L.H. Tang

Nucleotides and phospholipids compete for binding to the C terminus of KATP channels.

Inwardly rectifying, ATP-sensitive K+ channels (KATP) couple metabolism to either cell excitability (Kir6.x) or potassium secretion (Kir1.1). Phosphatidylinositol phospholipids, like PI(4,5)P2, antagonize nucleotide inhibition of KATP channels enhancing the coupling of metabolic events to cell electrical or transport activity. The mechanism by which phospholipids relieve ATP block is unclear. We have shown that maltose-binding fusion proteins (MBP) containing the COOH termini of KATP channels (Kir1.1, Kir6.1, and Kir6.2) form functional tetramers that directly bind at least two ATP molecules with negative cooperativity. Here we show that purified phosphatidylinositol phospholipids compete for 2,4,6,-trinitrophenyl (TNP)-ATP binding to the COOH termini of KATP channels with EC50 values for PIP2 between 6–8 μM. The phospholipid potency profile was PIP3 > PIP2 = PIP > PI, suggesting that net phospholipid charge was important. A role for head group charge was supported by polycations (neomycin, spermine, and polylysine) reversing the effect of PIP2 on TNP-ATP binding to the Kir1.1 channel COOH terminal fusion protein. In contrast, the water-soluble charged hydrolytic product of PIP2, inositol(1,4,5)P3 (IP3), had no effect on TNP-ATP binding, suggesting that the acyl chain of PIP2 was also necessary for its effect on TNP-ATP binding. Indeed, neutral and charged lipids had weak, but significant, effects on TNP-ATP binding. Whereas μM concentrations of PIP2 could compete with TNP-ATP, we found that mM concentrations of MgATP were required to compete with PIP2 for binding to these KATP channel COOH termini. Thus the COOH termini of KATP channels form a nucleotide- and phospholipid-modulated channel gate on which ATP and phospholipids compete for binding.

The Carboxyl Termini of KATP Channels Bind Nucleotides

ATP-sensitive potassium (KATP) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of KATP channel activity may involve direct binding of this nucleotide to the pore-forming inward rectifier (Kir) subunit despite the lack of known nucleotide-binding motifs. To examine this possibility, we assessed the binding of the fluorescent ATP analogue, 2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)adenosine 5′-triphosphate (TNP-ATP) to maltose-binding fusion proteins of the NH2- and COOH-terminal cytosolic regions of the three known KATP channels (Kir1.1, Kir6.1, and Kir6.2) as well as to the COOH-terminal region of an ATP-insensitive inward rectifier K+ channel (Kir2.1). We show direct binding of TNP-ATP to the COOH termini of all three known KATP channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of KATP channels because this nucleotide did not bind to the NH2 termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to KATP COOH termini of Kir1.1, Kir6.1, and Kir6.2 were similar. Binding was abolished by denaturing with 4 m urea or SDS and enhanced by reduction in pH. TNP-ATP to protein stoichiometries were similar for all KATP COOH-terminal proteins with 1 mol of TNP-ATP binding/mole of protein. Competition of TNP-ATP binding to the Kir1.1 COOH terminus by MgATP was complex with both Mg2+ and MgATP effects. Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of KATPtetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer.

Localization of the ATP/Phosphatidylinositol 4,5 Diphosphate-binding Site to a 39-Amino Acid Region of the Carboxyl Terminus of the ATP-regulated K+ Channel Kir1.1

