Putative ATP-binding Site Research Articles

Kinase activity of histone chaperone APLF maintains steady state of centrosomes in mouse embryonic stem cells

Our recent studies revealed the role of mouse Aprataxin PNK-like Factor (APLF) in development. Nevertheless, the comprehensive characterization of mouse APLF remains entirely unexplored. Based on domain deletion studies, here we report that mouse APLF's Acidic Domain and Fork Head Associated (FHA) domain can chaperone histones and repair DNA like the respective human orthologs. Immunofluorescence studies in mouse embryonic stem cells showed APLF co-localized with γ-tubulin within and around the centrosomes and govern the number and integrity of centrosomes via PLK4 phosphorylation. Enzymatic analysis established mouse APLF as a kinase. Docking studies identified three putative ATP binding sites within the FHA domain. Site-directed mutagenesis showed that R37 residue within the FHA domain is indispensable for the kinase activity of APLF thereby regulating the centrosome number. These findings might assist us comprehend APLF in different pathological and developmental conditions and reveal non-canonical kinase activity of proteins harbouring FHA domains that might impact multiple cellular processes.

Novel Dimeric hHv1 Model and Structural Bioinformatic Analysis Reveal an ATP-Binding Site Resulting in a Channel Activating Effect.

The human voltage-gated proton channel (hHv1) is a highly selective ion channel codified by the HVCN1 gene. It plays a fundamental role in several physiological processes such as innate and adaptive immunity, insulin secretion, and sperm capacitation. Moreover, in humans, a higher hHv1 expression/function has been reported in several types of cancer cells. Here we report a multitemplate homology model of the hHv1 channel, built and refined as a dimer in Rosetta. The model was then subjected to extensive Gaussian accelerated molecular dynamics (GaMD) for enhanced conformational sampling, and representative snapshots were extracted by clustering analysis. Combining different structure- and sequence-based methodologies, we predicted a putative ATP-binding site located on the intracellular portion of the channel. Furthermore, GaMD simulations of the ATP-bound dimeric hHv1 model showed that ATP interacts with a cluster of positively charged residues from the cytoplasmic N and C terminal segments. According to the in silico predictions, we found that 3 mM intracellular ATP significantly increases the H+ current mediated by the hHv1 channel expressed in HEK293 cells and measured by the patch-clamp technique in an inside-out configuration (2.86 ± 0.63 fold over control at +40 mV). When ATP was added on the extracellular side, it was not able to activate the channel supporting the idea that the ATP-binding site resides in the intracellular face of the hHV1 channel. In a physiological and pathophysiological context, this ATP-mediated modulation could integrate the cell metabolic state with the H+ efflux, especially in cells where hHv1 channels are relevant for pH regulation, such as pancreatic β-cells, immune cells, and cancer cells.

Mutagenic Analysis of the Putative ABCC6 Substrate-Binding Cavity Using a New Homology Model.

Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.

A Novel Model of the Dimeric Human Hv1 Channel Reveals a Putative ATP Binding Site

A multi-template model of the human voltage-gated proton channel (hHv1) was developed by combining the recently solved NMR structural ensemble of the hHv1 (PDBID: 5OQK) with the coiled-coil C-terminal domain (PDBID: 3A2A), bridging them through the chimeric mHv1cc structure (PDBID: 3WKV). The model was built and refined as a dimer within Rosetta's Membrane framework, using the symmetry extracted from the coiled-coil and including experimentally derived constraints during the relaxation. The dimeric model was then subjected to extensive Gaussian Accelerated Molecular Dynamics simulations in a POPC bilayer, and representative snapshots were extracted from the trajectory by clustering analysis. Blind docking simulations of an ATP molecule on this structural ensemble revealed a putative ATP binding site located on the intracellular portion of the permeation pathway, below the hydrophobic gasket. The same region was identified by two sequence-based ATP binding site predictors: ATPSite and ATPint; and a third meta-predictor which combines sequence and structure-based features: ATPBind. According to the in-silico predictions, we found that whole-cell hHv1 current measured in presence of intracellular 3 mM ATP (patch-clamp, Jurkat T cells, n=7) showed a higher amplitude (148.3±27.0 vs 35.6±4.6 pA at 20 mV p<0.05) and a hyperpolarized Vthreshold (−25.7±2.0 vs −12.0±3.7 mV, p<0.05), compared to control cells (without ATP, n=5). The combination of several in-silico methodologies allowed us to develop a novel model of dimeric hHv1 channel, and predict its direct modulation by ATP. Moreover, experimental results confirmed that ATP is able to activate the channel. In a physiological context, this ATP-mediated modulation could integrate the cell metabolic state with the H+ efflux, especially in cells where hHv1 channels are relevant for pH regulation such as pancreatic β-cells, cancer cells or immune cells.

