A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels.
ABSTRACT: ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ?60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using ? value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ?-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.
Project description:ATP-sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore-forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg-nucleotides complicates analysis of nucleotide inhibition of Kir6. 2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP-sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the beta-phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N-terminus preceding, and the C-terminus immediately following, the transmembrane domains. Some mutations in the C-terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single-channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C-terminus in KATP channel gating.
Project description:In the absence of intracellular nucleotides, ATP-sensitive potassium (KATP) channels exhibit spontaneous activity via a phosphatidylinositol-4,5-bisphosphate (PIP2)-dependent gating process. Previous studies show that stability of this activity requires subunit-subunit interactions in the cytoplasmic domain of Kir6.2; selective mutagenesis and disease mutations at the subunit interface result in time-dependent channel inactivation. Here, we report that mutation of the central glycine in the pore-lining second transmembrane segment (TM2) to proline in Kir6.2 causes KATP channel inactivation. Unlike C-type inactivation, a consequence of selectivity filter closure, in many K(+) channels, the rate of inactivation in G156P channels was insensitive to changes in extracellular ion concentrations or ion species fluxing through the pore. Instead, the rate of G156P inactivation decreased with exogenous application of PIP2 and increased when PIP2-channel interaction was inhibited with neomycin or poly-L-lysine. These findings indicate the G156P mutation reduces the ability of PIP2 to stabilize the open state of KATP channels, similar to mutations in the cytoplasmic domain that produce inactivation. Consistent with this notion, when PIP2-dependent open state stability was substantially increased by addition of a second gain-of-function mutation, G156P inactivation was abolished. Importantly, bath application and removal of Mg(2+)-free ATP or a nonhydrolyzable analog of ATP, which binds to the cytoplasmic domain of Kir6.2 and causes channel closure, recover G156P channel from inactivation, indicating crosstalk between cytoplasmic and transmembrane domains. The G156P mutation provides mechanistic insight into the structural and functional interactions between the pore and cytoplasmic domains of Kir6.2 during gating.
Project description:KEY POINTS:?-Cell KATP channels are partially open in the absence of metabolic substrates, whereas cardiac KATP channels are closed. Using cloned channels heterologously expressed in Xenopus oocytes we measured the effect of MgADP on the MgATP concentration-inhibition curve immediately after patch excision. MgADP caused a far more striking reduction in ATP inhibition of Kir6.2/SUR1 channels than Kir6.2/SUR2A channels; this effect declined rapidly after patch excision. Exchanging the final 42 amino acids of SUR was sufficient to switch the Mg-nucleotide regulation of Kir6.2/SUR1 and Kir6.2/SUR2A channels, and partially switch their sensitivity to metabolic inhibition. Deletion of the C-terminal 42 residues of SUR abolished MgADP activation of both Kir6.2/SUR1 and Kir6.2/SUR2A channels. We conclude that the different metabolic sensitivity of Kir6.2/SUR1 and Kir6.2/SUR2A channels is at least partially due to their different regulation by Mg-nucleotides, which is determined by the final 42 amino acids. ABSTRACT:ATP-sensitive potassium (KATP ) channels couple the metabolic state of a cell to its electrical activity and play important physiological roles in many tissues. In contrast to ?-cell (Kir6.2/SUR1) channels, which open when extracellular glucose levels fall, cardiac (Kir6.2/SUR2A) channels remain closed. This is due to differences in the SUR subunit rather than cell metabolism. As ATP inhibition and MgADP activation are similar for both types of channels, we investigated channel inhibition by MgATP in the presence of 100 ?m MgADP immediately after patch excision [when the channel open probability (PO ) is near maximal]. The results were strikingly different: 100 ?m MgADP substantially reduced MgATP inhibition of Kir6.2/SUR1, but had no effect on MgATP inhibition of Kir6.2/SUR2A. Exchanging the final 42 residues of SUR2A with that of SUR1 switched the channel phenotype (and vice versa), and deleting this region abolished Mg-nucleotide activation. This suggests the C-terminal 42 residues are important for the ability of MgADP to influence ATP inhibition at Kir6.2. This region was also necessary, but not sufficient, for activation of the KATP channel in intact cells by metabolic inhibition (azide). We conclude that the ability of MgADP to impair ATP inhibition at Kir6.2 accounts, in part, for the differential metabolic sensitivities of ?-cell and cardiac KATP channels.
