Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane Cl- channel regulated by cyclic AMP-dependent phosphorylation and by intracellular ATP. Mutations in CFTR cause cystic fibrosis partly through loss of cAMP-regulated Cl- permeability from the plasma membrane of affected epithelia. The most common mutation in cystic fibrosis is deletion of phenylalanine at residue 508 (CFTR delta F508) (ref. 10). Studies on the biosynthesis and localization of CFTR delta F508 indicate that the mutant protein is not processed correctly and, as a result, is not delivered to the plasma membrane. These conclusions are consistent with earlier functional studies which failed to detect cAMP-stimulated Cl- channels in cells expressing CFTR delta F508 (refs 16, 17). Chloride channel activity was detected, however, when CFTR delta F508 was expressed in Xenopus oocytes, Vero cells and Sf9 insect cells. Because oocytes and Sf9 cells are typically maintained at lower temperatures than mammalian cells, and because processing of nascent proteins can be sensitive to temperature, we tested the effect of temperature on the processing of CFTR delta F508. Here we show that the processing of CFTR delta F508 reverts towards that of wild-type as the incubation temperature is reduced. When the processing defect is corrected, cAMP-regulated Cl- channels appear in the plasma membrane. These results reconcile previous contradictory observations and suggest that the mutant most commonly associated with cystic fibrosis is temperature-sensitive.     

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.     

Oxygen uptake occurs faster than sodium pumping in bee retina after a light flash

When neurones are active there is an entry of Na+, which must subsequently be pumped out, and an increase in their oxygen consumption rate (Qo2). The Na+ pump derives its energy from ATP, splitting it into ADP and Pi, and it has reasonably been proposed that the changes in concentrations of ATP, ADP and Pi lead to a stimulation of the O2 consumption by the mitochondria and hence to a restoration of the stock of ATP. Here we present evidence suggesting that Qo2 must be controlled differently in the retinal photoreceptor cells of the honeybee drone. Stimulation of drone photoreceptors with a flash of light causes an entry of Na+ (ref. 4) and a transient increase in Qo2 that indicates respiration of the right order of magnitude to provide ATP to pump the Na+ out. We report intracellular recordings of changes in intracellular sodium (Nai+) and potassium (Ki+) in response to single light flashes and have compared the time course of extra oxygen consumption (delta Qo2) with these ion changes and other indices of Na+ pumping. We found that the time course of pumping seems to lag behind the time course of delta Qo2. It follows that the mitochondrial respiration must be stimulated by some signal which is generated earlier than the rise in ADP produced by the Na+ pump.     

Voltage-dependent ATP-sensitive potassium channels of skeletal muscle membrane

It has been known for some years that skeletal muscle develops a high potassium permeability in conditions that produce rigor, where ATP concentrations are low and intracellular Ca2+ is high. It has seemed natural to attribute this high permeability to K channels that are opened by internal Ca2+, especially as the presence of such channels has been demonstrated in myotubes and in the transverse tubular membrane system of adult skeletal muscle. However, as we show here, the surface membrane of frog muscle contains potassium channels that open at low internal concentrations of ATP (less than 2 mM). ATP induces closing of these channels without being split, apparently holding the channels in one of a number of closed states. The channels have at least two open states whose dwell times are voltage-dependent. Surprisingly, we find that these may be the most common K channels of the surface membrane of skeletal muscle.     

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.     

Subnanometre-resolution structure of the intact Thermus thermophilus H+-driven ATP synthase

Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7?? resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases.     

LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability

The molecular mechanisms that regulate basal or background entry of divalent cations into mammalian cells are poorly understood. Here we describe the cloning and functional characterization of a Ca2+- and Mg2+-permeable divalent cation channel, LTRPC7 (nomenclature compatible with that proposed in ref. 1), a new member of the LTRPC family of putative ion channels. Targeted deletion of LTRPC7 in DT-40 B cells was lethal, indicating that LTRPC7 has a fundamental and nonredundant role in cellular physiology. Electrophysiological analysis of HEK-293 cells overexpressing recombinant LTRPC7 showed large currents regulated by millimolar levels of intracellular Mg.ATP and Mg.GTP with the permeation properties of a voltage-independent divalent cation influx pathway. Analysis of several cultured cell types demonstrated small magnesium-nucleotide-regulated metal ion currents (MagNuM) with regulation and permeation properties essentially identical to the large currents observed in cells expressing recombinant LTRPC7. Our data indicate that LTRPC7, by virtue of its sensitivity to physiological Mg.ATP levels, may be involved in a fundamental process that adjusts plasma membrane divalent cation fluxes according to the metabolic state of the cell.     

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.     