Identification of Amino Acids Essential for Viral Replication in the HCMV Helicase-Primase Complex.

Promising new inhibitors that target the viral helicase-primase complex have been reported to block replication of herpes simplex and varicella-zoster viruses, but they have no activity against human cytomegalovirus (HCMV), another herpesvirus. The HCMV helicase-primase complex (pUL105-pUL102-pUL70) is essential for viral DNA replication and could thus be a relevant antiviral target. The roles of the individual subunits composing this complex remain to be defined. By using sequence alignment of herpesviruses homologs, we identified conserved amino acids in the putative pUL105 ATP binding site and in the putative pUL70 zinc finger pattern. Mutational analysis of several of these amino acids both in pUL105 and pUL70, proved that they are crucial for viral replication. We also constructed, by homology modeling, a theoretical structure of the pUL105 N-terminal domain which indicates that the mutated conserved amino acids in this domain could be involved in ATP hydrolysis.

ATP as an Allosteric Modulator and Chelator of Fe3+ from Fe3+‐transferrin. Is ATP a Major Fe3+ Carrier

The objectives of this study were to examine two chelators in the removal of Fe3+ from Fe3+‐transferrin. One was pyrophosphate (PPi) as it is a reasonably effective chelator in this reaction, and the other was ATP in that it consists of PPi attached to adenosine monophosphate. We reasoned that ATP might bind to a nucleotide binding site if present and in close proximity to the metal center, to enhance Fe3+ removal and give insight into the mechanism of Fe3+ release. In this study we used uv/vis spectroscopy to examine the removal of Fe3+ from Fe3+‐transferrin by time‐course and Vi reactions. At acidic pH we determined association constants of Fe3+‐transferrin alone, and Fe3+‐transferrin in the presence of ATP. We also employed molecular modeling to identify and characterize a putative ATP binding site. Our results show that sigmoidal kinetics were obtained as a function of pH when ATP was the chelator. At pH 7.5 the kinetic data showed there was no removal of Fe3+ by ATP. ATP was found to exert positive heterotropic effects at acidic pH and resulted in the increase in the association constant of Fe3+‐transferrin. These effects were not seen with PPi. We concluded that there were two destabilizations of Fe3+‐transferrin necessary for Fe3+ release; the first due to a low pH induced conformational change in the protein and the second due to ATP, also at low pH, where ATP served as an allosteric modulator. Essentially 100% of Fe3+ was released from both the N‐ and C‐lobes at pH 5.0 in the presence of ATP in ~ 3 minutes as determined by time‐course reaction. Molecular modeling studies identified a putative 25 amino acid ATP binding channel in the N‐lobe of human serum transferrin. These conserved amino acids were identified on each open face of the NI/NII subdomains of apo‐transferrin with the channel forming upon NI/NII subdomain closure as Fe3+ is acquired. At neutral pH ATP bound approximately 9 Å from Fe3+ in the metal center. At acidic pH the protein conformational change positioned ATP immediately adjacent to Fe3+ for rapid chelation. In that ATP is an extracellular, allosteric signaling molecule a mechanism of Fe3+ release from Fe3+‐transferrin is proposed. Here, extracellular ATP is acquired by transferrin in the serum at neutral pH where chelation does not occur. Upon binding to the transferrin receptor and internalization into an acidic endosome, a pH induced conformational change along with positive heterotropic effects occur to allow ATP to efficiently chelate Fe3+. We conclude that transferrin is an allosteric protein, ATP is an allosteric modulator and effective chelator, and that ATP is likely a major Fe3+ carrier in higher organisms.Support or Funding InformationState of Iowa grant GIVFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization.

Heat shock protein 90α plays a key role in myosin folding and thick filament assembly in muscle cells. To assess the structure and function of Hsp90α and its potential regulation by post-translational modification, we developed a combined knockdown and rescue assay in zebrafish embryos to systematically analyze the effects of various mutations on Hsp90α function in myosin thick filament organization. DNA constructs expressing the Hsp90α1 mutants with altered putative ATP binding, phosphorylation, acetylation or methylation sites were co-injected with Hsp90α1 specific morpholino into zebrafish embryos. Myosin thick filament organization was analyzed in skeletal muscles of the injected embryos by immunostaining. The results showed that mutating the conserved D90 residue in the Hsp90α1 ATP binding domain abolished its function in thick filament organization. In addition, phosphorylation mimicking mutations of T33D, T33E and T87E compromised Hsp90α1 function in myosin thick filament organization. Similarly, K287Q acetylation mimicking mutation repressed Hsp90α1 function in myosin thick filament organization. In contrast, K206R and K608R hypomethylation mimicking mutations had not effect on Hsp90α1 function in thick filament organization. Given that T33 and T87 are highly conserved residues involved post-translational modification (PTM) in yeast, mouse and human Hsp90 proteins, data from this study could indicate that Hsp90α1 function in myosin thick filament organization is potentially regulated by PTMs involving phosphorylation and acetylation.

