Salt and water balance after sweat loss: A study of Bikram yoga.

Bikram yoga is practiced in a room heated to 105°F with 40% humidity for 90 min. During the class a large volume of water and electrolytes are lost in the sweat, specifically, sodium is lost, the main cation of the extracellular fluid. There is little known about the volume of sweat and the amount of sodium lost in sweat during Bikram yoga or the optimum quantity of fluid required to replace these losses. The participants who took part in this small feasibility study were five females with a mean age of 47.4 ± 4.7 years and 2.6 ± 1.6 years of experience at Bikram yoga. The total body weight, water consumed, serum sodium concentration, serum osmolality, and serum aldosterone levels were all measured before and after a Bikram yoga practice. Sweat sodium chloride concentration and osmolality were measured at the end of the practice. The mean estimated sweat loss was 1.54 ± 0.65 L, while the amount of water consumed during Bikram yoga was 0.38 ± 0.22 L. Even though only 25% of the sweat loss was replenished with water intake during the Bikram yoga class, we did not observe a change in serum sodium levels or serum osmolality. The sweat contained 82 ± 16 mmol/L of sodium chloride for an estimated total of 6.8 ± 2.1 g of sodium chloride lost in the sweat. The serum aldosterone increased 3.5-fold from before to after Bikram yoga. There was a decrease in the extracellular body fluid compartment of 9.7%. Sweat loss in Bikram yoga predominately produced a volume depletion rather than the dehydration of body fluids. The sweating-stimulated rise in serum aldosterone levels will lead to increased sodium reabsorption from the kidney tubules and restore the extracellular fluid volume over the next 24 hr.

Calcium loss in sweat does not stimulate PTH release: A study of Bikram hot yoga.

It has been hypothesized that sweat loss during exercise causes a disruption in calcium homeostasis that activates bone resorption and over time leads to low bone mineral density. The purpose of this small pilot study was to determine whether dermal calcium loss from a bout of excessive sweating during light intensity physical activity triggers an increase in biomarkers of bone resorption. Biochemical markers related to bone homeostasis were measured before and after a 90 min Bikram hot yoga practice performed in a room heated to 105 °F with 40 % humidity. Participants were five females with a mean age of 47.4 ± 4.7 years. Nude body weight, serum total calcium (Ca2+), free ionized calcium, albumin, parathyroid hormone (PTH) and CTX-I were measured before and after a Bikram hot yoga practice. Mean estimated sweat loss was 1.54 ± 0.65 L, which elicited a 1.9 ± 0.9 % decrease in participant's body weight. Mean Ca2+ concentration in sweat was 2.9 ± 1.7 mg/dl and the estimated mean total calcium lost was 41.3 ± 16.4 mg. Serum ionized Ca2+ increased from 4.76 ± 0.29 mg/dl to 5.35 ± 0.36 mg/dl after the Bikram hot yoga practice (p = 0.0118). Serum PTH decreased from pre- 33.9 ± 3.3 pg/ml to post- 29.9 ± 2.1 pg/ml yoga practice (p = 0.0015) when adjusted for hemoconcentration (PTHADJ), implying a decrease in PTH secretion. We conclude that calcium loss in sweat during 90 min of Bikram hot yoga did not trigger an increase in PTH secretion and did not initiate bone resorption.

Sodium-Glucose Cotransporter-2 (SGLT2) Inhibitors: A Clinician's Guide.

