Intracellular ATP directly blocks K+ channels in pancreatic B-cells

It is known that glucose-induced depolarization of pancreatic B-cells is due to reduced membrane K+-permeability and is coupled to an increase in the rate of glycolysis, but there has been no direct evidence linking specific metabolic processes or products to the closing of membrane K+ channels. During patch-clamp studies of proton inhibition of Ca2+-activated K+ channels [GK(Ca)] in B-cells, we identified a second K+-selective channel which is rapidly and reversibly inhibited by ATP applied to the cytoplasmic surface of the membrane. This channel is spontaneously active in excised patches and frequently coexists with GK(Ca) channels yet is insensitive to membrane potential and to intracellular free Ca2+ and pH. Blocking of the channel is ATP-specific and appears not to require metabolism of the ATP. This ATP-sensitive K+ channel [GK(ATP)] may be a link between metabolism and membrane K+-permeability in pancreatic B-cells.     

Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR

The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane--similar to the catalytic function of the active site of an enzyme--and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.     

Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle

The recent development of techniques for recording currents through single ionic channels has led to the identification of a K+-specific channel that is activated by cytoplasmic Ca2+. The channel has complex properties, being activated by depolarizing voltages and having a voltage-sensitivity that is modulated by cytoplasmic Ca2+ levels. The conduction behaviour of the channel is also unusual, its high ionic selectivity being displayed simultaneously with a very high unitary conductance. Very little is known about the biochemistry of this channel, largely due to the lack of a suitable ligand for use as a biochemical probe for the channel. We describe here a protein inhibitor of single Ca2+-activated K+ channels of mammalian skeletal muscle. This inhibitor, a minor component of the venom of the Israeli scorpion, Leiurus quinquestriatus, reversibly blocks the large Ca2+-activated K+ channel in a simple biomolecular reaction. We have partially purified the active component, a basic protein of relative molecular mass (Mr) approximately 7,000.     

Molecular mechanism of ATP binding and ion channel activation in P2X receptors

P2X receptors are trimeric ATP-activated ion channels permeable to Na+, K+ and Ca2+. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body β-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.     

ATP敏感性钾通道与心肌低氧损伤

ATP敏感性钾通道是一种受细胞内ATP浓度，NDPs，KCOs,SUs等多种因素调节启闭的钾通道。在低氧条件下，ATP敏感性钾通道被激活，引起动作电位时程缩短和细胞外钾离子蓄积，减少钙离子内流，对心肌有一定的保护作用；但过度的钾离子外流，对心肌则有损害，甚于诱发心律失常。提示：ATP敏感性钾通道的开放，可能是低氧引起心肌损伤的一种内源性机制。     

Heteromultimeric channels formed by rat brain potassium-channel proteins

An important step towards understanding the molecular basis of the functional diversity of voltage-gated K+ channels in the mammalian brain has been the discovery of a family of genes encoding rat brain K+ channel-forming (RCK) proteins. All species of these RCK proteins form homomultimeric voltage-gated K+ channels with distinct functional characteristics in Xenopus laevis oocytes following injection of the respective cRNAs. RCK-specific mRNAs are coexpressed in several regions of the brain, suggesting that RCK proteins also assemble into heteromultimeric K+ channels. In addition expression experiments with fractionated poly(A)+ mRNA have suggested that heteromultimeric K+ channels may occur in mammalian brain. We report here that heteromultimeric K+ channels composed of two different RCK proteins (RCK1 and RCK4) assemble after cotransfection of HeLa cells with the corresponding cDNAs and after coinjection of the corresponding cRNAs into Xenopus oocytes. The heteromultimeric RCK1, 4 channel mediates a transient potassium outward current, similar to the RCK4 channel but inactivates more slowly, has a larger conductance and is more sensitive to block by dendrotoxin and tetraethylammonium chloride.     

Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic B cells

Insulin is produced and secreted by the B cells in the endocrine pancreas. In vivo, insulin secretion is under the control of a number of metabolic, neural and hormonal substances. It is now clear that stimulation of insulin release by fuel secretagogues, such as glucose, involves the closure of K+ channels that are sensitive to the intracellular ATP concentration (KATP channels). This leads to membrane depolarization and the generation of Ca2(+)-dependent action potentials. The mechanisms whereby hormones and neurotransmitters such as adrenaline, galanin and somatostatin, which are released by intraislet nerve endings and the pancreatic D cells, produce inhibition of insulin secretion are not clear. Here we show that adrenaline suppresses B-cell electrical activity (and thus insulin secretion) by a G protein-dependent mechanism, which culminates in the activation of a sulphonylurea-insensitive low-conductance K+ channel distinct from the KATP channel.     

