting with ATP. These results support the idea that K185 interacts with the beta-phosphate of ATP. Thus both N- and C-termini may contribute to the ATP binding site.     

Molecular determinants of cardiac K(ATP) channel activation by epoxyeicosatrienoic acids

We have previously reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, are potent stereospecific activators of the cardiac K(ATP) channel. The epoxide group in EET is critical for reducing channel sensitivity to ATP, thereby activating the channel. This study is to identify the molecular sites on the K(ATP) channels for EET-mediated activation. We investigated the effects of EETs on Kir6.2delta C26 with or without the coexpression of SUR2A and on Kir6.2 mutants of positively charged residues known to affect channel activity coexpressed with SUR2A in HEK293 cells. The ATP IC50 values were significantly increased in Kir6.2 R27A, R50A, K185A, and R201A but not in R16A, K47A, R54A, K67A, R192A, R195A, K207A, K222A, and R314A mutants. Similar to native cardiac K(ATP) channel, 5 microM 11,12-EET increased the ATP IC50 by 9.6-fold in Kir6.2/SUR2A wild type and 8.4-fold in Kir6.2delta C26. 8,9- and 14,15-EET regioisomers activated the Kir6.2 channel as potently as 11,12-EET. 8,9- and 11,12-EET failed to change the ATP sensitivity of Kir6.2 K185A, R195A, and R201A, whereas their effects were intact in the other mutants. 14,15-EET had a similar effect with K185A and R201A mutants, but instead of R195A, it failed to activate Kir6.2R192A. These results indicate that activation of Kir6.2 by EETs does not require the SUR2A subunit, and the region in the Kir6.2 C terminus from Lys-185 to Arg-201 plays a critical role in EET-mediated Kir6.2 channel activation. Based on computer modeling of the Kir6.2 structure, we infer that the EET-Kir6.2 interaction may allosterically change the ATP binding site on Kir6.2, reducing the channel sensitivity to ATP.     

Molecular determinants of KATP channel inhibition by ATP.

ATP-sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore-forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg-nucleotides complicates analysis of nucleotide inhibition of Kir6. 2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP-sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the beta-phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N-terminus preceding, and the C-terminus immediately following, the transmembrane domains. Some mutations in the C-terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single-channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C-terminus in KATP channel gating.     

Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit

ATP-sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore-forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP-binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C-terminus of one subunit, and with R50 in the N-terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP-dependent channel inhibition. The model also suggests that interactions between the C-terminus of one subunit and the 'slide helix' of the adjacent subunit may be involved in ATP-dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state.     

Regulation of cloned ATP-sensitive K channels by adenine nucleotides and sulfonylureas: interactions between SUR1 and positively charged domains on Kir6.2

K(ATP) channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 "PIP2-insensitive" mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the K(ATP) channel. In contrast, the R301C and R314C mutants, which are also "PIP2-insensitive," remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the K(ATP) channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since K(ATP) channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.     

Identification of the PIP2-binding site on Kir6.2 by molecular modelling and functional analysis

ATP-sensitive potassium (K(ATP)) channels couple cell metabolism to electrical activity by regulating K(+) fluxes across the plasma membrane. Channel closure is facilitated by ATP, which binds to the pore-forming subunit (Kir6.2). Conversely, channel opening is potentiated by phosphoinositol bisphosphate (PIP(2)), which binds to Kir6.2 and reduces channel inhibition by ATP. Here, we use homology modelling and ligand docking to identify the PIP(2)-binding site on Kir6.2. The model is consistent with a large amount of functional data and was further tested by mutagenesis. The fatty acyl tails of PIP(2) lie within the membrane and the head group extends downwards to interact with residues in the N terminus (K39, N41, R54), transmembrane domains (K67) and C terminus (R176, R177, E179, R301) of Kir6.2. Our model suggests how PIP(2) increases channel opening and decreases ATP binding and channel inhibition. It is likely to be applicable to the PIP(2)-binding site of other Kir channels, as the residues identified are conserved and influence PIP(2) sensitivity in other Kir channel family members.     

Direct photoaffinity labeling of Kir6.2 by [gamma-(32)P]ATP-[gamma]4-azidoanilide

ATP-sensitive potassium (K(ATP)) channels are under complex regulation by intracellular ATP and ADP. The potentiatory effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We have previously reported that Kir6.2 can be directly labeled by 8-azido-[gamma-(32)P]ATP. However, the binding affinity of 8-azido-ATP to Kir6.2 was low probably due to modification at 8' position of adenine. Here we demonstrate that Kir6.2 can be directly photoaffinity labeled with higher affinity by [gamma-(32)P]ATP-[gamma]4-azidoanilide ([gamma-(32)P]ATP-AA), containing an unmodified adenine ring. Photoaffinity labeling of Kir6.2 by [gamma-(32)P]ATP-AA is not affected by the presence of Mg(2+), consistent with Mg(2+)-independent ATP inhibition of K(ATP) channels. Interestingly, SUR1, which can be strongly and specifically photoaffinity labeled by 8-azido-ATP, was not photoaffinity labeled by ATP-AA. These results identify key differences in the structure of the nucleotide binding sites on SUR1 and Kir6.2.     