This comprehensive review covers the historical background, physiology, application in type 2 diabetes, novel uses, cardiovascular benefits, side effects and contraindications of sodium-glucose cotransporter-2 (SGLT2) inhibitors. SGLT2 inhibitors are an insulin-independent class of oral antihyperglycemic medication that clinicians use in the treatment of type 2 diabetes. Multiple landmark clinical trials support the effectiveness of SGLT2 inhibitors in reducing blood glucose levels, but it is important to understand when to properly utilize them. SGLT2 inhibitors are the most beneficial as an adjunct medication in addition to metformin in patients with a history of cardiovascular or renal disease who need further hemoglobin A1c reduction. The novel mechanism of action also demands clinicians be aware of the side effects not typically experienced with other oral antihyperglycemic drugs, such as genital tract infections, lower leg amputations, electrolyte disturbances and bone fractures. On top of their benefits in type 2 diabetes, novel uses for SGLT2 inhibitors are being uncovered. Diabetic patients with non-alcoholic fatty liver disease, who are at an increased risk of cirrhosis and hepatocellular carcinoma, experience a clinically significant reduction in serum alanine aminotransferase levels. SGLT2 inhibitors are also effective at lowering body weight in obese individuals and decreasing systolic blood pressure. Dual SGLT1/SGLT2 inhibitors are currently being investigated as possibly the first oral medication for type 1 diabetes.

Glucose-dependent growth arrest of leukemia cells by MCT1 inhibition: Feeding Warburg's sweet tooth and blocking acid export as an anticancer strategy.

This study aims to investigate the utilization of The Warburg Effect, cancer's "sweet tooth" and natural greed for glucose to enhance the effect of monocarboxylate transporter inhibition on cellular acidification. By simulating hyperglycemia with high glucose we may increase the effectiveness of inhibition of lactate and proton export on the dysregulation of cell pH homeostasis causing cell death or disruption of growth in cancer cells. MCT1 and MCT4 expression was determined in MCF7 and K562 cell lines using RT-PCR. Cell viability, growth, intracellular pH and cell cycle analysis was measured in the cell lines grown in 5 mM and 25 mM glucose containing media in the presence and absence of the MCT1 inhibitor AR-C155858 (1 µM) and the NHE1 inhibitor cariporide (10 µM). The MCT1 inhibitor, AR-C155858 had minimal effect on the viability, growth and intracellular pH of MCT4 expressing MCF7 cells. AR-C155858 had no effect on the viability of the MCT1 expressing K562 cells, but decreased intracellular pH and cell proliferation, by a glucose-dependent mechanism. Inhibition of NHE1 on its own had a no effect on cell growth, but together with AR-C155858 showed an additive effect on inhibition of cell growth. In cancer cells that only express MCT1, increased glucose concentrations in the presence of an MCT1 inhibitor decreased intracellular pH and reduced cell growth by G1 phase cell-cycle arrest. Thus we propose a transient hyperglycemic-clamp in combination with proton export inhibitors be evaluated as an adjunct to cancer treatment in clinical studies.

Abnormal social behavior in mice with tyrosinemia type I is associated with an increase of myelin in the cerebral cortex.

Hereditary tyrosinemia type I (HT1) is caused by mutations in the fumarylacetoacetate hydrolase (FAH) gene, the template for the final enzyme in the tyrosine catabolism pathway. If left untreated this deficiency of functional FAH leads to a buildup of toxic metabolites that can cause liver disease, kidney dysfunction and high mortality. The current treatment with the drug NTBC prevents the production of these metabolites and has consequently increased the survival rate in HT1 children. As a result of this increased survival, long term complications of HT1 are now being observed, including slower learning, impaired cognition and altered social behavior. We studied a mouse model of HT1 to gain insight into the effects of HT1 and treatment with NTBC on social behavior in mice. We showed that mice with HT1 display abnormal social behavior in that they spend more time in the absence of another mouse and do not discriminate between a novel mouse and an already familiar mouse. This altered behavior was due to HT1 and not treatment with NTBC. Quantification of cerebral cortex myelin in mice with HT1 showed a two to threefold increase in myelin expression. Our findings suggest that absence of FAH expression in the brain produces an altered brain biochemistry resulting in increased expression of myelin. This increase in myelination could lead to abnormal action potential velocity and altered neuronal connections that provide a mechanism for the altered learning, social behavior and cognitive issues recently seen in HT1.

Tyrosinemia type I and not treatment with NTBC causes slower learning and altered behavior in mice.

