Interaction of Vanadate with the Cloned Beta Cell KATP Channel

134

143

ATP-sensitive potassium (K ATP )1 channels are inwardly rectifying potassium channels, which are inhibited by ATP and stimulated by MgADP (1-3). They play important roles by linking cellular metabolic level to membrane potential by sensing intracellular ATP and ADP levels in various tissues such as pancreatic ␤-cells, heart, brain, skeletal muscle, and vascular smooth muscle. The K ATP channel is a hetero-octamer composed of sulfonylurea receptor (SURx) and Kir6.x subunits in 4:4 stoichiometry (4 -7). SURx is a member of the ATP-binding cassette (ABC) superfamily including P-glycoprotein (MDR1), multidrug resistance-associated protein (MRP1), and the cystic fibrosis transmembrane conductance regulator (CFTR) (8, 9), all of which have two nucleotide-binding folds (NBFs) per molecule; Kir6.x is a member of the inwardly rectifying potassium channel family (10 -12). Both SURx and Kir6.x have a number of subtypes as follows: SUR1, SUR2A, and SUR2B and Kir6.1 and Kir6.2. SUR1 has been cloned as a high affinity binding protein for sulfonylurea (9), the most commonly used drug for treatment of patients with type 2 diabetes. SUR2A shares 68% amino acid identity with SUR1, and SUR2B is a splicing variant of SUR2A differing only in its C-terminal 42 amino acids (13,14). The Cterminal 42 amino acids of SUR2B are similar to those of SUR1. Kir6.1 and Kir6.2 share 71% amino acid identity with each other, both of which have two putative transmembrane domains and an ion pore-forming (H5) region.Pancreatic ␤-cell K ATP channels, composed of SUR1 and Kir6.2, regulate insulin secretion by altering the ␤-cell membrane potential (1,3,15,16). Coexpression of SUR2A/Kir6.2, SUR2B/Kir6.2, and SUR2B/Kir6.1 has been reported to reconstitute cardiac, smooth muscle, and vascular smooth muscle K ATP channels, respectively (2, 3). These channels have different sensitivities to ATP and show different responses to sulfonylurea drugs and potassium channel openers (10,11,14,17,18). The IC 50 (ATP) of SUR1/Kir6.2 K ATP channels is about 10 M, whereas those of SUR2A/Kir6.2 and SUR2B/Kir6.2 are about 100 and 300 M, respectively (10,11,14). SUR2B/Kir6.1 K ATP channels are not inhibited by ATP but are stimulated by ADP and ATP (17). SUR1/Kir6.2 K ATP channels are inhibited by glibenclamide at K i ϳ10 nM, and SUR2A/Kir6.2, SUR2B/ Kir6.2, and SUR2B/Kir6.1 K ATP channels are inhibited with K i values in the low micromolar range (10,11,14,17). Both SUR1/Kir6.2 and SUR2B/Kir6.2 K ATP channels are stimulated by diazoxide, but SUR2A/Kir6.2 K ATP channels are not (10,11,14). The differences between these channels may be caused, at least in part, by differences in SUR subtype. However, it is not clear how SUR subtypes cause the different properties of K ATP channel subtypes.We have already shown that NBF1 of SUR1 is a Mg 2ϩ -independent high affinity ATP-binding site, that NBF2 is a Mg 2ϩ -dependent low affinity ATP-binding site, and that MgADP binding at NBF2 stabilizes the 8-azido-ATP binding at

Section: Discussionsupporting

confidence: 81%

Different Binding Properties and Affinities for ATP and ADP among Sulfonylurea Receptor Subtypes, SUR1, SUR2A, and SUR2B

Matsuo

Tanabe

Kioka

et al. 2000

134

143

“…These results suggest that the catalytic mechanism of the NBFs of SUR1 may be different from that of the NBFs of MDR1 and CFTR. This is in agreement with experiments demonstrating that orthovanadate does not influence K ATP channel activity (38,39).…”

