Molecular basis and structural insight of vascular K(ATP) channel gating by S-glutathionylation

The vascular ATP-sensitive K(+) (K(ATP)) channel is targeted by a variety of vasoactive substances, playing an important role in vascular tone regulation. Our recent studies indicate that the vascular K(ATP) channel is inhibited in oxidative stress via S-glutathionylation. Here we show evidence for the molecular basis of the S-glutathionylation and its structural impact on channel gating. By comparing the oxidant responses of the Kir6.1/SUR2B channel with the Kir6.2/SUR2B channel, we found that the Kir6.1 subunit was responsible for oxidant sensitivity. Oxidant screening of Kir6.1-Kir6.2 chimeras demonstrated that the N terminus and transmembrane domains of Kir6.1 were crucial. Systematic mutational analysis revealed three cysteine residues in these domains: Cys(43), Cys(120), and Cys(176). Among them, Cys(176) was prominent, contributing to >80% of the oxidant sensitivity. The Kir6.1-C176A/SUR2B mutant channel, however, remained sensitive to both channel opener and inhibitor, which indicated that Cys(176) is not a general gating site in Kir6.1, in contrast to its counterpart (Cys(166)) in Kir6.2. A protein pull-down assay with biotinylated glutathione ethyl ester showed that mutation of Cys(176) impaired oxidant-induced incorporation of glutathione (GSH) into the Kir6.1 subunit. In contrast to Cys(176), Cys(43) had only a modest contribution to S-glutathionylation, and Cys(120) was modulated by extracellular oxidants but not intracellular GSSG. Simulation modeling of Kir6.1 S-glutathionylation suggested that after incorporation to residue 176, the GSH moiety occupied a space between the slide helix and two transmembrane helices. This prevented the inner transmembrane helix from undergoing conformational changes necessary for channel gating, retaining the channel in its closed state.     

Modeling K(ATP) channel gating and its regulation

ATP-sensitive potassium (K_ATP) channels couple cell metabolism to plasmalemmal potassium fluxes in a variety of cell types. The activity of these channels is primarily determined by intracellular adenosine nucleotides, which have both inhibitory and stimulatory effects. The role of K_ATP channels has been studied most extensively in pancreatic beta-cells, where they link glucose metabolism to insulin secretion. Many mutations in K_ATP channel subunits (Kir6.2, SUR1) have been identified that cause either neonatal diabetes or congenital hyperinsulinism. Thus, a mechanistic understanding of K_ATP channel behavior is necessary for modeling beta-cell electrical activity and insulin release in both health and disease. Here, we review recent advances in the K_ATP channel structure and function. We focus on the molecular mechanisms of K_ATP channel gating by adenosine nucleotides, phospholipids and sulphonylureas and consider the advantages and limitations of various mathematical models of macroscopic and single-channel K_ATP currents. Finally, we outline future directions for the development of more realistic models of K_ATP channel gating.     

The molecular basis of the specificity of action of K(ATP) channel openers

K(ATP) channels incorporate a regulatory subunit of the ATP-binding cassette (ABC) transporter family, the sulfonylurea receptor (SUR), which defines their pharmacology. The therapeutically important K(+) channel openers (e.g. pinacidil, cromakalim, nicorandil) act specifically on the SUR2 muscle isoforms but, except for diazoxide, remain ineffective on the SUR1 neuronal/pancreatic isoform. This SUR1/2 dichotomy underpinned a chimeric strategy designed to identify the structural determinants of opener action, which led to a minimal set of two residues within the last transmembrane helix of SUR. Transfer of either residue from SUR2A to SUR1 conferred opener sensitivity to SUR1, while the reverse operation abolished SUR2A sensitivity. It is therefore likely that these residues form part of the site of interaction of openers with the channel. Thus, openers would target a region that, in other ABC transporters, is known to be tightly involved with the binding of substrates and other ligands. This first glimpse of the site of action of pharmacological openers should permit rapid progress towards understanding the structural determinants of their affinity and specificity.     