Tyrosinemia type I is a recessive inborn error of metabolism caused by mutations in the fumarylacetoacetate hydrolase (FAH) gene, coding for the final enzyme in the metabolism of tyrosine. This renders FAH nonfunctional and without treatment, toxic metabolites accumulate causing liver and kidney damage. Introduction of the drug NTBC in 2002 offered a treatment which inhibits an upstream enzyme, preventing the production of the toxic metabolites. There is now a long-term survival rate of greater than 90 % in children, but there are reports of lower cognitive function and IQ as well as schooling and behavioral problems in these children. We studied a mouse model of tyrosinemia type I to gain insight into the effects of tyrosinemia type I and treatment with NTBC on mouse learning, memory, and behavior. In the Barnes maze, visual and spatial cues can be used by mice to remember the location of a dark escape box. The primary time, distance, and strategy taken by the mice to locate the escape box is a measure of learning and memory. Our findings show that mice with tyrosinemia type I were slower to learn than wild-type mice treated with NTBC and made more mistakes, but were capable of learning and storing long-term memory. After learning the location of the target hole, mice with tyrosinemia type I respond differently to a change in location and were less flexible in learning the new target hole location. Our findings suggest that this slower learning and cognitive difference is caused by tyrosinemia type I and not by the treatment with NTBC.

Kir1.1 (ROMK) and Kv7.1 (KCNQ1/KvLQT1) are essential for normal gastric acid secretion: importance of functional Kir1.1.

Potassium channels comprise the apical leak pathway supplying extracellular K(+) for exchange with protons by the gastric H(+), K(+)-ATPase and provide potential therapeutic targets for inhibiting gastric acid secretion. The Kir1.1 (ROMK) potassium channel mediates the high capacity K(+) recycling necessary for NaCl reabsorption in the thick ascending limb of the kidney, and this channel exhibits functional and regulatory characteristic well suited for K(+) recycling by gastric parietal cells. We report here that Kir1.1 channels are required for gastric acid secretion and that this channel participates with Kv7.1 (KCNQ1/KvLQT1) in the potassium recycling process. We show that Kir1.1 colocalizes with the ß-subunit of H(+), K(+)-ATPase in gastric parietal cells of Kir1.1 wild-type mice. In Kir1.1-deficient mice, gastric mucosal morphology, as well as parietal cell number, proliferation index, and ultrastructure were normal but secretagogue-stimulated gastric acid secretion in whole stomach and perfused gastric glands was absent. Luminal application of potassium-restored acid secretion in perfused gastric glands from Kir1.1-deficient as well as barium-blocked wild-type mice. In wild-type mice, both luminal Tertiapin-Q, an inhibitor of Kir1.1, as well as XE991, an inhibitor of Kv7.1, reduced proton secretion. We propose that Kir1.1 and Kv7.1 channels collaborate in potassium and current recycling across the apical pole of parietal cells.

Glucose stimulates calcium-activated chloride secretion in small intestinal cells.

The sodium-coupled glucose transporter-1 (SGLT1)-based oral rehydration solution (ORS) used in the management of acute diarrhea does not substantially reduce stool output, despite the fact that glucose stimulates the absorption of sodium and water. To explain this phenomenon, we investigated the possibility that glucose might also stimulate anion secretion. Transepithelial electrical measurements and isotope flux measurements in Ussing chambers were used to study the effect of glucose on active chloride and fluid secretion in mouse small intestinal cells and human Caco-2 cells. Confocal fluorescence laser microscopy and immunohistochemistry measured intracellular changes in calcium, sodium-glucose linked transporter, and calcium-activated chloride channel (anoctamin 1) expression. In addition to enhancing active sodium absorption, glucose increased intracellular calcium and stimulated electrogenic chloride secretion. Calcium imaging studies showed increased intracellular calcium when intestinal cells were exposed to glucose. Niflumic acid, but not glibenclamide, inhibited glucose-stimulated chloride secretion in mouse small intestines and in Caco-2 cells. Glucose-stimulated chloride secretion was not seen in ileal tissues incubated with the intracellular calcium chelater BAPTA-AM and the sodium-potassium-2 chloride cotransporter 1 (NKCC1) blocker bumetanide. These observations establish that glucose not only stimulates active Na absorption, a well-established phenomenon, but also induces a Ca-activated chloride secretion. This may explain the failure of glucose-based ORS to markedly reduce stool output in acute diarrhea. These results have immediate potential to improve the treatment outcomes for acute and/or chronic diarrheal diseases by replacing glucose with compounds that do not stimulate chloride secretion.