Intracellular ATP and membrane-associated phosphatidylinositol phospholipids, like PIP2(PI(4,5)P2), regulate the activity of ATP-sensitive K+ (KATP) and Kir1.1 channels by direct interaction with the pore-forming subunits of these channels. We previously demonstrated direct binding of TNP-ATP (2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)-ATP) to the COOH-terminal cytosolic domains of the pore-forming subunits of Kir1.1 and Kir6.x channels. In addition, PIP2 competed for TNP-ATP binding on the COOH termini of Kir1.1 and Kir6.x channels, providing a mechanism that can account for PIP2 antagonism of ATP inhibition of these channels. To localize the ATP-binding site within the COOH terminus of Kir1.1, we produced and purified maltose-binding protein (MBP) fusion proteins containing truncated and/or mutated Kir1.1 COOH termini and examined the binding of TNP-ATP and competition by PIP2. A truncated COOH-terminal fusion protein construct, MBP_1.1CΔC170, containing the first 39 amino acid residues distal to the second transmembrane domain was sufficient to bind TNP-ATP with high affinity. A construct containing the remaining COOH-terminal segment distal to the first 39 amino acid residues did not bind TNP-ATP. Deletion of 5 or more amino acid residues from the NH2-terminal side of the COOH terminus abolished nucleotide binding to the entire COOH terminus or to the first 49 amino acid residues of the COOH terminus. PIP2 competed TNP-ATP binding to MBP_1.1CΔC170 with an EC50 of 10.9 μm. Mutation of any one of three arginine residues (R188A/E, R203A, and R217A), which are conserved in Kir1.1 and KATP channels and are involved in ATP and/or PIP2 effects on channel activity, dramatically reduced TNP-ATP binding to MBP_1.1ΔC170. In contrast, mutation of a fourth conserved residue (R212A) exhibited slightly enhanced TNP-ATP binding and increased affinity for PIP2 competition of TNP-ATP (EC50 = 5.7 μm). These studies suggest that the first 39 COOH-terminal amino acid residues form an ATP-PIP2 binding domain in Kir1.1 and possibly the Kir6.x ATP-sensitive K+ channels.

Novel nucleotide-binding sites in ATP-sensitive potassium channels formed at gating interfaces

The coupling of cell metabolism to membrane electrical activity is a vital process that regulates insulin secretion, cardiac and neuronal excitability and the responses of cells to ischemia. ATP‐sensitive potassium channels (KATP; Kir6.x) are a major part of this metabolic–electrical coupling system and translate metabolic signals such as the ATP:ADP ratio to changes in the open or closed state (gate) of the channel. The localization of the nucleotide‐binding site (NBS) on Kir6.x channels and how nucleotide binding gates these KATP channels remain unclear. Here, we use fluorescent nucleotide binding to purified Kir6.x proteins to define the peptide segments forming the NBS on Kir6.x channels and show that unique N‐ and C‐terminal interactions from adjacent subunits are required for high‐affinity nucleotide binding. The short N‐ and C‐terminal segments comprising the novel intermolecular NBS are next to helices that likely move with channel opening/closing, suggesting a lock‐and‐key model for ligand gating.

Helicobacter pylori Lipopolysaccharide Alters ECL Cell DNA Synthesis via a CD14 Receptor and Polyamine Pathway in Mastomys

Chronic Helicobacter pylori infection is associated with alterations in gastric mucosal cell proliferation. Despite the recognition that bacterial lipopolysaccharide (LPS) is present in biologically active quantities in the gastric mucosa, the mechanisms by which it stimulates cells are largely unknown. We have previously established a gastric enterochromaffin-like (ECL) cell neoplasia model in the African rodent species Mastomys and identified that tumor ECL cell proliferation is associated with polyamine biosynthesis and ornithine decarboxylase (ODC) activity. In addition, we have shown that H. pylori LPS exhibits a specific mitogenic effect on naive ECL cells in vitro. The aim of this study was to evaluate whether H. pylori has a direct effect on tumor ECL cell proliferation in vitro and further to evaluate the possible molecular mechanisms for this effect. ECL cell neoplasia was generated in Mastomys by endogenous hypergastrinemia induced by H2 blockade (loxtidine 1 g/kg/day) and tumor ECL cells prepared. The DNA synthesis in 24-hour cultured tumor cells was measured by bromodeoxyuridine uptake and ODC activity by 14CO2 formation from 14C-ornithine. The putative LPS receptor, CD14, was evaluated by reverse-transcription polymerase chain reaction. Our results demonstrated: (1) H. pylori LPS (10–12 to 10–7 M) stimulated basal DNA synthesis (2.2-fold) with an estimated EC50 of 10–10 M; (2) this proliferative response correlated with an increase in ODC activity (1.4-fold, EC50 ∼10–10 M) which could be inhibited by a specific ODC inhibitor, difluoromethyl ornithine, at 10–9 M; (3) the CD14 receptor was identified in both naive and transformed ECL cells by reverse-transcription polymerase chain reaction, and (4) the effects of LPS were inhibited by blocking the CD14 receptor with its specific monoclonal antibody (1:100). Thus, H. pylori LPS appears to influence tumor ECL cell proliferation by activation of the intracellular polyamine pathway and ODC activity via a CD14 receptor on the ECL cell.