Project description:Neuroendocrine-type ATP-sensitive K+ (KATP) channels are metabolite sensors coupling membrane potential with metabolism, thereby linking insulin secretion to plasma glucose levels. They are octameric complexes, (SUR1/Kir6.2)4, comprising sulfonylurea receptor 1 (SUR1 or ABCC8) and a K+-selective inward rectifier (Kir6.2 or KCNJ11). Interactions between nucleotide-, agonist-, and antagonist-binding sites affect channel activity allosterically. Although it is hypothesized that opening these channels requires SUR1-mediated MgATP hydrolysis, we show here that ATP binding to SUR1, without hydrolysis, opens channels when nucleotide antagonism on Kir6.2 is minimized and SUR1 mutants with increased ATP affinities are used. We found that ATP binding is sufficient to switch SUR1 alone between inward- or outward-facing conformations with low or high dissociation constant, KD , values for the conformation-sensitive channel antagonist [3H]glibenclamide ([3H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's KD for [3H]GBM. This reduction required the Kir N terminus (KNtp), consistent with KNtp occupying a "transport cavity," thus positioning it to link ATP-induced SUR1 conformational changes to channel gating. Moreover, ATP/GBM site coupling was constrained in WT SUR1/WT Kir6.2 channels; ATP-bound channels had a lower KD for [3H]GBM than ATP-bound SUR1. This constraint was largely eliminated by the Q1179R neonatal diabetes-associated mutation in helix 15, suggesting that a "swapped" helix pair, 15 and 16, is part of a structural pathway connecting the ATP/GBM sites. Our results suggest that ATP binding to SUR1 biases KATP channels toward open states, consistent with SUR1 variants with lower KD values causing neonatal diabetes, whereas increased KD values cause congenital hyperinsulinism.
Project description:Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels are molecular sensors of cell metabolism. These hetero-octameric channels, comprising four inward rectifier K+ channel subunits (Kir6.1 or Kir6.2) and four sulfonylurea receptor (SUR1 or SUR2A/B) subunits, detect metabolic changes via three classes of intracellular adenine nucleotide (ATP/ADP) binding site. One site, located on the Kir subunit, causes inhibition of the channel when ATP or ADP is bound. The other two sites, located on the SUR subunit, excite the channel when bound to Mg nucleotides. In pancreatic ? cells, an increase in extracellular glucose causes a change in oxidative metabolism and thus turnover of adenine nucleotides in the cytoplasm. This leads to the closure of KATP channels, which depolarizes the plasma membrane and permits Ca2+ influx and insulin secretion. Many of the molecular details regarding the assembly of the KATP complex, and how changes in nucleotide concentrations affect gating, have recently been uncovered by several single-particle cryo-electron microscopy structures of the pancreatic KATP channel (Kir6.2/SUR1) at near-atomic resolution. Here, the author discusses the detailed picture of excitatory and inhibitory ligand binding to KATP that these structures present and suggests a possible mechanism by which channel activation may proceed from the ligand-binding domains of SUR to the channel pore.
Project description:ATP-sensitive potassium (KATP) channels in the pancreatic beta 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 Ca2+ influx 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 beta 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 nucleotide-binding 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. Mg2+ 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 Mg2+.
Project description:Chloroquine and related compounds can inhibit inwardly rectifying potassium channels by multiple potential mechanisms, including pore block and allosteric effects on channel gating. Motivated by reports that chloroquine inhibition of cardiac ATP-sensitive inward rectifier K(+) current (I(KATP)) is antifibrillatory in rabbit ventricle, we investigated the mechanism of chloroquine inhibition of ATP-sensitive potassium (K(ATP)) channels (Kir6.2/SUR2A) expressed in human embryonic kidney 293 cells, using inside-out patch-clamp recordings. We found that chloroquine inhibits the Kir6.2/SUR2A channel by interacting with at least two different sites and by two mechanisms of action. A fast-onset effect is observed at depolarized membrane voltages and enhanced by the N160D mutation in the central cavity, probably reflecting direct channel block resulting from the drug entering the channel pore from the cytoplasmic side. Conversely, a slow-onset, voltage-independent inhibition of I(KATP) is regulated by chloroquine interaction with a different site and probably involves disruption of interactions between Kir6.2/SUR2A and phosphatidylinositol 4,5-bisphosphate. Our findings reveal multiple mechanisms of K(ATP) channel inhibition by chloroquine, highlighting the numerous convergent regulatory mechanisms of these ligand-dependent ion channels.