N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2

Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (P(o)) and bursting patterns of activity observed in full-length K(ATP) channels. However, the nature of TMD0-Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in K(ATP) channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-K(ATP) channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced P(o), exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP(2). Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP(2). These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP(2). Moreover, they uncover a novel mechanism of K(ATP) channel inactivation involving aberrant functional coupling between SUR1 and Kir6.2.     

Mutations in the linker domain of NBD2 of SUR inhibit transduction but not nucleotide binding

ATP-sensitive potassium (K(ATP)) channels are composed of an ATP-binding cassette (ABC) protein (SUR1, SUR2A or SUR2B) and an inwardly rectifying K(+) channel (Kir6.1 or Kir6.2). Like other ABC proteins, the nucleotide binding domains (NBDs) of SUR contain a highly conserved "signature sequence" (the linker, LSGGQ) whose function is unclear. Mutation of the conserved serine to arginine in the linker of NBD1 (S1R) or NBD2 (S2R) did not alter the ability of ATP or ADP (100 microM) to displace 8-azido-[(32)P]ATP binding to SUR1, or abolish ATP hydrolysis at NBD2. We co-expressed Kir6.2 with wild-type or mutant SUR in Xenopus oocytes and recorded the resulting currents in inside-out macropatches. The S1R mutation in SUR1, SUR2A or SUR2B reduced K(ATP) current activation by 100 microM MgADP, whereas the S2R mutation in SUR1 or SUR2B (but not SUR2A) abolished MgADP activation completely. The linker mutations also reduced (S1R) or abolished (S2R) MgATP-dependent activation of Kir6.2-R50G co-expressed with SUR1 or SUR2B. These results suggest that the linker serines are not required for nucleotide binding but may be involved in transducing nucleotide binding into channel activation.     

Engineered interaction between SUR1 and Kir6.2 that enhances ATP sensitivity in KATP channels

The ATP-sensitive potassium (K(ATP)) channel consisting of the inward rectifier Kir6.2 and SUR1 (sulfonylurea receptor 1) couples cell metabolism to membrane excitability and regulates insulin secretion. Inhibition by intracellular ATP is a hallmark feature of the channel. ATP sensitivity is conferred by Kir6.2 but enhanced by SUR1. The mechanism by which SUR1 increases channel ATP sensitivity is not understood. In this study, we report molecular interactions between SUR1 and Kir6.2 that markedly alter channel ATP sensitivity. Channels bearing an E203K mutation in SUR1 and a Q52E in Kir6.2 exhibit ATP sensitivity ～100-fold higher than wild-type channels. Cross-linking of E203C in SUR1 and Q52C in Kir6.2 locks the channel in a closed state and is reversible by reducing agents, demonstrating close proximity of the two residues. Our results reveal that ATP sensitivity in K(ATP) channels is a dynamic parameter dictated by interactions between SUR1 and Kir6.2.     

Protons activate homomeric Kir6.2 channels by selective suppression of the long and intermediate closures

The ATP-sensitive K+ channels (KATP) play an important role in regulating membrane excitability. These channels are regulated by H+ in addition to ATP, ADP, and phospholipids. To understand how protons affect the single-channel properties, Kir6.2DeltaC36 currents were studied in excised inside-out patches. We chose to study the homomeric Kir6.2 channel with 36 amino acids deleted at the C-terminal end, as there are ADP/ATP-binding sites in the SUR subunit, which may obscure the understanding of the channel-gating process. In the absence of ATP, moderate intracellular acidosis (pH 6.8) augmented P(open) with small suppression (by approximately 10%) of the single-channel conductance. The long and intermediate closures were selectively inhibited, leading to a shortening of the mean closed time without significant changes in the mean open time. Stronger acidification (相似文献   

Activation of adenosine triphosphate-sensitive potassium channels by acyl coenzyme A esters involves multiple phosphatidylinositol 4,5-bisphosphate-interacting residues

