Focused issue on KATP channels
KATP channel interaction with adenine nucleotides

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Abstract

ATP-sensitive potassium (KATP) channels are regulated by adenine nucleotides to convert changes in cellular metabolic levels into membrane excitability. Hence, elucidation of interaction of SUR and Kir6.x with adenine nucleotides is an important issue to understand the molecular mechanisms underlying the metabolic regulation of the KATP channels. We analyzed direct interactions with adenine nucleotides of each subunit of KATP channels. Kir6.2 binds adenine nucleotides in a Mg2+-independent manner. SUR has two NBFs which are not equivalent: NBF1 is a Mg2+-independent high affinity nucleotide binding site, whereas NBF2 is a Mg-dependent low affinity site. Although SUR has ATPase activity at NBF2, it is not used to transport substrates against the concentration gradient unlike other ABC proteins. The ATPase cycle at NBF2 serves as a sensor of cellular metabolism. This may explain the low ATP hydrolysis rate compared to other ABC proteins. Based on studies of photoaffinity labeling, a model of KATP channel regulation is proposed, in which KATP channel activity is regulated by SUR via monitoring the intracellular MgADP concentration. KATP channel activation is expected to be induced by the cooperative interaction of ATP binding at NBF1 and MgADP binding at NBF2.

Introduction

ATP-sensitive potassium (KATP) channels link the metabolism of the cell to the membrane potential [1], [2], [3], [4]. KATP channels are expressed in various tissues including pancreatic β-cells, neurons, cardiac muscle, skeletal muscle and smooth muscle, and play important physiological roles in those tissues (Table 1). Pancreatic KATP channels play a key role in glucose-stimulated insulin secretion. Cardiac KATP channels are regulatory components in the general adaptation syndrome, and protect the myocardium from lethal injury during ischemia. KATP channels protect neuron cells against neuronal damage during metabolic stress in brain. Vascular smooth muscle KATP channels are thought to play a role in regulation of vascular tone. Electrophysiological studies have suggested that an increase in the ATP/ADP ratio inhibits KATP channel activity, while a decrease in the ratio stimulates activity. However, regulation by adenine nucleotides and pharmacological agents was very complex and even paradoxical. We analyzed direct interactions with adenine nucleotides of each subunit of KATP channels to unveil the regulatory mechanism of KATP channels.

Section snippets

SUR as an ABC protein

KATP channels are hetero-octamers composed of sulfonylurea receptor (SUR) and Kir6.x subunits in a 4:4 stoichiometry [5], [6], [7] (Table 1). SUR is a member of the ABCC subfamily of ATP binding cassette (ABC) proteins and have three subtypes: SUR1, SUR2A, and SUR2B, where SUR2A and SUR2B are splicing variants [8], [9], [10]. The ABC proteins are characterized by well-conserved nucleotide binding folds (NBFs) and multispanning transmembrane domains. Like other members of the eukaryote ABC

Interaction of Kir6.2 with adenine nucleotides

Kir6.x, which forms a pore of the KATP channels as a tetramer, is a member of the inwardly rectifying potassium channel family and has two subtypes: Kir6.1 and Kir6.2 [28], [29]. Although only fully functional octameric complexes reach the plasma membrane, truncation of the last 26–36 amino acids from Kir6.2 (Kir6.2ΔC) allows this subunit to reach the surface membrane in the absence of SUR, and to form KATP channels [30]. This suggested that ATP-induced inhibition of the KATP channels is via

Interaction of SUR with adenine nucleotides

Because ATP-induced inhibition of the KATP channels is via Kir6.2 subunit, the nucleotide interaction with SUR was expected to modulate the ability of ATP to keep pore impermeable for potassium ions. There was a possibility that SUR functioned as a transporter of some endogenous substrates, which regulated Kir6.x channels from outside of the cells in an autocrine manner. Another possibility was that SUR functioned as a direct regulator of Kir6.x channel. MgADP stimulates Kir6.2/SUR1 channel

Cooperative nucleotide binding of two NBFs of SUR

As described above, 8-azido-ATP continues to bind to NBF1 stably in the presence of Mg2+ for more than 15 min at 4 °C [66]. We expected that the strong and stable ATP-binding to NBF1 would make it possible to investigate the functional interaction between the two NBFs of SUR1. Two procedures, a “pre-incubation procedure” and a “post-incubation procedure” were used, to analyze the interactions of SUR1 with adenine nucleotides (Fig. 1). First, the membrane proteins were pre-incubated with ADP at