Arterial dilations in response to calcitonin gene-related peptide involve activation of K+ channels

Calcitonin gene-related peptide (CGRP) is a 37-amino-acid peptide produced by alternative processing of messenger RNA from the calcitonin gene. CGRP is one of the most potent vasodilators known. It occurs in and is released from perivascular nerves and has been detected in the blood stream, suggesting that it is important in the control of blood flow. The mechanism by which it dilates arteries is not known. Here, we report that arterial dilations in response to CGRP are partially reversed by blockers of the ATP-sensitive potassium channel (K(ATP)), glibenclamide and barium. We also show that CGRP hyperpolarizes arterial smooth muscle and that blockers of K(ATP) channels reverse this hyperpolarization. Finally, we show that CGRP opens single K+ channels in patches on single smooth muscle cells from the same arteries. We propose that activation of K(ATP) channels underlies a substantial part of the relaxation produced by CGRP.     

Crystal structure of a voltage-gated sodium channel in two potentially inactivated states

In excitable cells, voltage-gated sodium (Na(V)) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance. Inactivation is a hallmark of Na(V) channel function and is critical for control of membrane excitability, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type Na(V)Ab channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2?? resolution. Compared to previous structures of Na(V)Ab channels with cysteine mutations in the pore-lining S6 helices (ref. 4), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type Na(V)Ab models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type Na(V)Ab, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in Na(V) channel gating and inactivation. These potential inactivated-state structures provide new insights into Na(V) channel gating and novel avenues to drug development and therapy for a range of debilitating Na(V) channelopathies.     

Crystal structure and mechanism of a calcium-gated potassium channel

Ion channels exhibit two essential biophysical properties; that is, selective ion conduction, and the ability to gate-open in response to an appropriate stimulus. Two general categories of ion channel gating are defined by the initiating stimulus: ligand binding (neurotransmitter- or second-messenger-gated channels) or membrane voltage (voltage-gated channels). Here we present the structural basis of ligand gating in a K(+) channel that opens in response to intracellular Ca(2+). We have cloned, expressed, analysed electrical properties, and determined the crystal structure of a K(+) channel (MthK) from Methanobacterium thermoautotrophicum in the Ca(2+)-bound, opened state. Eight RCK domains (regulators of K(+) conductance) form a gating ring at the intracellular membrane surface. The gating ring uses the free energy of Ca(2+) binding in a simple manner to perform mechanical work to open the pore.     

Evolution of phosphofructokinase--gene duplication and creation of new effector sites

Phosphofructokinases (PFK; EC 2.7.1.11) are tetrameric enzymes that have a key role in the regulation of glycolysis; as such, they are subject to allosteric activation and inhibition by various metabolites. Eukaryotic PFKs are about twice the size of prokaryotic enzymes and are regulated by a wider repertoire of effectors: for example, the subunit molecular weights of rabbit muscle (RM) PFK and Bacillus stearothermophilus (Bs) PFK are 82,000 and 36,000, respectively. Both enzymes are activated by ADP (or AMP), but RM-PFK is also activated by fructose bisphosphates (FBP) and inhibited by ATP and citrate. This, together with other evidence, has led to speculation that mammalian PFKs have evolved by duplication of a prokaryotic gene, although previous peptide analysis failed to reveal internal homology in RM-PFK. Here we demonstrate clear homology among the N- and C-halves of RM-PFK and Bs-PFK, thus establishing an evolutionary relationship by series gene duplication and divergence. Furthermore, detailed knowledge of the Bs-PFK structure provides the basis for inferences concerning the structural organization of RM-PFK and the evolution of new effector sites in the enzyme tetramer.     

Sequence and expression of a frog brain complementary DNA encoding a kainate-binding protein

Excitatory amino acids (EAAs) are important neurotransmitters in the vertebrate central nervous system. Electrophysiological and ligand-binding studies indicate that at least three different receptor subtypes for EAAs exist--N-methyl-D-aspartate, kainate and quisqualate receptor subtypes--on the basis of the preferred agonist of the receptors. We recently purified a kainate-binding protein (KBP) from frog (Rana pipiens berlandieri) brain by domoic acid (a high-affinity kainate analogue) affinity chromatography, and showed that the kainate-binding activity was associated with a protein of relative molecular mass 48,000 (Mr 48 K). The pharmacological properties and the anatomical distribution of KBP were consistent with those of a kainate receptor-ionophore complex. We have now isolated a complementary DNA encoding KBP of Mr 48 K. The deduced amino-acid sequence of the KBP has similar hydrophobic profiles to those found in other ligand-gated ion channel subunits, and shows some amino-acid sequence similarities to the corresponding regions of brain nicotinic acetylcholine receptor subunits. Localization of the KBP messenger RNAs by in situ hybridization histochemistry is compatible with the results of immunohistochemistry and receptor autoradiography studies. COS-7 cells transfected with the cDNA encoding the KBP show high-affinity kainate-binding activity with pharmacological properties similar to those of the biochemically purified KBP. These results provide the first molecular characterization of an EAA-binding site and raise the possibility that the KBP cDNA encodes a ligand-binding subunit of a kainate receptor-ionophore complex.     