Na+/K+-ATPase α-subunit in swimming crab Portunus trituberculatus: molecular cloning, characterization, and expression under low salinity stress

Na+/K+-ATPases are membrane-associated enzymes responsible for the active transport of Na+ and K+ ions across cell membranes, generating chemical and electrical gradients. These enzymes’ α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na+/K+-ATPase α-subunit cDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end methods. Analysis of the nucleotide sequence revealed that the cDNA had a full-length of 3 833 base pairs (bp), with an open reading frame of 3 120 bp, 5′ untranslated region (UTR) of 317 bp, and 3′ UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na+/K+-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of amino acid sequences showed that the P. trituberculatus α-subunit possessed an overall identity of 75%–99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit’s transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab’s Na+/K+-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.

Synthesis and biological evaluation of novobiocin analogues as potential heat shock protein 90 inhibitors

Recent studies have shown that novobiocin (NB), a member of the coumermycin (CA) family of antibiotics with demonstrated DNA gyrase inhibitory activity, inhibits Heat shock protein 90 (HSP90) by binding weakly to a putative ATP-binding site within its C-terminus. To develop more potent HSP90 inhibitors that target this site and to define structure–activity relationships (SARs) for this class of compounds, we have synthesized twenty seven 3-amido-7-noviosylcoumarin analogues starting from NB and CA. These were evaluated for evidence of HSP90 inhibition using several biological assays including inhibition of cell proliferation and cell cycle arrest, induction of the heat shock response, inhibition of luciferase-refolding in vitro, and depletion of the HSP90 client protein c-erbB-2/HER-2/neu (HER2). This SAR study revealed that a substantial increase in biological activity can be achieved by introduction of an indole-2-carboxamide group in place of 4-hydroxy-isopentylbenzamido group at C-3 of NB in addition to removal/derivatization of the 4-hydroxyl group from the coumarin ring. Methylation of the 4-hydroxyl group in the coumarin moiety moderately increased biological activity as shown by compounds 11 and 13. Our most potent new analogue 19 demonstrated biological activities consistent with known HSP90-binding agents, but with greater potency than NB.

Activation of ATP Binding for the Autophosphorylation of DosS, a Mycobacterium tuberculosis Histidine Kinase Lacking an ATP Lid Motif

The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity.

The Mechanism by which ATP Binding Opens IKs Channels

In cardiac myocytes KCNQ1 and KCNE1 form the IKs channel that is important in terminating action potentials. KCNQ1 has a common Kv channel membrane topology with the central pore domain (PD, S5-S6) and the voltage-sensing domain (VSD, S1-S4). We have demonstrated that ATP directly binds to the cytosolic C-terminus of KCNQ1 to regulate IKs channel function. Here we study if ATP regulates VSD movements or pore opening. using inside-out patch clamp techniques, we found that the steady state G-V relationship and current kinetics of the WT IKs channel are not changed by [ATP]. Consistent with these results, mutations in the putative ATP binding site reduce ATP sensitivity of the current amplitude but do not alter G-V relationship or activation kinetics. Furthermore, using voltage clamp fluorometry, we measured ionic current and VSD movements of the WT and mutant channels in which ATP binding is eliminated; and our results show that florescence signals remain the same, while the ionic current is completely absent in the mutant channels due to the lack of ATP binding. These results suggest that ATP does not regulate VSD movements but is required for pore opening by directly pulling the activation gate to open from the binding site located downstream from S6. Supporting this mechanism, a mutation located between S6 and the putative ATP binding site is found to alter the coupling between ATP binding and channel opening by reducing ATP sensitivity and shifting the G-V relationship to more positive voltages at a given [ATP]; however, increasing [ATP] shifts the G-V back toward negative voltages. The effects of this mutation can be explained by an allosteric model for the coupling between ATP binding and channel opening in which the mutation reduces the allosteric factor for the coupling.