ATP-sensitive potassium (K(ATP)) channels are crucial to pancreatic endocrine function and their activation by acyl coenzyme A esters (acyl CoAs) may disrupt hormone secretion, contributing to the pathophysiology of type 2 diabetes. The molecular mechanism of this activation is potentially important in our further understanding of this disease. We use excised patch-clamp techniques to assess the effects of N- and C-terminal Kir6.2 mutations on the activation of recombinant K(ATP) channels by palmitoyl CoA. We demonstrate that several residues previously shown to be involved in channel activation by the structurally related lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) also play a role in activation by acyl CoAs, including R54, R176, R192, and R301. Mutation of these residues caused decreased open probability in the absence of ATP and slower and greater relative activation by both PIP(2) and acyl CoAs. By contrast, K185Q, which probably alters ATP binding, had no effect on either PIP(2) or palmitoyl CoA activation. These findings suggest that activation by the two classes of lipids involves multiple common residues. We use the crystal structure of a related channel, KirBac1.1, as a template to locate the residues of interest in this study within a putative three-dimensional model of Kir6.2. We propose a model in which these residues mediate both direct electrostatic interactions and allosteric modulations of open state stability.     

Inhibition of ATP binding to the carboxyl terminus of Kir6.2 by epoxyeicosatrienoic acids

Epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid (AA), are potent and stereospecific activators of cardiac ATP-sensitive K(+)(K(ATP)) channels. EETs activate K(ATP) channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to K(ATP) channel protein. A fluorescent ATP analog, 2,4,6-trinitrophenyl (TNP)-ATP, which increases its fluorescence emission significantly upon binding with proteins, was used for binding studies with glutathione-S-transferase (GST) Kir6.2 fusion proteins. TNP-ATP bound to GST fusion protein containing the C-terminus of Kir6.2 (GST-Kir6.2C), but not to the N-terminus of Kir6.2, or to GST alone. 11,12-EET (5 muM) did not change TNP-ATP binding K(D) to GST-Kir6.2C, but B(max) was reduced by half. The effect of 11,12-EET was dose-dependent, and 8,9- and 14,15-EETs were as effective as 11,12-EET in inhibiting TNP-ATP binding to GST-Kir6.2C. AA and 11,12-dihydroxyeicosatrienoic acid (11,12-DHET), the parent compound and metabolite of 11,12-EET, respectively, were not effective inhibitors of TNP-ATP binding to GST-Kir6.2C, whereas the methyl ester of 11,12-EET was. These findings suggest that the epoxide group in EETs is important for modulation of ATP binding to Kir6.2. We conclude that EETs bind to the C-terminus of K(ATP) channels, inhibiting binding of ATP to the channel.     

The role of NH2-terminal positive charges in the activity of inward rectifier KATP channels

Approximately half of the NH(2) terminus of inward rectifier (Kir) channels can be deleted without significant change in channel function, but activity is lost when more than approximately 30 conserved residues before the first membrane spanning domain (M1) are removed. Systematic replacement of the positive charges in the NH(2) terminus of Kir6.2 with alanine reveals several residues that affect channel function when neutralized. Certain mutations (R4A, R5A, R16A, R27A, R39A, K47A, R50A, R54A, K67A) change open probability, whereas an overlapping set of mutants (R16A, R27A, K39A, K47A, R50A, R54A, K67A) change ATP sensitivity. Further analysis of the latter set differentiates mutations that alter ATP sensitivity as a consequence of altered open state stability (R16A, K39A, K67A) from those that may affect ATP binding directly (K47A, R50A, R54A). The data help to define the structural determinants of Kir channel function, and suggest possible structural motifs within the NH(2) terminus, as well as the relationship of the NH(2) terminus with the extended cytoplasmic COOH terminus of the channel.     

Conformational dynamics of the ligand-binding domain of inward rectifier K channels as revealed by molecular dynamics simulations: toward an understanding of Kir channel gating

Inward rectifier (Kir) potassium channels are characterized by two transmembrane helices per subunit, plus an intracellular C-terminal domain that controls channel gating in response to changes in concentration of various ligands. Based on the crystal structure of the tetrameric C-terminal domain of Kir3.1, it is possible to build a homology model of the ATP-binding C-terminal domain of Kir6.2. Molecular dynamics simulations have been used to probe the dynamics of Kir C-terminal domains and to explore the relationship between their dynamics and possible mechanisms of channel gating. Multiple simulations, each of 10 ns duration, have been performed for Kir3.1 (crystal structure) and Kir6.2 (homology model), in both their monomeric and tetrameric forms. The Kir6.2 simulations were performed with and without bound ATP. The results of the simulations reveal comparable conformational stability for the crystal structure and the homology model. There is some decrease in conformational flexibility when comparing the monomers with the tetramers, corresponding mainly to the subunit interfaces in the tetramer. The beta-phosphate of ATP interacts with the side chain of K185 in the Kir6.2 model and simulations. The flexibility of the Kir6.2 tetramer is not changed greatly by the presence of bound ATP, other than in two loop regions. Principal components analysis of the simulated dynamics suggests loss of symmetry in both the Kir3.1 and Kir6.2 tetramers, consistent with "dimer-of-dimers" motion of subunits in C-terminal domains of the corresponding Kir channels. This is suggestive of a gating model in which a transition between exact tetrameric symmetry and dimer-of-dimers symmetry is associated with a change in transmembrane helix packing coupled to gating of the channel. Dimer-of-dimers motion of the C-terminal domain tetramer is also supported by coarse-grained (anisotropic network model) calculations. It is of interest that loss of exact rotational symmetry has also been suggested to play a role in gating in the bacterial Kir homolog, KirBac1.1, and in the nicotinic acetylcholine receptor channel.     