Interaction of disease related mutants of SUR1 and Kir6.2 with adenine nucleotides

Altered function of KATP channels is responsible for human diseases, because KATP channels play important physiological roles. It has been reported that Kir6.2 polymorphism (E23K) is associated with type 2 diabetes [76], [77], [78]. This mutation lies within N terminal cytosolic region of Kir6.2, and reduced ATP sensitivity of reconstituted KATP channels. Heterozygous missense mutations were identified in patients with permanent neonatal diabetes [79]. Among them, R201H mutation, which lies in

Differences between SUR1, SUR2A and SUR2B on the interaction with adenine nucleotides

Pancreatic, cardiac, and vascular smooth muscle KATP channels, which consist of different subtypes of SUR, differ in their responses to cellular metabolic state (Fig. 2). Under normal conditions, pancreatic β-cell KATP channels stay open to maintain membrane potential, and close when elevation of blood glucose concentration results in increased intracellular concentration of ATP to trigger insulin secretion [1], [2], [84], [90]. On the other hand, cardiac muscle KATP channels remain closed

SUR as an intracellular ADP sensor and the role of ATP hydrolysis

Based on the studies analyzing nucleotide-binding properties, we propose a model for the open and closed states of KATP channels (Fig. 3). Because MgADP binding at NBF2 seems to be essential for channel activation, we can assume that SUR binding MgADP at NBF2 activates the channel. Zingman et al. [55] have demonstrated that the KATP channel closes when the ATPase cycle of SUR2A is trapped by beryllium fluoride in a prehydrolytic state, which mimics the MgATP binding form, and that KATP channel

Conclusion

KATP channels are regulated by adenine nucleotides to convert changes in cellular metabolic levels into membrane excitability. Hence, elucidation of interaction of SUR and Kir6.x with adenine nucleotides is an important issue to understand the molecular mechanisms underlying the metabolic regulation of the KATP channels. Kir6.2 binds adenine nucleotides in a Mg2+-independent manner. SUR has two NBFs, which are not equivalent: NBF1 is a Mg2+-independent high-affinity nucleotide binding site,

Acknowledgement

We thank Dr. Andre Terzic for reading the manuscript.

References (104)

  • C. Wang et al.

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

    FEBS Lett

    (2002)
  • C.G. Vanoye et al.

    The carboxyl termini of KATP channels bind nucleotides

    J Biol Chem

    (2002)
  • S.J. Tucker et al.

    Mapping of the physical interaction between the intracellular domains of an inwardly rectifying potassium channel, Kir6.2

    J. Biol. Chem.

    (1999)
  • K. Ueda et al.

    MgADP antagonism to Mg2+-independent ATP binding of the sulfonylurea receptor SUR1

    J. Biol. Chem.

    (1997)
  • I.L. Urbatsch et al.

    P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site

    J. Biol. Chem.

    (1995)
  • Y. Taguchi et al.

    Anti-cancer drugs and glutathione stimulate vanadate-induced trapping of nucleotide in multidrug resistance-associated protein (MRP)

    FEBS Lett.

    (1997)
  • M. Matsuo et al.

    NEM modification prevents high-affinity ATP binding to the first nucleotide binding fold of the sulphonylurea receptor, SUR1

    FEBS Lett.

    (1999)
  • M. Matsuo et al.

    ATP binding properties of the nucleotide binding folds of SUR1

    J. Biol. Chem.

    (1999)
  • M. Matsuo et al.

    Different binding properties and affinities for ATP and ADP among sulfonylurea receptor subtypes, SUR1, SUR2A, and SUR2B

    J Biol Chem

    (2000)
  • L.V. Zingman et al.

    Signaling in channel/enzyme multimers: ATPase transitions in SUR module gate ATP-sensitive K+ conductance

    Neuron

    (2001)
  • A.B. Shapiro et al.

    ATPase activity of purified and reconstituted P-glycoprotein from Chinese hamster ovary cells

    J. Biol. Chem.

    (1994)
  • I.L. Urbatsch et al.

    Effects of lipids on ATPase activity of purified Chinese hamster P-glycoprotein

    Arch. Biochem. Biophys.

    (1995)
  • R. Callaghan et al.