Parametrisation of the free energy of ATP binding to wild-type and mutant Kir6.2 potassium channels

ATP-sensitive K+ (KATP) channels, comprised of pore-forming Kir6.x and regulatory SURx subunits, play important roles in many cellular functions; because of their sensitivity to inhibition by intracellular ATP, KATP channels provide a link between cell metabolism and membrane electrical activity. We constructed structural homology models of Kir6.2 and a series of Kir6.2 channels carrying mutations within the putative ATP-binding site. Computational docking was carried out to determine the conformation of ATP in its binding site. The Linear Interaction Energy (LIE) method was used to estimate the free-energy of ATP binding to wild-type and mutant Kir6.2 channels. Comparisons of the theoretical binding free energies for ATP with those determined from mutational experiments enabled the identification of the most probable conformation of ATP bound to the Kir6.2 channel. A set of LIE parameters was defined that may enable prediction of the effects of additional Kir6.2 mutations within the ATP binding site on the affinity for ATP.

Alanine substitution scanning of pannexin1 reveals amino acid residues mediating ATP sensitivity

Pannexin1 is a prime candidate to represent an ATP release channel. The pannexin1 channel can be activated by extracellular ATP through purinergic receptors P2X7 or P2Y. Recent studies have shown that the Pannexin1 channel is inhibited by its own permeant ion, ATP, and also by P2X7 receptor agonists and antagonists. However, the dose dependence of this inhibition indicated that significant inhibition was prominent at ATP concentrations higher than required for activation of purinergic receptors, including P2X7 and P2Y2. The inhibitory effect of ATP is largely decreased when R75 in the first extracellular loop of Pannexin1 is mutated to alanine, indicating that ATP regulates this channel presumably through binding. To further investigate the structural property of the putative ATP binding site, we performed alanine-scanning mutagenesis of the extracellular loops of pannexin1. Mutations on W74, S237, S240, I247 and L266 in the extracellular loops 1 and 2 severely impaired the inhibitory effect of BzATP, indicating that they might be the essential amino acids in the putative binding site. Mutations on R75, S82, S93, L94, D241, S249, P259 and I267 moderately (≥50%) decreased BzATP sensitivity, suggesting their supporting roles in the binding. Mutations of other residues did not change the BzATP potency compared to wild-type except for some nonfunctional mutants. These data demonstrate that several amino acid residues on the extracellular loops of Pannexin1 mediate ATP sensitivity. Cells expressing mutant Panx1W74A exhibited an enhanced release of ATP, consistent with the removal of a negative feedback loop controlling ATP release.

A Putative Extracellular Salt Bridge at the Subunit Interface Contributes to the Ion Channel Function of the ATP-gated P2X2 Receptor

The recent crystal structure of the ATP-gated P2X4 receptor revealed a static view of its architecture, but the molecular mechanisms underlying the P2X channels activation are still unknown. By using a P2X2 model based on the x-ray structure, we sought salt bridges formed between charged residues located in a region that directly connects putative ATP-binding sites to the ion channel. To reveal their significance for ion channel activation, we made systematic charge exchanges and measured the effects on ATP sensitivity. We found that charge reversals at the interfacial residues Glu(63) and Arg(274) produced gain-of-function phenotypes that were cancelled upon paired charge swapping. These results suggest that a putative intersubunit salt bridge formed between Glu(63) and Arg(274) contributes to the ion channel function. Engineered cysteines E63C and R274C formed redox-dependent cross-links in the absence of ATP. By contrast, the presence of ATP reduced the rate of disulfide bond formation, indicating that ATP binding might trigger relative movement of adjacent subunits at the level of Glu(63) and Arg(274), allowing the transmembrane helices to open the channel.

Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensitizes Host Cells to Apoptosis

The bacterial outer membrane porin PorB of Neisseria gonorrhoeae is an essential virulence factor of these pathogenic gram-negative bacteria which finally lead to the apoptotic degradation of host cells. In course of the infection process PorB is known to be transferred to the mitochondrial membranes of infected cells where it induces the dissipation of mitochondrial membrane potential by a so far unknown mechanism. Using single channel electrophysiology and spectroscopic techniques we were able to reveal the molecular mechanisms underlying this regulated process. Based on a detailed study of the single channel characteristics of wildtype PorB and the mutant PorBK98Q lacking the putative ATP-binding site, we could identify the prerequisites for the formation of open pores in the mitochondrial inner membrane at physiological membrane potentials. Spontaneous incorporation of the β-barrel protein into membranes was followed by using CD-spectroscopy revealing large structural rearrangements during membrane insertion. We are now able to explain the molecular mechanisms by which targeting of bacterial toxin PorB to mitochondria of infected cells is setting up host cells to apoptosis.