Pharmacological and kinetic analysis of K channel gating currents

We have measured gating currents from the squid giant axon using solutions that preserve functional K channels and with experimental conditions that minimize Na channel contributions to these currents. Two pharmacological agents were used to identify a component of gating current that is associated with K channels. Low concentrations of internal Zn2+ that considerably slow K channel ionic currents with no effect on Na channel currents altered the component of gating current associated with K channels. At low concentrations (10-50 microM) the small, organic, dipolar molecule phloretin has several reported specific effects on K channels: it reduces K channel conductance, shifts the relationship between channel conductance and membrane voltage (Vm) to more positive potentials, and reduces the voltage dependence of the conductance-Vm relation. The K channel gating charge movements were altered in an analogous manner by 10 microM phloretin. We also measured the dominant time constants of the K channel ionic and gating currents. These time constants were similar over part of the accessible voltage range, but at potentials between -40 and 0 mV the gating current time constants were two to three times faster than the corresponding ionic current values. These features of K channel function can be reproduced by a simple kinetic model in which the channel is considered to consist of two, two-state, nonidentical subunits.     

Conformational dynamics of the ligand-binding domain of inward rectifier K channels as revealed by molecular dynamics simulations: toward an understanding of Kir channel gating

Inward rectifier (Kir) potassium channels are characterized by two transmembrane helices per subunit, plus an intracellular C-terminal domain that controls channel gating in response to changes in concentration of various ligands. Based on the crystal structure of the tetrameric C-terminal domain of Kir3.1, it is possible to build a homology model of the ATP-binding C-terminal domain of Kir6.2. Molecular dynamics simulations have been used to probe the dynamics of Kir C-terminal domains and to explore the relationship between their dynamics and possible mechanisms of channel gating. Multiple simulations, each of 10 ns duration, have been performed for Kir3.1 (crystal structure) and Kir6.2 (homology model), in both their monomeric and tetrameric forms. The Kir6.2 simulations were performed with and without bound ATP. The results of the simulations reveal comparable conformational stability for the crystal structure and the homology model. There is some decrease in conformational flexibility when comparing the monomers with the tetramers, corresponding mainly to the subunit interfaces in the tetramer. The beta-phosphate of ATP interacts with the side chain of K185 in the Kir6.2 model and simulations. The flexibility of the Kir6.2 tetramer is not changed greatly by the presence of bound ATP, other than in two loop regions. Principal components analysis of the simulated dynamics suggests loss of symmetry in both the Kir3.1 and Kir6.2 tetramers, consistent with "dimer-of-dimers" motion of subunits in C-terminal domains of the corresponding Kir channels. This is suggestive of a gating model in which a transition between exact tetrameric symmetry and dimer-of-dimers symmetry is associated with a change in transmembrane helix packing coupled to gating of the channel. Dimer-of-dimers motion of the C-terminal domain tetramer is also supported by coarse-grained (anisotropic network model) calculations. It is of interest that loss of exact rotational symmetry has also been suggested to play a role in gating in the bacterial Kir homolog, KirBac1.1, and in the nicotinic acetylcholine receptor channel.     

How ATP inhibits the open K(ATP) channel

ATP-sensitive potassium (K(ATP)) channels are composed of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits. Binding of ATP to Kir6.2 leads to inhibition of channel activity. Because there are four subunits and thus four ATP-binding sites, four binding events are possible. ATP binds to both the open and closed states of the channel and produces a decrease in the mean open time, a reduction in the mean burst duration, and an increase in the frequency and duration of the interburst closed states. Here, we investigate the mechanism of interaction of ATP with the open state of the channel by analyzing the single-channel kinetics of concatenated Kir6.2 tetramers containing from zero to four mutated Kir6.2 subunits that possess an impaired ATP-binding site. We show that the ATP-dependent decrease in the mean burst duration is well described by a Monod-Wyman-Changeux model in which channel closing is produced by all four subunits acting in a single concerted step. The data are inconsistent with a Hodgkin-Huxley model (four independent steps) or a dimer model (two independent dimers). When the channel is open, ATP binds to a single ATP-binding site with a dissociation constant of 300 microM.     