ATP modulation of ATP-sensitive potassium channel ATP sensitivity varies with the type of SUR subunit

ATP-sensitive potassium (K(ATP)) channels comprise Kir and SUR subunits. Using recombinant K(ATP) channels expressed in Xenopus oocytes, we observed that MgATP (100 microm) block of Kir6.2/SUR2A currents gradually declined with time, whereas inhibition of Kir6.2/SUR1 or Kir6.2DeltaC36 currents did not change. The decline in Kir6.2/SUR2A ATP sensitivity was not observed in Mg(2+) free solution and was blocked by the phosphatidylinositol (PI) 3-kinase inhibitors LY 294002 (10 microm) and wortmannin (100 microm), and by neomycin (100 microm). These results suggest that a MgATP-dependent synthesis of membrane phospholipids produces a secondary decrease in the ATP sensitivity of Kir6.2/SUR2A. Direct application of the phospholipids PI 4,5-bisphosphate and PI 3,4,5-trisphosphate in the presence of 100 microm MgATP activated all three types of channel, but the response was faster for Kir6.2/SUR2A. Chimeric studies indicate that the different responses of Kir6.2/SUR2A and Kir6.2/SUR1 are mediated by the first six transmembrane domains of SUR. The MgATP-dependent loss of ATP sensitivity of Kir6.2/SUR2A was enhanced by the actin filament disrupter cytochalasin and blocked by phalloidin (which stabilizes the cytoskeleton). Phalloidin did not block the effect of PI 3,4,5-trisphosphate. This suggests that MgATP may cause disruption of the cytoskeleton, leading to enhanced membrane phospholipid levels (or better targeting to the K(ATP) channel) and thus to decreased channel ATP sensitivity.     

Critical protein domains and amino acid residues for gating the KIR6.2 channel by intracellular ATP

K(ATP) channels couple intermediary metabolism to cellular excitability. Such a property relies on the inherent ATP-sensing mechanism known to be located in the Kir6 subunit. However, the molecular basis for the ATP sensitivity remains unclear. Here we showed evidence for protein domains and amino acid residues essential for the channel gating by intracellular ATP. Chimerical channels were constructed using protein domains of Kir6.2 and Kir1.1, expressed in HEK293 cells, and studied in inside-out patches. The N and C termini, although important, were inadequate for channel gating by intracellular ATP. Full ATP sensitivity also required M1 and M2 helices. Cytosolic portions of the M1 and M2 sequences were crucial, in which six amino acid residues were identified, i.e., Thr76, Met77, Ala161, Iso162, Leu164, and Cys166. Site-specific mutation of any of them reduced the ATP sensitivity. Construction of these residues together with the N/C termini produced ATP sensitivity identical to the wild-type channels. The requirement for specific membrane helices suggests that the Kir6.2 gating by ATP is not shared by even two closest relatives in the K(+) channel family, although the general gating mechanisms involving membrane helices appear to be conserved in all K(+) channels.     

Phosphatidylinositol 4,5-bisphosphate (PIP2) modulation of ATP and pH sensitivity in Kir channels. A tale of an active and a silent PIP2 site in the N terminus

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.     

Compromised ATP binding as a mechanism of phosphoinositide modulation of ATP-sensitive K+ channels

Inhibition of ATP-sensitive K(+) (K(ATP)) channels by ATP, a process presumably initiated by binding of ATP to the pore-forming subunit, Kir6.2, is reduced in the presence of phosphoinositides (PPIs). Previous studies led to the hypothesis that PPIs compromise ATP binding. Here, this hypothesis was tested using purified Kir6.2. We show that PPIs bind purified Kir6.2 in an isomer-specific manner, that biotinylated ATP analogs photoaffinity label purified Kir6.2, and that this labeling is weakened in the presence of PPIs. Patch-clamp measurements confirmed that these ATP analogs inhibited Kir6.2 channels, and that PPIs decreased the level of inhibition. These results indicate that interaction of PPIs with Kir6.2 impedes ATP-binding activity. The PPI regulation of ATP binding revealed in this study provides a putative molecular mechanism that is potentially pivotal to the nucleotide sensitivity of K(ATP) channels.