MgATP activates the b cell KATP channel by interaction with its SUR 1 subunit

ATP-sensitive potassium (KATP) channels in the pancreatic b cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca21 inf lux and exocytosis. Metabolic regulation of KATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The b cell KATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotidebinding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate KATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg21 caused an apparent reduction in the inhibitory action of ATP on wild-type KATP channels, and MgATP actually activated KATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates KATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg21. ATP-sensitive K1 (KATP) channels couple cell metabolism to electrical activity. They thereby regulate insulin secretion from pancreatic b cells, participate in the response to cardiac and cerebral ischemia, control vascular smooth muscle tone, modulate transmitter release at brain synapses, and mediate K1 f luxes across epithelial cells (1–3). It is thought that metabolic regulation is achieved, at least in part, by variation in the intracellular concentrations of the adenine nucleotides ATP and ADP. These nucleotides influence KATP channel activity by interacting with two physically distinct sites on the KATP channel: interaction with one site leads to channel inhibition whereas interaction with the other site causes an increase in channel activity (4, 5). ADP interacts with both sites, being stimulatory at low concentrations and inhibitory at high concentrations. Although it is well established that ATP interacts with the inhibitory site, it is not known whether it is also effective at the stimulatory site. The properties of the two sites differ in their requirement for Mg21, the divalent cation being required for the potentiatory but not the inhibitory action of ADP (4, 6, 7). The b cell KATP channel consists of two types of subunit (8, 9): an inwardly rectifying K1 channel subunit (Kir6.2) and a sulfonylurea receptor subunit (SUR1), which assemble in a 4:4 stoichiometry to form an octameric channel (10–12). Both subunits are required to form a fully functional KATP channel. The Kir6.2 subunit serves as the KATP channel pore, whereas SUR1 endows Kir6.2 with sensitivity to sulfonylureas and K1 channel openers (5, 13). There is evidence that the inhibitory effect of ATP (and ADP) results from interaction of the nucleotide with Kir6.2 (5). By contrast, the potentiatory effects of MgADP are mediated through the sulfonylurea receptor subunit (5, 14–16). Sequence and hydropathy analysis suggest that SUR1 has two cytosolic nucleotide-binding domains (NBDs) and multiple transmembrane domains (17, 18). The NBDs of several other members of the ABC transporter family, to which SUR1 belongs, are known to be involved in ATP binding and hydrolysis (19). In the case of the cystic fibrosis transmembrane conductance regulator, CFTR, the energy of ATP hydrolysis is used to drive a conformational change that opens an intrinsic Cl2 channel (20–22). There is evidence that both NBDs of SUR1 are involved in channel activation by MgADP (15, 16). The fact that MgGTP, like MgADP and MgGDP, stimulates KATP channel activity (23) raises the possibility that MgATP also may have a stimulatory effect, mediated by the NBDs, which normally is obscured by the inhibitory effect of the nucleotide. This possibility is supported by the fact that the sensitivity of native b cell KATP channels to ATP inhibition is increased by the removal of Mg21 ions (24, 25). Originally, this finding was interpreted to indicate that the b cell KATP channel is inhibited by the free base, ATP42, whose concentration is reduced in solutions containing Mg21. An alternative explanation, however, is that, like MgADP, MgATP has a small stimulatory effect on KATP channel activity that causes an apparent reduction in ATP sensitivity in the presence of Mg21. In this paper we show that the effect of Mg21 on the ATP sensitivity of the b cell KATP channel is mediated by the sulfonylurea receptor subunit. We further show that this effect requires interaction of MgATP with the NBDs of SUR1. Our results suggest that, like MgADP, MgATP has dual stimulatory and inhibitory actions on the KATP channel. Removal of Mg21 enhances the apparent ATP sensitivity by abolishing the stimulatory effect. MgATP is less effective at increasing KATP channel activity than MgADP. It is possible that the stimulatory effect of MgATP also contributes to the metabolic regulation of KATP channel activity.