A polyamine pathway-mediated mitogenic mechanism in enterochromaffin-like cells of Mastomys

We have previously demonstrated that in Mastomys species proliferation of gastric enterochromaffin-like (ECL) cells is predominantly regulated by gastrin and by transforming growth factor-α (TGF-α) in the naive and neoplastic state, respectively. In this study we examined whether these intracellular mitogenic responses are mediated by polyamines and ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis. An ECL cell preparation of high purity was used to measure the effect of the polyamine derivatives putrescine, spermidine, and spermine on DNA synthesis by bromodeoxyuridine uptake. Both putrescine and spermidine augmented gastrin-stimulated, but not basal, DNA synthesis in naive cells. This proliferative response correlated with an increase in ODC activity that was partially inhibited (20%) by difluoromethylornithine (DFMO), an inhibitor of ODC (IC50, 30 pM). In contrast, all polyamines increased both basal and TGF-α-stimulated DNA synthesis as well as ODC activity in tumor ECL cells. DFMO completely inhibited the proliferative response of TGF-α (IC50, 3 pM). Thus polyamine biosynthesis is involved in proliferation of ECL cells and in particular the mitogenesis of tumor cells, suggesting a role for this pathway in the regulation of ECL cell transformation.We have previously demonstrated that in Mastomys species proliferation of gastric enterochromaffin-like (ECL) cells is predominantly regulated by gastrin and by transforming growth factor-alpha (TGF-alpha) in the naive and neoplastic state, respectively. In this study we examined whether these intracellular mitogenic responses are mediated by polyamines and ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis. An ECL cell preparation of high purity was used to measure the effect of the polyamine derivatives putrescine, spermidine, and spermine on DNA synthesis by bromodeoxyuridine uptake. Both putrescine and spermidine augmented gastrin-stimulated, but not basal, DNA synthesis in naive cells. This proliferative response correlated with an increase in ODC activity that was partially inhibited (20%) by difluoromethylornithine (DFMO), an inhibitor of ODC (IC50, 30 pM). In contrast, all polyamines increased both basal and TGF-alpha-stimulated DNA synthesis as well as ODC activity in tumor ECL cells. DFMO completely inhibited the proliferative response of TGF-alpha (IC50, 3 pM). Thus polyamine biosynthesis is involved in proliferation of ECL cells and in particular the mitogenesis of tumor cells, suggesting a role for this pathway in the regulation of ECL cell transformation.

Rolle von KATP-Kanälen bei der Ischämie intestinaler Organe

Introduction: Metabolic regulation and metabolic stress (ischaemia) are thought to be mediated by metabolically induced changes in the intracellular levels of the adenine nucleotides ATP and ADP. These nucleotides respectively inhibit and activate KATP channels which in turn play an important regulatory role in many tissues. In the pancreas, for example they control the regulated release of insulin. KATP-channels consist of two subunits: the sulfonylurea receptor (SUR) and an inwardly rectifying potassium channel (KIR). The aim of this work was to examine the molecular regulation of KATP-channels by nucleotides. It also provides information concerning the influence of ischaemic conditions (where there is a change in the relative concentration of ATP/ADP as a consequence of increased metabolic rate) on KATP-channel function. Methods: To assess the binding of ATP to these recombinant fusion proteins we used fluorescent ATP, TNP-ATP (molecular probes). MBP fusion proteins were constructed containing the C-Termini of KIR1.1, KIR6.1 and KIR6.2. Additionally nicotinamide-adenosine dinucleotides were tested on KIR1.1, KIR6.1 and KIR6.2 in the presence of TNP-ATP. Results: The Carboxy-Termini of KATP-channels bind nucleotides. KIR1.1, KIR6.1 and KIR6.2 have different affinity to TNP-ATP explained in the dissociation constants (Kd). TNP-ATP binding on the KIR-subunits was significantly enhanced by lowering pH from 7.5 to pH 6.5.