Transmitter-like action of ATP on patched membranes of cultured myoblasts and myotubes

The concept of purinergic neurotransmission, first proposed by Burnstock, has been confirmed in various cell types. We show here, by the patch-clamp method, that external ATP in micromolar concentrations (1-100 microM) activates cation channels in the membranes of fusion-competent myoblasts and myotubes. In cell-attached membrane patches of myoblasts and myotubes the mean number of simultaneously activated channels increases with time after external ATP application. In myoblasts only one population of channels having a mean single-channel conductance of gamma=43 pS was found, while in myotubes two populations with gamma 1=48 pS and gamma 2=20 pS were observed. Treatment of myotube membranes with acetylcholine (ACh) or carbachol resulted in two populations of channels which had conductance values and voltage-dependent mean channel lifetimes similar to those produced in response to ATP. The results show that embryonic skeletal muscle cells contain cation channels sensitive to ATP and provide evidence for a neurotransmitter-like action of ATP on these cells.     

P2X receptors as cell-surface ATP sensors in health and disease

P2X receptors are membrane ion channels activated by the binding of extracellular adenosine triphosphate (ATP). For years their functional significance was consigned to distant regions of the autonomic nervous system, but recent work indicates several further key roles, such as afferent signalling, chronic pain, and in autocrine loops of endothelial and epithelial cells. P2X receptors have a molecular architecture distinct from other ion channel protein families, and have several unique functional properties.     

Altered chloride ion channel kinetics associated with the delta F508 cystic fibrosis mutation.

Cystic fibrosis is associated with a defect in epithelial chloride ion transport which is caused by mutations in a membrane protein called CFTR (cystic fibrosis transmembrane conductance regulator). Heterologous expression of CFTR produces cyclicAMP-sensitive Cl(-)-channel activity. Deletion of phenylalanine at amino-acid position 508 in CFTR (delta F508 CFTR) is the most common mutation in cystic fibrosis. It has been proposed that this mutation prevents glycoprotein maturation and its transport to its normal cellular location. We have expressed both CFTR and delta F508 CFTR in Vero cells using recombinant vaccinia virus. Although far less delta F508 CFTR reached the plasma membrane than normal CFTR, sufficient delta F508 CFTR was expressed at the plasma membrane to permit functional analysis. delta F508 CFTR expression induced a reduced activity of the cAMP-activated Cl- channel, with conductance, anion selectivity and open-time kinetics similar to those of CFTR, but with much greater closed times, resulting in a large decrease of open probability. The delta F508 mutation thus seems to have two major consequences, an abnormal translocation of the CFTR protein which limits membrane insertion, and an abnormal function in mediating Cl- transport.     

Cell-free expression of functional Shaker potassium channels.

The functional activity of ion channels and other membrane proteins requires that the proteins be correctly assembled in a transmembrane configuration. Thus, the functional expression of ion channels, neurotransmitter receptors and complex membrane-limited signalling mechanisms from complementary DNA has required the injection of messenger RNA or transfection of DNA into Xenopus oocytes or other target cells that are capable of processing newly translated protein into the surface membrane. These approaches, combined with voltage-clamp analysis of ion channel currents, have been especially powerful in the identification of structure-function relationships in ion channels. But oocytes express endogenous ion channels, neurotransmitter receptors and receptor-channel subunits, complicating the interpretation of results in mRNA-injected eggs. Furthermore, it is difficult to control experimentally the membrane lipids and post-translational modifications that underlie the regulation and modulation of ion channels in intact cells. A cell-free system for ion channel expression is ideal for good experimental control of protein expression and modulatory processes. Here we combine cell-free protein translation, microsomal membrane processing of nascent channel proteins, and reconstitution of newly synthesized ion channels into planar lipid bilayers to synthesize, glycosylate, process into membranes, and record in vitro the activity of functional Shaker potassium channels.     

Direct modulation of Aplysia S-K+ channels by a 12-lipoxygenase metabolite of arachidonic acid

Lipoxygenase metabolites of arachidonic acid have recently been shown to modulate the activity of ion channels in nerve and muscle cells. The mechanism of action of these metabolites is, however, unknown. In sensory neurons of Aplysia, the S-K- channel is under the dual modulatory control of 5-hydroxytryptamine (5-HT), which decreases the number of active S channels through cyclic AMP-dependent phosphorylation, and the neuropeptide FMRFamide, which increases the probability of S-channel opening through the 12-lipoxygenase metabolite 12-hydroperoxyeicosatetraenoic acid (12-HPETE). Here we report that the increase in the probability of S-channel opening with FMRFamide is mimicked by application of 12-HPETE to cell-free membrane patches that lack ATP and GTP. Thus, 12-HPETE can act directly to modulate S-channel activity, independently of protein phosphorylation or dephosphorylation, G-protein activation or cyclic nucleotides.     

细胞膜上的离子通道

离子通道是细胞膜上控制离子进出的功能蛋白,在细胞生命活动中发挥重要作用．离子通道具有对离子的选择性、通透的饱和性和开关的可控制性等特点;膜电压的变化、机械刺激和某些信号分子都可以调控离子通道开关;离子通道担负着离子吸收、渗透压调控、电冲动的形成和信号转导等重要的生理功能．离子通道的结构或功能失常会导致一些严重的疾病,对离子通道进行研究,寻找和设计调控离子通道的有效药物是治疗相关疾病的重要手段。     

A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom

Venomous animals produce small protein toxins that inhibit ion channels with high affinity. In several well-studied cases the inhibitory proteins are water-soluble and bind at a channel's aqueous-exposed extracellular surface. Here we show that a voltage-sensor toxin (VSTX1) from the Chilean Rose Tarantula (Grammostola spatulata) reaches its target by partitioning into the lipid membrane. Lipid membrane partitioning serves two purposes: to localize the toxin in the membrane where the voltage sensor resides and to exploit the free energy of partitioning to achieve apparent high-affinity inhibition. VSTX1, small hydrophobic poisons and anaesthetic molecules reveal a common theme of voltage sensor inhibition through lipid membrane access. The apparent requirement for such access is consistent with the recent proposal that the sensor in voltage-dependent K+ channels is located at the membrane-protein interface.     