An NH2-terminal multi-basic RKR motif is required for the ATP-dependent regulation of hIK1.

We previously demonstrated that the ATP/PKA-dependent activation of the human intermediate conductance, Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal motif. The NH2-terminus of hIK1 contains a multi-basic 13RRRKR17 motif, known to be important in the trafficking and function of ion channels. While individual mutations within this domain have no effect on channel function, the triple mutation (15RKR17/AAA), as well as additional double mutations, result in a near complete loss of functional channels, as assessed by whole-cell patch-clamp. However, cell-surface immunoprecipitation studies confirmed expression of these mutated channels at the plasma membrane. To elucidate the functional consequences of the (15)RKR(17)/AAA mutation we performed inside-out patch clamp recordings where we observed no difference in Ca2+ affinity between the wild-type and mutated channels. However, in contrast to wild-type hIK1, channels expressing the 15RKR17/AAA mutation exhibited rundown, which could not be reversed by the addition of ATP. Wild-type hIK1 channel activity was reduced by alkaline phosphatase both in the presence and absence of ATP, indicative of a phosphorylation event, whereas the 15RKR17/AAA mutation eliminated this effect of alkaline phosphatase. Further, single channel analysis demonstrated that the 15RKR17/AAA mutation resulted in a four-fold lower channel open probability (P(o)), in the presence of saturating Ca2+ and ATP, compared to wild-type hIK1. In conclusion, these results represent the first demonstration for a role of the NH2-terminus in the second messenger-dependent regulation of hIK1 and, in combination with our previous findings, suggest that this regulation is dependent upon a close NH2/C-terminal association.

Subunit-subunit interactions are critical for proton sensitivity of ROMK: evidence in support of an intermolecular gating mechanism.

The tetrameric K channel ROMK provides an important pathway for K secretion by the mammalian kidney, and the gating of this channel is highly sensitive to changes in cytosolic pH. Although charge-charge interactions have been implicated in pH sensing by this K channel tetramer, the molecular mechanism linking pH sensing and the gating of ion channels is poorly understood. The x-ray crystal structure KirBac1.1, a prokaryotic ortholog of ROMK, has suggested that channel gating involves intermolecular interactions of the N- and C-terminal domains of adjacent subunits. Here we studied channel gating behavior to changes in pH using giant patch clamping of Xenopus laevis oocytes expressing WT or mutant ROMK, and we present evidence that no single charged residue provides the pH sensor. Instead, we show that N-C- and C-C-terminal subunit-subunit interactions form salt bridges, which function to stabilize ROMK in the open state and which are modified by protons. We identify a highly conserved C-C-terminal arginine-glutamate (R-E) ion pair that forms an intermolecular salt bridge and responds to changes in proton concentration. Our results support the intermolecular model for pH gating of inward rectifier K channels.

WNK3, a kinase related to genes mutated in hereditary hypertension with hyperkalaemia, regulates the K+ channel ROMK1 (Kir1.1).