ATP Binding to the C Terminus of the Arabidopsis thaliana Nitrate/Proton Antiporter, AtCLCa, Regulates Nitrate Transport into Plant Vacuoles

Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO(3)(-)/H(+) exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine beta-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of nitrate influx into the vacuole at cytosolic physiological nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.

Functional and biochemical evidence for an inter-subunit crosslink in the putative ATP binding site in P2X receptors

Functional and biochemical evidence for an inter-subunit crosslink in the putative ATP binding site in P2X receptors

Synaptic Vesicle Protein 2 Binds Adenine Nucleotides

Synaptic vesicle protein 2 (SV2) is required for normal calcium-regulated secretion of hormones and neurotransmitters. Neurons lacking the two most widely expressed isoforms, SV2A and SV2B, have a reduced readily releasable pool of synaptic vesicles, indicating that SV2 contributes to vesicle priming. The presence of putative ATP-binding sites in SV2 suggested that SV2 might be an ATP-binding protein. To explore this, we examined the binding of the photoaffinity reagent 8-azido-ATP[gamma] biotin to purified, recombinant SV2 in the presence and absence of other nucleotides. Our results indicate that SV2A and SV2B bind nucleotides, with the highest affinity for adenine-containing nucleotides. SV2A contains two binding sites located in the cytoplasmic domains preceding the first and seventh transmembrane domains. These results suggest that SV2-mediated vesicle priming could be regulated by adenine nucleotides, which might provide a link between cellular energy levels and regulated secretion.

Adenine nucleotides decrease the apparentKmof endogenous natriuretic peptide receptors for GTP

Natriuretic peptide receptors A (NPR-A) and B (NPR-B) mediate most effects of natriuretic peptides by synthesizing cGMP. ATP increases the activity of these receptors by an unknown mechanism. We recently reported that a nonhydrolyzable form of ATP, adenylyl imidodiphosphate (AMPPNP), stabilizes but is not required for the activation of NPR-A and NPR-B in membranes from highly overexpressing cells. Here, we repeated these studies on receptors expressed in endogenous settings. Kinetic analysis indicated that both AMPPNP and ATP dramatically decrease the apparent K(m) of both receptors for GTP but had little effect on the V(max). The EC(50) for AMPPNP decreased as substrate concentration increased whereas the magnitude of the effect was greater at lower GTP concentrations. ATP increased the activity of a mutant receptor containing glutamates substituted for all known phosphorylation sites similarly to the wild-type receptor, consistent with a phosphorylation independent mechanism. Finally, the putative ATP binding sites were investigated. Mutation of the ATP modulatory domain region had no effect, but mutation of K535A dramatically diminished ANP-dependent cyclase activity in a manner that was unresponsive to ATP. Mutation of the highly conserved 630-KSS to AAA (all alanines) resulted in an expressed receptor that had no detectable guanylyl cyclase activity. We conclude that ATP is not required for the initial activation of NPRs but does increase activity over time by reducing the apparent K(m) for GTP.

A mutation in the ATP-binding site of the Kir6.2 subunit of the KATP channel alters coupling with the SUR2A subunit.

Mutations in the pore-forming subunit of the ATP-sensitive K(+) (K(ATP)) channel Kir6.2 cause neonatal diabetes. Understanding the molecular mechanism of action of these mutations has provided valuable insight into the relationship between the structure and function of the K(ATP) channel. When Kir6.2 containing a mutation (F333I) in the putative ATP-binding site is coexpressed with the cardiac type of regulatory K(ATP) channel subunit, SUR2A, the channel sensitivity to ATP inhibition is reduced and the intrinsic open probability (P(o)) is increased. However, the extent of macroscopic current activation by MgADP was unaffected. Here we examine rundown and MgADP activation of wild-type and Kir6.2-F333I/SUR2A channels using single-channel recording, noise analysis and spectral analysis. We also compare the effect of mutating the adjacent residue, G334, on rundown and MgADP activation. All three approaches indicated that rundown of Kir6.2-F333I/SUR2A channels is due to a reduction in the number of active channels in the patch and that MgADP reactivation involves recruitment of inactive channels. In contrast, rundown and MgADP reactivation of wild-type and Kir6.2-G334D/SUR2A channels, and of Kir6.2-F333I/SUR1 channels, involve a gradual change in P(o). Our results suggest that F333 in Kir6.2 interacts functionally with SUR2A to modulate channel rundown and MgADP activation. This interaction is fairly specific as it is not disturbed when the adjacent residue (G334) is mutated. It is also not a consequence of the enhanced P(o) of Kir6.2-F333I/SUR2A channels, as it is not found for other mutant channels with high P(o) (Kir6.2-I296L/SUR2A).