Molecular basis for K(ATP) assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter

K(ATP) channels are large heteromultimeric complexes containing four subunits from the inwardly rectifying K+ channel family (Kir6.2) and four regulatory sulphonylurea receptor subunits from the ATP-binding cassette (ABC) transporter family (SUR1 and SUR2A/B). The molecular basis for interactions between these two unrelated protein families is poorly understood. Using novel trafficking-based interaction assays, coimmunoprecipitation, and current measurements, we show that the first transmembrane segment (M1) and the N terminus of Kir6.2 are involved in K(ATP) assembly and gating. Additionally, the transmembrane domains, but not the nucleotide-binding domains, of SUR1 are required for interaction with Kir6.2. The identification of specific transmembrane interactions involved in K(ATP) assembly may provide a clue as to how ABC proteins that transport hydrophobic substrates evolved to regulate other membrane proteins.     

The HERG K<Superscript>+</Superscript> channel: progress in understanding the molecular basis of its unusual gating kinetics

The HERG K+ channel has very unusual kinetic behaviour that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarisation as well as in preventing lethal ventricular arrhythmias. Extensive mutagenesis of the HERG K+ channel has allowed identification of which regions of the channel are important for the unusual kinetic behaviour of the channel. Furthermore, structural studies on scorpion toxins that potently inhibit HERG are beginning to provide clues as to the structural differences between HERG and other voltage-gated K+ channels.     

Physical and molecular basis of ion channel gating: Can electrostatic interactions close the ion channel?

We analyzed voltage-dependent ion channel structure and conformational changes corresponding to channel gating. During the gating, S4 segments, as well as other parts of the channel, undergo a set of conformational modifications. These changes are accompanied by complicated movements of positive charges that are mostly located in the S4 segments. These charges electrostatically interact with the ions passing through the channel. The interaction energy depends on the conformational state of the channel, i.e., on the mutual positions of the permeant ions and these charges. Analyzing and making energetical estimations, we propose a hypothesis: the closed state of the ion channel corresponds to the S4 position when electrostatic interaction between positively charged groups of the S4 segments and permeant ions is strong enough to close the pathway for these ions.     

Tectonics of a K+ channel: The importance of the N-terminus for channel gating

The small K⁺ channel Kcv represents the pore module of complex potassium channels. It was found that its gating can be modified by sensor domains, which are N-terminally coupled to the pore. This implies that the short N-terminus of the channel can transmit conformational changes from upstream sensors to the channel gates. To understand the functional role of the N-terminus in the context of the entire channel protein, we apply combinatorial screening of the mechanical coupling and long-range interactions in the Kcv potassium channel by reduced molecular models. The dynamics and mechanical connections in the channel complex show that the N-terminus is indeed mechanically connected to the pore domain. This includes a long rang coupling to the pore and the inner and outer transmembrane domains. Since the latter domains host the two gates of the channel, the data support the hypothesis that mechanical perturbation of the N-terminus can be transmitted to the channel gates. This effect is solely determined by the topology of the channel; sequence details only have an implicit effect on the coarse-grained dynamics via the fold and not through biochemical details at a smaller scale. This observation has important implications for engineering of synthetic channels on the basis of a K⁺ channel pore.     

Molecular basis of the voltage-dependent gating of TREK-1, a mechano-sensitive K(+) channel

TREK-1 is a member of the mammalian two P domain K(+) channel family. Mouse TREK-1 activity, in transiently transfected COS cells, is reduced at negative resting membrane potentials by both an external Mg(2+) block and an intrinsic voltage-dependent gating mechanism leading to a strong outward rectification. Deletional and chimeric analysis demonstrates that the carboxy terminal domain of TREK-1, but not the PKA phosphorylation site S333, is responsible for voltage-dependent gating. Since the same region is also critically required for TREK-1 mechano-gating, both mechanisms might be functionally linked. Preferential opening of TREK-1 at depolarized potentials will greatly affect action potential duration, recovery from inactivation and neuronal repetitive firing activity.     