The serine-threonine kinase WNK3 modulates Cl- transport into and out of cells through its regulation of SLC12A cation/Cl- cotransporters, implicating it as (one of) the long-sought Cl-/volume-sensitive kinase(s). Integrators in homeostatic systems regulate structurally diverse but functionally coupled elements. For example, the related kinase WNK4 regulates the Na-Cl co-transporter (NCC), paracellular Cl- flux, and the K+ channel ROMK1 (Kir1.1) to maintain renal NaCl and K+ homeostasis; mutations in PRKWNK4, encoding WNK4, cause a Mendelian disease featuring hypertension and hyperkalemia. It is known that WNK3 is expressed in the nephron's distal convoluted tubule (DCT) and stimulates NCC activity. Here, we show that WNK3 is also expressed in cortical and outer medullary collecting duct principal cells. Accordingly, we tested WNK3's effect on the mediators of NaCl and K+ handling in these nephron segments--the epithelial sodium channel (ENaC), paracellular Cl- flux, and ROMK1--using established model systems. WNK3 did not alter paracellular Cl- flux in tetracycline-responsive MDCK II cells, nor affect amiloride-sensitive currents when co-expressed with ENaC in Xenopus laevis oocytes. However, additional co-expression studies in oocytes revealed WNK3 inhibited the renal-specific K+ channel ROMK1 activity greater than 5.5-fold (p < .0001) by altering its plasmalemmal surface expression; WNK3 did not affect ROMK1's conductance or open/closed probability. In contrast, WNK3 had no effect on the activity of the cardiac long-QT syndrome K+ channel KCNQ1/KCNE1 when co-expressed in oocytes. Inhibition of ROMK1 is independent of WNK3's catalytic activity and is mediated by WNK3's carboxyl terminus--a mechanism distinct from its known kinase-dependent activation of NCC. A kinase-inactivating point mutation, or a missense mutation homologous to one in WNK4 that causes disease produced a gain-of-function effect, enhancing WNK3's inhibition of ROMK1 greater than 2.5-fold relative to wild type kinase (p < .0001). The magnitude and specificity of WNK3's effects at both NCC and ROMK1, its co-expression with its targets in the distal nephron, and the established in vivo effect of WNK4 at these same targets provide evidence that WNK3's action is physiologically relevant. WNK3 is likely a component of one of the mechanisms that determines the balance between renal NaCl reabsorption and K+ secretion.

Phosphorylation-regulated endoplasmic reticulum retention signal in the renal outer-medullary K+ channel (ROMK).

The renal outer-medullary K+ channel (ROMK; Kir1.1) mediates K+ secretion in the renal mammalian nephron that is critical to both sodium and potassium homeostasis. The posttranscriptional expression of ROMK in the plasma membrane of cells is regulated by delivery of protein from endoplasmic reticulum (ER) to the cell surface and by retrieval by dynamin-dependent endocytic mechanisms in clathrin-coated pits. The S44 in the NH(2) terminus of ROMK1 can be phosphorylated by PKA and serum- and glucocorticoid-inducible kinase-1, and this process increases surface expression of functional channels. We present evidence that phosphorylation of S44 modulates channel expression by increasing its cell surface delivery consequent to suppression of a COOH-terminal ER retention signal. This phosphorylation switch of the ER retention signal could provide a pool of mature and properly folded channels for rapid delivery to the plasma membrane. The x-ray crystal structures of inward rectifier K+ channels have shown a close apposition of the NH(2) terminus with the distal COOH terminus of the adjacent subunit in the channel homotetramer, which is important to channel gating. Thus, NH(2)-terminal phosphorylation modifying a COOH-terminal ER retention signal in ROMK1 could serve as a checkpoint for proper subunit folding critical to channel gating.

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 (K(ATP); 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 K(ATP) 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.

Paracellular Cl- permeability is regulated by WNK4 kinase: insight into normal physiology and hypertension.

Paracellular ion flux across epithelia occurs through selective and regulated pores in tight junctions; this process is poorly understood. Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension and hyperkalemia. Whereas WNK4 is known to regulate several transcellular transporters and channels involved in NaCl and K+ homeostasis, its localization to tight junctions suggests it might also regulate paracellular flux. We performed electrophysiology on mammalian kidney epithelia with inducible expression of various WNK4 constructs. Induction of wild-type WNK4 reduced transepithelial resistance by increasing absolute chloride permeability. PHAII-mutant WNK4 produced markedly larger effects, whereas kinase-mutant WNK4 had no effect. The electrochemical and pharmacologic properties of these effects indicate they are attributable to the paracellular pathway. The effects of WNK4 persist when induction is delayed until after tight-junction formation, demonstrating a dynamic effect. WNK4 did not alter the flux of uncharged solutes, or the expression or localization of selected tight-junction proteins. Transmission and freeze-fracture electron microscopy showed no effect of WNK4 on tight-junction structure. These findings implicate WNK signaling in the coordination of transcellular and paracellular flux to achieve NaCl and K+ homeostasis, explain PHAII pathophysiology, and suggest that modifiers of WNK signaling may be potent antihypertensive agents.