Modulation of ATP-sensitive K+ channels in skeletal muscle by intracellular protons

Since their discovery in cardiac muscle, ATP-sensitive K+(KATP) channels have been identified in pancreatic beta-cells, skeletal muscle, smooth muscle and central neurons. The activity of KATP channels is inhibited by the presence of cytosolic ATP. Their wide distribution indicates that they could have important physiological roles that may vary between tissues. In muscle cells the role of K+ channels is to control membrane excitability and the duration of the action potential. In anoxic cardiac ventricular muscle KATP channels are believed to be responsible for shortening the action potential, and it has been proposed that a fall in ATP concentration during metabolic exhaustion increases the activity of KATP channels in skeletal muscle, which may reduce excitability. But the intracellular concentration of ATP in muscle is buffered by creatine phosphate to 5-10 mM, and changes little, even during sustained activity. This concentration is much higher than the intracellular ATP concentration required to half block the KATP-channel current in either cardiac muscle (0.1 mM) or skeletal muscle (0.14 mM), indicating that the open-state probability of KATP channels is normally very low in intact muscle. So it is likely that some additional means of regulating the activity of KATP channels exists, such as the binding of nucleotides other than ATP. Here I present evidence that a decrease in intracellular pH (pHi) markedly reduces the inhibitory effect of ATP on these channels in excised patches from frog skeletal muscle. Because sustained muscular activity can decrease pHi by almost 1 unit in the range at which KATP channels are most sensitive to pHi, it is likely that the activity of these channels in skeletal muscle is regulated by intracellular protons under physiological conditions.     

Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution.

Ion transport proteins must remove an ion's hydration shell to coordinate the ion selectively on the basis of its size and charge. To discover how the K+ channel solves this fundamental aspect of ion conduction, we solved the structure of the KcsA K+ channel in complex with a monoclonal Fab antibody fragment at 2.0 A resolution. Here we show how the K+ channel displaces water molecules around an ion at its extracellular entryway, and how it holds a K+ ion in a square antiprism of water molecules in a cavity near its intracellular entryway. Carbonyl oxygen atoms within the selectivity filter form a very similar square antiprism around each K+ binding site, as if to mimic the waters of hydration. The selectivity filter changes its ion coordination structure in low K+ solutions. This structural change is crucial to the operation of the selectivity filter in the cellular context, where the K+ ion concentration near the selectivity filter varies in response to channel gating.     

ATP-sensitive K+ channel in the mitochondrial inner membrane.

Mitochondria take up and extrude various inorganic and organic ions, as well as larger substances such as proteins. The technique of patch clamping should provide real-time information on such transport and on energy transduction in oxidative phosphorylation. It has been applied to detect microscopic currents from mitochondrial membranes and conductances of ion channels in the 5-1,000 pS range in the outer and inner membranes. These pores are not, however, selective for particular ions. Here we use fused giant mitoplasts prepared from rat liver mitochondria to identify a small conductance channel highly selective for K+ in the inner mitochondrial membrane. This channel can be reversibly inactivated by ATP applied to the matrix side under inside-out patch configuration; it is also inhibited by 4-aminopyridine and by glybenclamide. The slope conductance of the unitary currents measured at negative membrane potentials was 9.7 +/- 1.0 pS (mean +/- s.d., n = 6) when the pipette solution contained 100 mM K+ and the bathing solution 33.3 mM K+. Our results indicate that mitochondria depolarize by generating a K+ conductance when ATP in the matrix is deficient.     

Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide

Muscle contraction involves the cyclic interaction of the myosin cross-bridges with the actin filament, which is coupled to steps in the hydrolysis of ATP. While bound to actin each cross-bridge undergoes a conformational change, often referred to as the "power stroke", which moves the actin filament past the myosin filaments; this is associated with the release of the products of ATP hydrolysis and a stronger binding of myosin to actin. The association of a new ATP molecule weakens the binding again, and the attached cross-bridge rapidly dissociates from actin. The nucleotide is then hydrolysed, the conformational change reverses, and the myosin cross-bridge reattaches to actin. X-ray crystallography has determined the structural basis of the power stroke, but it is still not clear why the binding of actin weakens that of the nucleotide and vice versa. Here we describe, by fitting atomic models of actin and the myosin cross-bridge into high-resolution electron cryo-microscopy three-dimensional reconstructions, the molecular basis of this linkage. The closing of the actin-binding cleft when actin binds is structurally coupled to the opening of the nucleotide-binding pocket.     