Molecular modelling of K(ATP) channel blockers-ADP/ ATP carrier interactions

The modelling of molecule-molecule interactions has been widely accepted as a tool for drug discovery and development studies. However, this powerful technique is unappreciated in physiological and biochemical studies, where it could be extremely useful for understanding the mechanisms of action of various compounds in cases when experimental data are controversial due to complexity of the investigated systems. In this study, based on the biochemical data suggesting involvement of mitochondrial ADP/ATP carrier in K+ and H+ transport to mitochondrial matrix molecular modelling is applied to elucidate the possible interactions between the ADP/ATP carrier and its putative ligands--K(ATP) channel blockers glybenclamide, tolbutamide and 5-hydroxydecanoate. Results revealed that K(ATP) channel blockers could bind to the specific location proximal to H1, H4, H5 and H6 transmembrane helices within the cavity of the ADP/ ATP carrier. Analysis of the predicted binding site suggests that K(ATP) channel blockers could interfere with both the ADP/ATP translocation and possible cation flux through the ADP/ATP carrier, and supports the hypothesis that the ADP/ATP carrier is a target of K(ATP) channel modulators.     

ATP interaction with the open state of the K(ATP) channel

The mechanism of ATP-sensitive potassium (K(ATP)) channel closure by ATP is unclear, and various kinetic models in which ATP binds to open or to closed states have previously been presented. Effects of phosphatidylinositol bisphosphate (PIP2) and multiple Kir6.2 mutations on ATP inhibition and open probability in the absence of ATP are explainable in kinetic models where ATP stabilizes a closed state and interaction with an open state is not required. Evidence that ATP can in fact interact with the open state of the channel is presented here. The mutant Kir6.2[L164C] is very sensitive to Cd2+ block, but very insensitive to ATP, with no significant inhibition in 1 mM ATP. However, 1 mM ATP fully protects the channel from Cd2+ block. Allosteric kinetic models in which the channel can be in either open or closed states with or without ATP bound are considered. Such models predict a pedestal in the ATP inhibition, i.e., a maximal amount of inhibition at saturating ATP concentrations. This pedestal is predicted to occur at >50 mM ATP in the L164C mutant, but at >1 mM in the double mutant L164C/R176A. As predicted, ATP inhibits Kir6.2[L164C/R176A] to a maximum of approximately 40%, with a clear plateau beyond 2 mM. These results indicate that ATP acts as an allosteric ligand, interacting with both open and closed states of the channel.     

Structure-function relationships in the beta-cell K(ATP) channel

The ATP-sensitive potassium (K(ATP)) channel plays a key role in controlling beta-cell membrane potential and insulin secretion. The channels are composed of two subunits, Kir6.2, which forms the channel pore, and SUR1, which contains binding sites for nucleotides and sulphonylureas and acts as a channel regulator. Our current studies are aimed at delineating the molecular interactions involved in assembly and ligand binding by K(ATP) channel proteins. We have employed a complementation approach in which SUR1 half-molecules are co-expressed in insect cells using a baculovirus system. Together with data from truncated SUR1 molecules and a fusion protein in which SUR1 is linked to Kir6.2, we have interpreted our findings in terms of a model for the structure of the K(ATP) channel. The main features of the model are: (i) the C-terminal end of SUR1 is close to the N-terminus of Kir6.2; (ii) the two nucleotide binding domains (NBDs) of SUR1--NBD1 and NBD2--are in proximity; (iii) transmembrane helix 12 of SUR1 is orientated in such a way that it can make contact with Kir6.2; (iv) formation of the glibenclamide binding site requires that the two cytosolic loops (CLs) CL3 and CL8 are located close to each other; (v) there are homomeric interactions between the NBD1 domains of neighbouring subunits. We suggest that binding of glibenclamide leads to conformational changes in CL3 and CL8 leading to rearrangement of transmembrane helices. These effects are transmitted to Kir6.2 to result in channel closure.     