WNK4 regulates the balance between renal NaCl reabsorption and K+ secretion.

A key question in systems biology is how diverse physiologic processes are integrated to produce global homeostasis. Genetic analysis can contribute by identifying genes that perturb this integration. One system orchestrates renal NaCl and K+ flux to achieve homeostasis of blood pressure and serum K+ concentration. Positional cloning implicated the serine-threonine kinase WNK4 in this process; clustered mutations in PRKWNK4, encoding WNK4, cause hypertension and hyperkalemia (pseudohypoaldosteronism type II, PHAII) by altering renal NaCl and K+ handling. Wild-type WNK4 inhibits the renal Na-Cl cotransporter (NCCT); mutations that cause PHAII relieve this inhibition. This explains the hypertension of PHAII but does not account for the hyperkalemia. By expression in Xenopus laevis oocytes, we show that WNK4 also inhibits the renal K+ channel ROMK. This inhibition is independent of WNK4 kinase activity and is mediated by clathrin-dependent endocytosis of ROMK, mechanisms distinct from those that characterize WNK4 inhibition of NCCT. Most notably, the same mutations in PRKWNK4 that relieve NCCT inhibition markedly increase inhibition of ROMK. These findings establish WNK4 as a multifunctional regulator of diverse ion transporters; moreover, they explain the pathophysiology of PHAII. They also identify WNK4 as a molecular switch that can vary the balance between NaCl reabsorption and K+ secretion to maintain integrated homeostasis.

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 PIP(2) (PI(4,5)P(2)), regulate the activity of ATP-sensitive K(+) (K(ATP)) 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, PIP(2) competed for TNP-ATP binding on the COOH termini of Kir1.1 and Kir6.x channels, providing a mechanism that can account for PIP(2) 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 PIP(2). A truncated COOH-terminal fusion protein construct, MBP_1.1CDeltaC170, 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 NH(2)-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. PIP(2) competed TNP-ATP binding to MBP_1.1CDeltaC170 with an EC(50) of 10.9 microm. Mutation of any one of three arginine residues (R188A/E, R203A, and R217A), which are conserved in Kir1.1 and K(ATP) channels and are involved in ATP and/or PIP(2) effects on channel activity, dramatically reduced TNP-ATP binding to MBP_1.1DeltaC170. In contrast, mutation of a fourth conserved residue (R212A) exhibited slightly enhanced TNP-ATP binding and increased affinity for PIP(2) competition of TNP-ATP (EC(50) = 5.7 microm). These studies suggest that the first 39 COOH-terminal amino acid residues form an ATP-PIP(2) binding domain in Kir1.1 and possibly the Kir6.x ATP-sensitive K(+) channels.

The carboxyl termini of K(ATP) channels bind nucleotides.

ATP-sensitive potassium (K(ATP)) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of K(ATP) 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 NH(2)- and COOH-terminal cytosolic regions of the three known K(ATP) 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 K(ATP) channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of K(ATP) channels because this nucleotide did not bind to the NH(2) termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to K(ATP) 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 K(ATP) 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 Mg(2+) 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 K(ATP) tetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer.

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

Inwardly rectifying, ATP-sensitive K+ channels (K(ATP)) couple metabolism to either cell excitability (Kir6.x) or potassium secretion (Kir1.1). Phosphatidylinositol phospholipids, like PI(4,5)P2, antagonize nucleotide inhibition of K(ATP) 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 K(ATP) 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 K(ATP) channels with EC50 values for PIP2 between 6-8 microM. 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 microM 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 K(ATP) channel COOH termini. Thus the COOH termini of K(ATP) channels form a nucleotide- and phospholipid-modulated channel gate on which ATP and phospholipids compete for binding.