Atomic structure of a Na+- and K+-conducting channel

Ion selectivity is one of the basic properties that define an ion channel. Most tetrameric cation channels, which include the K+, Ca2+, Na+ and cyclic nucleotide-gated channels, probably share a similar overall architecture in their ion-conduction pore, but the structural details that determine ion selection are different. Although K+ channel selectivity has been well studied from a structural perspective, little is known about the structure of other cation channels. Here we present crystal structures of the NaK channel from Bacillus cereus, a non-selective tetrameric cation channel, in its Na+- and K+-bound states at 2.4 A and 2.8 A resolution, respectively. The NaK channel shares high sequence homology and a similar overall structure with the bacterial KcsA K+ channel, but its selectivity filter adopts a different architecture. Unlike a K+ channel selectivity filter, which contains four equivalent K+-binding sites, the selectivity filter of the NaK channel preserves the two cation-binding sites equivalent to sites 3 and 4 of a K+ channel, whereas the region corresponding to sites 1 and 2 of a K+ channel becomes a vestibule in which ions can diffuse but not bind specifically. Functional analysis using an 86Rb flux assay shows that the NaK channel can conduct both Na+ and K+ ions. We conclude that the sequence of the NaK selectivity filter resembles that of a cyclic nucleotide-gated channel and its structure may represent that of a cyclic nucleotide-gated channel pore.     

Control of CFTR chloride conductance by ATP levels through non-hydrolytic binding.

Site-specific mutation and membrane reconstitution experiments provide compelling evidence that the product of the gene which is at fault in the disease cystic fibrosis, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is a small-conductance chloride channel activated by phosphorylation. As transport of chloride ions is passive, the predicted presence of two nucleotide-binding domains in CFTR seems as puzzling as a report that ATP hydrolysis is essential to activate the channel. We now find that in the sweat duct, which expresses high levels of CFTR and has a very high Cl- conductance, intracellular concentrations of ATP must be about normal (5 mM) for activation of this conductance, apparently by a non-hydrolytic, perhaps allosteric, mechanism. This passive dependence on ATP should mean that even a modest depletion of cell energy levels will significantly lower the energy demands of electrolyte transport by decreasing chloride conductance. We believe this direct coupling between cellular ATP levels and chloride channel activity is an adaptive mechanism to protect the tissue from damage resulting from excessive energy depletion.     

Kluyveromyces lactis killer toxin inhibits adenylate cyclase of sensitive yeast cells

K1 killer toxin secreted by the K1 strain of Saccharomyces cerevisiae, has been well characterized. It is a simple protein of molecular weight (MW) 11,470 (ref. 3), encoded by a double-stranded, linear RNA plasmid, called M RNA, of MW 1.1-1.7 x 10(6) (refs 4-6). It is lethal to sensitive Saccharomyces cerevisiae which does not carry M RNA. Leakage of K+ and ATP is the first distinct response in sensitive cells, and the toxic action is thought to be due to its action as a protonophore or K+ ionophore. Recently, a further killer toxin has been found in Kluyveromyces lactis IFO 1267, and it is associated with the presence of the double-stranded linear DNA plasmids, pGK1-1 (MW 5.4 x 10(6)) and pGK1-2 (MW 8.4 x 10(6)). It has been shown, by curing pGK1-1 or deletion mapping, that the structural gene for the killer toxin and immunity-determining gene reside on the smaller plasmid. Moreover, the plasmids could be transferred from K. lactis to S. cerevisiae by protoplast fusion and protoplast transformation. As the K. lactis toxin is encoded by a DNA plasmid and has a relatively wider action spectrum than K1 killer toxin, the mode of action of the toxin is highly interesting. Here we report that K. lactis toxin inhibits adenylate cyclase in sensitive yeast cells and brings about arrest of the cells at the G1 stage.     

Homology of Ti alpha-subunit of a T-cell antigen-MHC receptor with immunoglobulin

Human T-cell receptors for antigen and major histocompatibility complex (MHC) determinants have now been defined on inducer, suppressor, and class 1 and class 2 MHC-specific cytotoxic T lymphocytes as T3-associated clonotypic molecules (Ti) of relative molecular mass 90,000 (90K) composed of one 49-54K alpha- and one 43K beta-subunit which are disulphide-linked. In the case of the Ti beta-subunit, N-terminal amino acid sequencing and molecular cloning techniques led recently to identification of the Ti beta-gene and showed that T-specific V, D, J and C segments fuse to form an active beta-gene. So far, however, there have been little structural data available on the Ti alpha-subunit. Here we have derived the amino acid sequence of a portion of the Ti alpha-subunit by CNBr fragmentation. Sequence analysis reveals approximately 40% homology between the Ti alpha-subunit fragment and the third framework of the variable region of immunoglobulin light and heavy chains, supporting the notion that the Ti alpha-subunit is a member of the immunoglobulin-Ti beta-gene family.