Focused issue on KATP channelsKATP channel interaction with adenine nucleotides
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.
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Production and purification of ATP-sensitive potassium channel particles for cryo-electron microscopy
2021, Methods in EnzymologyCitation Excerpt :The requirement of Mg2 + in this process has prompted studies to examine nucleotide interactions and hydrolysis at the NBDs. These studies led to a proposal that hydrolysis of MgATP to MgADP at NBD2, which contains the consensus ATPase site, stabilizes ATP binding at NBD1, which carries a degenerate ATPase site, and drives dimerization of the NBDs to promote channel opening at Kir6.2 (Masia & Nichols, 2008; Matsuo, Kimura, & Ueda, 2005; Zingman et al., 2001). It is worth noting that direct measurements of MgATP hydrolysis using purified SUR or recombinant SUR NBDs indicate relatively poor hydrolysis efficiency (de Wet et al., 2007; Masia, Enkvetchakul, & Nichols, 2005), suggesting that increased MgADP binding at NBD2 as intracellular [ADP] rises may be sufficient to induce or stabilize conformational changes at the NBDs to stimulate channel opening.
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2017, Noradrenergic Signaling and AstrogliaThe Nucleotide-Binding Sites of SUR1: A Mechanistic Model
2015, Biophysical JournalCitation Excerpt :This suggests that NBS2, but not NBS1, hydrolyzes ATP. Detailed discussions of experiments that addressed nucleotide interactions with the NBSs of SUR can be found elsewhere (25,26). In spite of the structural complexity of the KATP channel, with eight subunits and 12 nucleotide-binding sites (one inhibitory site on each Kir6.x subunit and two stimulatory sites on each SUR), the main features of nucleotide regulation of the KATP channel can be explained with a simplified equilibrium gating model (Fig. 3).
Reinterpreting the action of ATP analogs on KATP channels
2013, Journal of Biological ChemistryCitation Excerpt :SUR1, the channel regulatory subunit, is an enzyme, a member of the ATP-binding cassette (ABC) family of proteins that utilize the energy of ATP binding and hydrolysis to transport substrates across cell membranes (17, 18). A current model of KATP channel regulation assumes that a post-hydrolytic, ADP-bound enzymatic intermediate or conformation of SUR1 stimulates channel openings, i.e. that hydrolysis is essential for activation (Ref. 19 and reviewed in (20–22). This model is based in part on studies demonstrating that nonhydrolyzable ATP analogs such as AMP-PNP and AMP-PCP fail to stimulate channel activity, whereas the slowly hydrolyzable ATPγS stimulates less efficiently than ATP (23–30).
Syntaxin-1A interacts with distinct domains within nucleotide-binding folds of sulfonylurea receptor 1 to inhibit β-cell ATP-sensitive potassium channels
2011, Journal of Biological ChemistryCitation Excerpt :Each NBF (see Fig. 1A) contains three characteristic motifs: Walker A (WA), linker or signature sequence, and Walker B (WB). It is primarily through these motifs that the NBFs are able to coordinate molecules of ATP, a process essential to proper nucleotide regulation of the channel (9–11). It has been well studied that the SNARE proteins form a complex necessary for driving the fusion of plasma and vesicle membranes during exocytosis (for reviews, see Refs. 12–14).