The biophysical and molecular basis of TRPV1 proton gating

The capsaicin receptor TRPV1, a member of the transient receptor potential family of non-selective cation channels is a polymodal nociceptor. Noxious thermal stimuli, protons, and the alkaloid irritant capsaicin open the channel. The mechanisms of heat and capsaicin activation have been linked to voltage-dependent gating in TRPV1. However, until now it was unclear whether proton activation or potentiation or both are linked to a similar voltage-dependent mechanism and which molecular determinants underlie the proton gating. Using the whole-cell patch-clamp technique, we show that protons activate and potentiate TRPV1 by shifting the voltage dependence of the activation curves towards more physiological membrane potentials. We further identified a key residue within the pore region of TRPV1, F660, to be critical for voltage-dependent proton activation and potentiation. We conclude that proton activation and potentiation of TRPV1 are both voltage dependent and that amino acid 660 is essential for proton-mediated gating of TRPV1.     

ATP敏感性钾通道与2型糖尿病

ATP敏感性钾通道（KATP）是调节葡萄糖代谢平衡的关键因子。KATP通道的遗传变异可改变β-细胞电活性、葡萄糖代谢平衡,增加2型糖尿病易感性,因此,编码该通道的基因可作为2型糖尿病的易感标记。Kir6．2的E23K多态性在高加索人群中与2型糖尿病易感性增加和肥胖相关。E23K多态性在高加索人群中较常见,提示其可能具有进化优势。     

Assembly, maturation, and turnover of K(ATP) channel subunits

ATP-sensitive K(+), or K(ATP), channels are comprised of K(IR)6.x and sulfonylurea receptor (SUR) subunits that assemble as octamers, (K(IR)/SUR)(4). The assembly pathway is unknown. Pulse-labeling studies show that when K(IR)6.2 is expressed individually, its turnover is biphasic; approximately 60% is lost with t((1/2)) approximately 36 min. The remainder converts to a long-lived species (t((1/2)) approximately 26 h) with an estimated half-time of 1.2 h. Expressed alone, SUR1 has a long half-life, approximately 25.5 h. When K(IR)6.2 and SUR1 are co-expressed, they associate rapidly and the fast degradation of K(IR)6.2 is eliminated. Based on changes in the glycosylation state of SUR1, the half-time for the maturation of K(ATP) channels, including completion of assembly, transit to the Golgi, and glycosylation, is approximately 2.2 h. Estimation of the turnover rates of mature, fully glycosylated SUR1 associated with K(IR)6.2 and of K(IR)6.2 associated with Myc-tagged SUR1 gave similar values for the half-life of K(ATP) channels, a mean value of approximately 7.3 h. K(ATP) channel subunits in INS-1 beta-cells displayed qualitatively similar kinetics. The results imply the octameric channels are stable. Two mutations, K(IR)6.2 W91R and SUR1 DeltaF1388, identified in patients with the severe form of familial hyperinsulinism, profoundly alter the rate of K(IR)6.2 and SUR1 turnover, respectively. Both mutant subunits associate with their respective partners but dissociate freely and degrade rapidly. The data support models of channel formation in which K(IR)6.2-SUR1 heteromers assemble functional channels and are inconsistent with models where SUR1 can only assemble with K(IR)6.2 tetramers.     

Redox regulation of the mitochondrial K(ATP) channel in cardioprotection

The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK(ATP) regulation. mK(ATP) activity was measured by both osmotic swelling and Tl(+) flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK(ATP) in cardioprotection by nitroxyl. Key findings were as follows: (i) mK(ATP) was activated by O(2)(-) and H(2)O(2) but not other peroxides. (ii) mK(ATP) was inhibited by NADPH. (iii) mK(ATP) was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK(ATP)-dependent manner. Overall, these results suggest that the mK(ATP) channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK(ATP) may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.