Detecting the breakup of spiral waves in small-world networks of neurons due to channel block

The breakup of a spiral wave by blockade of sodium and potassium channels in a small-world network of Hodgkin-Huxley neurons is investigated in detail.The influence of ion channel block in poisoned excitable membrane patches of a certain size is measured,by varying channel noise and channel densities resulting from the change in conductance,For example,tetraethylammonium is known to cause a block(poisoning) of potassium channels,while tetrodotoxin blocks sodium channels.We observed the occurrence of spiral waves,which are ordered waves believed to play an important role in facilitating the propagation of electric signals across quiescent regions of the brain.In this paper,the effect of channel block was measured by the factors xK and xNa,which represent the ratios of unblocked,or active,ion channels,to the overall number of potassium or sodium ion channels,respectively.To quantify these observations,we use a simple but robust synchronization measure,which succinctly captures the transition from spiral waves to other collective states,such as broken segments resulting from the breakup of the spiral wave.The critical thresholds of channel block can be inferred from the abrupt changes occurring in plots of the synchronization measure against different values of xK and xNa.Notably,small synchronization factors can be tightly associated with states where the formation of spiral waves is robust to mild channel block.     

An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum

P2X receptors are membrane ion channels gated by extracellular ATP that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion channel activated by ATP (30-100 muM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists but was blocked by nanomolar concentrations of Cu2+ ions. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells that were unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation.     

CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains

ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR's two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2). Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP, and hydrolysis of the ATP causes dimer dissociation. Here, using single-channel recording methods on intact CFTR molecules, we directly follow opening and closing of the channel gates, and relate these occurrences to ATP-mediated events in the NBDs. We find that energetic coupling between two CFTR residues, expected to lie on opposite sides of its predicted NBD1-NBD2 dimer interface, changes in concert with channel gating status. The two monitored side chains are independent of each other in closed channels but become coupled as the channels open. The results directly link ATP-driven tight dimerization of CFTR's cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains. This establishes a molecular mechanism, involving dynamic restructuring of the NBD dimer interface, that is probably common to all members of the ABC protein superfamily.     

Cloned neuronal IK(A) channels reopen during recovery from inactivation

The kinetic behaviour and functional role of potassium ion (K+) channels mediating a fast-inactivating K+ current (IK(A)) has been widely discussed. Activating in the subthreshold range of excitation, IK(A) channels are assumed to reduce the excitatory effect of depolarizing membrane currents in a time-dependent manner. Here we report that IK(A) channels not only open in response to a depolarization but open again after repolarization of the membrane. Although the current in response to the depolarization is rapidly inactivating, the current elicited by repolarization declines slowly and produces long-lasting afterhyperpolarizations under current-clamp conditions. This implies an additional physiological role for IK(A) channels, particularly those that activate positive to the threshold of excitation. The underlying biophysical mechanism was studied by fast-application of peptides corresponding to the N-terminal end of the IK(A) channel proteins. It was found to be a voltage-dependent release of the inactivation gate.     

Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2

The regulation of ion channel activity by specific lipid molecules is widely recognized as an integral component of electrical signalling in cells. In particular, phosphatidylinositol 4,5-bisphosphate (PIP(2)), a minor yet dynamic phospholipid component of cell membranes, is known to regulate many different ion channels. PIP(2) is the primary agonist for classical inward rectifier (Kir2) channels, through which this lipid can regulate a cell's resting membrane potential. However, the molecular mechanism by which PIP(2) exerts its action is unknown. Here we present the X-ray crystal structure of a Kir2.2 channel in complex with a short-chain (dioctanoyl) derivative of PIP(2). We found that PIP(2) binds at an interface between the transmembrane domain (TMD) and the cytoplasmic domain (CTD). The PIP(2)-binding site consists of a conserved non-specific phospholipid-binding region in the TMD and a specific phosphatidylinositol-binding region in the CTD. On PIP(2) binding, a flexible expansion linker contracts to a compact helical structure, the CTD translates 6 ? and becomes tethered to the TMD and the inner helix gate begins to open. In contrast, the small anionic lipid dioctanoyl glycerol pyrophosphatidic acid (PPA) also binds to the non-specific TMD region, but not to the specific phosphatidylinositol region, and thus fails to engage the CTD or open the channel. Our results show how PIP(2) can control the resting membrane potential through a specific ion-channel-receptor-ligand interaction that brings about a large conformational change, analogous to neurotransmitter activation of ion channels at synapses.