Section: Discussionsupporting

confidence: 81%

ATP Binding Properties of the Nucleotide-binding Folds of SUR1

Matsuo

Kioka

Amachi

et al. 1999

103

Pancreatic beta cell ATP-sensitive potassium (K ATP ) channels regulate glucose-induced insulin secretion. The activity of the K ATP channel, composed of SUR1 and Kir6.2 subunits, is regulated by intracellular ATP and ADP, but the molecular mechanism is not clear. To distinguish the ATP binding properties of the two nucleotide-binding folds (NBFs) of SUR1, we prepared antibodies against NBF1 and NBF2, and the tryptic fragment of SUR1 was immunoprecipitated after photoaffinity labeling with 8-azido-[ ATP-sensitive potassium (K ATP ) 1 channels of pancreatic beta cells regulate insulin release by altering the beta cell membrane potential (1-4). Because the channels are inhibited by ATP and activated by MgADP, they act as sensors of intracellular nucleotides, but it is not known how they monitor concentrations of intracellular ATP and ADP. The K ATP channel is a hetero-octamer composed of SUR1 and Kir6.2 subunits (5-8). SUR1 is a member of the ABC superfamily including P-glycoprotein (MDR1), MRP1, and the cystic fibrosis transmembrane conductance regulator (CFTR) (9, 10), all of which have two nucleotide-binding folds (NBFs) in the molecule; Kir6.2 is an inwardly rectifying potassium channel (11,12).Electrophysiological studies have shown that K ATP channels are inhibited by ATP through binding to Kir6.2 and are activated by MgADP through binding to SUR1 (13,14). We have reported previously that mutations of either the Walker A or B motifs of NBF1, K719M, and D854N abolish the high-affinity 8-azido-ATP binding of SUR1, whereas the equivalent mutations in NBF2 do not affect ATP binding (15). We have also reported that MgADP and MgATP stabilize binding of prebound 8-azido-ATP to SUR1 and that mutations in the Walker A and B motifs of NBF2 abolish this stabilizing effect of MgADP and MgATP (16). These biochemical results suggest that SUR1 binds 8-azido-ATP strongly at NBF1 and MgADP at NBF2 and that the two NBFs cooperate in nucleotide binding. However, due probably to the high-affinity ATP binding of NBF1, we have not been able to detect nucleotide binding of NBF2 directly.The ATP binding/hydrolysis properties of the NBF are best studied with MDR1 among the ABC proteins, and its two NBFs are proposed to be equivalent in function (17). The covalent modification of the cysteine residue in the Walker A motif of either NBF has been shown to be sufficient to inactivate the ATPase activity (18 -21). Mutation of the Walker A lysine residue of either NBF, thought to interact with the ␤-and ␥-phosphates of ATP, abolishes the ATPase activity of MDR1 and its ability to confer multidrug resistance (22)(23)(24). Both NBFs of MDR1 can hydrolyze nucleotides (25-27), and mild trypsin digestion showed that two NBFs were labeled in equal proportion with 8-azido-ATP after hydrolysis in the presence of orthovanadate (28).SUR1 photoaffinity-labeled with 8-azido-[ 32 P]ATP was digested mildly with trypsin. Tryptic fragments were immunoprecipitated with antibodies against NBF1, NBF2, and a Cterminal sequence of SUR1. The ATP binding proper...

“…Our results indicate that these different responses are mediated by the first set of transmembrane domains of SUR (TMs 1-6). It is noteworthy that this region of SUR confers the different gating kinetics of Kir6.2/SUR1 and Kir6.2/SUR2A channels (46,47).…”

Section: Resultsmentioning

confidence: 99%

ATP Modulation of ATP-sensitive Potassium Channel ATP Sensitivity Varies with the Type of SUR Subunit

Song¹,

Ashcroft

2001

Self Cite

ATP-sensitive potassium (K ATP) ATP-sensitive potassium (K ATP )1 channels are widely distributed, being found in pancreatic B-cells, cardiac, smooth, and skeletal muscles, and neurones (1). They play important functional roles in all these tissues by linking cellular metabolism to electrical activity. Opening of the K ATP channel produces a voltage-independent K ϩ current that hyperpolarizes the cell and reduces its electrical excitability; conversely, K ATP channel closure usually decreases membrane excitability.The K ATP channel is an octamer, formed by the physical association of four inwardly rectifying potassium channel subunits (Kir6.2) and four regulatory sulfonylurea receptor subunits (2). K ATP channels in different tissues are composed of different Kir and SUR subunits. In most tissues, Kir6.2 acts as the pore-forming subunit (3, 4). Two different sulfonylurea receptor genes (SUR1 and SUR2) have been identified, and further diversity is created by alternative splicing of SUR2 (5-9). In this paper, the major isoform of SUR2 (6) is called SUR2A. It shares 67% sequence identity with SUR1. There is evidence that SUR1 serves as the regulatory subunit of the K ATP channel in pancreatic B-cells and some types of neurone (5, 10), whereas a similar role is played by SUR2A in cardiac and skeletal muscle (6).Binding sites for drugs and modulatory agents are found on both Kir6.2 and SUR subunits. The nucleotides ATP and ADP bind to an intracellular site on Kir6.2 and bring about K ATP channel closure (11, 12), whereas a range of magnesium nucleotides (including ATP and ADP) interact with the NBDs of SUR and thereby increase channel activity (13-16). The balance between these inhibitory and stimulatory effects results in channel inhibition by most concentrations of MgATP and high concentrations of MgADP (10 mM) and in channel activation by low micromolar concentrations of MgADP. It is thought that metabolic regulation of K ATP channel activity is mediated, in part, by changes in the intracellular concentrations of these nucleotides.Recent studies indicate that membrane phospholipids also play an important role in modulating K ATP channel activity. Thus, phosphatidylinositol 4,5-bisphosphate (PIP 2 ) increases the activity and reduces the ATP sensitivity of both native and cloned K ATP channels (17-21). Overexpression of PI5-kinase, which enhances PIP 2 levels, reduces the ATP sensitivity of the channel in membrane patches (21), whereas breakdown of PIP 2 by phospholipase C enhances the K ATP channel ATP sensitivity (22). This is in agreement with data indicating that PIP 2 binds directly to Kir channels (23). Interestingly, application of PIP 3 and PI 3-P also enhances K ATP channel activity (19,20,24).The rundown of both native and cloned K ATP channels that occurs in excised membrane patches is reversed by application of MgATP (25-27), and PIP 2 has also been implicated in this effect (25). It is postulated that PIP 2 is produced in plasma membrane by serial phosphorylation of PI and that this process is sequent...