    The functional purification of P-glycoprotein is dependent on maintenance of a lipid–protein interface. Biochim Biophys Acta (BBA)-

    Biomembr

    (1997)
  • Q. Mao et al.

    Purification of functional human P-glycoprotein expressed in Saccharomyces cerevisiae. Biochim Biophys Acta (BBA)-

    Biomembr

    (1997)
  • X.-B. Chang et al.

    ATPase activity of purified multidrug resistance-associated protein

    J. Biol. Chem.

    (1997)
  • C. Li et al.

    ATPase activity of the cystic fibrosis transmembrane conductance regulator

    J. Biol. Chem.

    (1996)
  • L.V. Zingman et al.

    Tandem function of nucleotide binding domains confers competence to sulfonylurea receptor in gating ATP-sensitive K+ channels

    J Biol Chem

    (2002)
  • G. Chang

    Structure of MsbA from Vibrio cholera: a multidrug resistance ABC transporter homolog in a closed conformation

    J Mol Biol

    (2003)
  • J. Chen et al.

    A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle

    Mol Cell

    (2003)
  • J.E. Moody et al.

    Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters

    J Biol Chem

    (2002)
  • E. Hough et al.

    Expression, purification, and evidence for the interaction of the two nucleotide-binding folds of the sulphonylurea receptor

    Biochem Biophys Res Commun

    (2002)
  • A.P. Babenko et al.

    The tolbutamide site of SUR1 and a mechanism for its functional coupling to KATP channel closure

    FEBS Lett.

    (1999)
  • M.V. Mikhailov et al.

    Molecular structure of the glibenclamide binding site of the beta-cell KATP channel

    FEBS Lett

    (2001)
  • J.C. Koster et al.

    Targeted overactivity of beta cell KATP channels induces profound neonatal diabetes

    Cell

    (2000)
  • N. Sharma et al.

    Familial hyperinsulinism and pancreatic beta-cell ATP-sensitive potassium channels

    Kidney Int

    (2000)
  • M. Matsuo et al.

    Functional analysis of a mutant sulfonylurea receptor, SUR1-R1420C, that is responsible for persistent hyperinsulinemic hypoglycemia of infancy

    J Biol Chem

    (2000)
  • A.P. Babenko et al.

    Two regions of sulfonylurea receptor specify the spontaneous bursting and ATP inhibition of KATP channel isoforms

    J. Biol. Chem.

    (1999)
  • N. Askenasy et al.

    Intermittent ischemia: energy metabolism, cellular volume regulation, adenosine and insights into preconditioning

    J. Mol. Cell. Cardiol.

    (1997)
  • A. Ghosh et al.

    The role of ATP and free ADP in metabolic coupling during fuel-stimulated insulin release from islet beta-cells in the isolated perfused rat pancreas

    J. Biol. Chem.

    (1991)
  • L. Aguilar-Bryan et al.

    Toward understanding the assembly and structure of KATP channels

    Physiol. Rev.

    (1998)
  • A.P. Babenko et al.

    A view of SUR/Kir6.X, KATP channels

    Annu. Rev. Physiol.

    (1998)
  • S.-L. Shyng et al.

    Octameric stoichiometry of the KATP channel complex

    J. Gen. Physiol.

    (1997)
  • M. Yamada et al.

    Sulfonylurea receptor 2B and Kir6.1 form a sulfonylurea-sensitive but ATP-insensitive K+ channel

    J. Physiol.

    (1997)
  • L. Aguilar-Bryan et al.

    Cloning of the β cell high-affinity sulfonylurea receptor: a regulator of insulin secretion

    Science

    (1995)
  • K. Ueda et al.

    Expression of a full-length cDNA for the human “MDR1” gene confers resistance to colchicine, doxorubicinm and vinblastine

    Proc. Natl. Acad. Sci. USA

    (1987)
  • A.T. Fojo et al.

    Expression of a multidrug-resistance gene in human tumors

    Proc. Natl. Acad. Sci. USA

    (1987)
  • J.R. Riordan et al.

    Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA

    Science

    (1989)
  • K. Ito et al.

    Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR

    Am. J. Physiol.

    (1997)
  • C.C. Paulusma et al.

    Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene

    Science

    (1996)
  • R. Allikmets et al.

    Mutation of the stargardt disease gene (ABCR) in age-related macular degeneration

    Science

    (1997)
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