Project description:ATP-sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore-forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP-binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C-terminus of one subunit, and with R50 in the N-terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP-dependent channel inhibition. The model also suggests that interactions between the C-terminus of one subunit and the 'slide helix' of the adjacent subunit may be involved in ATP-dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state.
Project description:Inward rectifier potassium (Kir) channels are expressed in almost all mammalian tissues and play critical roles in the control of excitability. Pancreatic ATP-sensitive K (KATP) channels are key regulators of insulin secretion and comprise Kir6.2 subunits coupled to sulfonylurea receptors. Because these channels are reversibly inhibited by cytoplasmic ATP, they link cellular metabolism with membrane excitability. Loss-of-function mutations in the pore-forming Kir6.2 subunit cause congenital hyperinsulinism as a result of diminished channel activity. Here, we show that several disease mutations, which disrupt intersubunit salt bridges at the interface of the cytoplasmic domains (CD-I) of adjacent subunits, induce loss of channel activity via a novel channel behavior: after ATP removal, channels open but then rapidly inactivate. Re-exposure to inhibitory ATP causes recovery from this inactivation. Inactivation can be abolished by application of phosphatidylinositol-4,5-bisphosphate (PIP2) to the cytoplasmic face of the membrane, an effect that can be explained by a simple kinetic model in which PIP2 binding competes with the inactivation process. Kir2.1 channels contain homologous salt bridges, and we find that mutations that disrupt CD-I interactions in Kir2.1 also reduce channel activity and PIP2 sensitivity. Kir2.1 channels also contain an additional CD-I salt bridge that is not present in Kir6.2 channels. Introduction of this salt bridge into Kir6.2 partially rescues inactivating mutants from the phenotype. These results indicate that the stability of the intersubunit CD-I is a major determinant of the inactivation process in Kir6.2 and may control gating in other Kir channels.
Project description:Highly reactive carbonyl methylglyoxal (MGO) is one of the metabolites excessively produced in diabetes. We have showed that prolonged exposure of vascular smooth muscle cells to MGO leads to instability of the mRNA encoding ATP-sensitive potassium (KATP) channel. In the present study we investigated the effects of MGO on the activity of KATP channels.Kir6.1/ SUR2B, Kir6.2/SUR2B or Kir6.2?36 (a truncated Kir6.2 isoform) alone was expressed in HEK293 cells. Whole-cell currents were recorded in the cells with an Axopatch 200B amplifier. Macroscopic currents and single-channel currents were recorded in giant inside-out patches and normal inside-out patches, respectively. Data were analyzed using Clampfit 9 software.The basal activity of Kir6.1/SUR2B channels was low. The specific KATP channel opener pinacidil (10 ?mol/L) could fully activate Kir6.1/SUR2B channels, which was inhibited by the specific KATP channel blocker glibenclamide (10 ?mol/L). MGO (0.1-10 mmol/L) dose-dependently activated Kir6.1/SUR2B channels with an EC50 of 1.7 mmol/L. The activation of Kir6.1/SUR2B channels by MGO was reversible upon washout, and could be inhibited completely by glibenclamide. Kir6.2?36 channels expressed in HEK293 cells could open automatically, and the channel activity was enhanced in the presence of MGO (3 mmol/L). Single channel recordings showed that MGO (3 mmol/L) markedly increased the open probability of Kir6.1/SUR2B channels, leaving the channel conductance unaltered.Acute application of MGO activates KATP channels through direct, non-covalent and reversible interactions with the Kir6 subunits.