Red cell sodium fluxes catalysed by the sodium pump in the absence of K+ and ADP

In the absence of extracellular Na+ or K+, the sodium pump catalyses an ouabain-sensitive "uncoupled" Na+ efflux1-4. With red cell ghosts Glynn and Karlish5 showed that this Na+ efflux is accompanied by ATP hydrolysis and that extracellular sodium ions, at low concentrations, inhibit this efflux as well as the associated ATP hydrolysis. At higher concentrations, extracellular sodium ions restore the hydrolysis of ATP3,6 but it is not known whether there is an associated increase in Na+ efflux and, perhaps, an influx. To answer this question we have used inside-out red cell membrane vesicles which are specially suitable for controlling the composition of the medium at the two membrane surfaces while measuring 22Na+ fluxes in both directions. We report here that the sodium pump can operate in a mode in which influx and efflux of sodium are associated with ATP hydrolysis. This mode is different from the Na-Na exchange described by Garrahan and Glynn7, and Glynn and Hoffman8, which requires ADP as well as ATP9 and is probably associated with ADP-ATP exchage rather than ATP hydrolysis10,11.     

Regulation and deregulation of cardiac Na(+)-Ca2+ exchange in giant excised sarcolemmal membrane patches

A plasmalemmal Na(+)-Ca2+ exchange mechanism is an important electrogenic determinant of contractility in cardiac cells. As in other cell types, calcium influx by Na(+)-Ca2+ exchange is secondarily activated by cytoplasmic calcium and probably ATP, but these modulatory mechanisms are either absent or altered in isolated cardiac sarcolemmal vesicles. Involvement of a calcium-dependent protein kinase in exchange regulation has been suggested but not verified. Here I describe measurements of outward Na(+)-Ca2+ exchange current, corresponding to calcium influx, in giant excised sarcolemmal patches from guinea pig myocytes. The exchange current is stimulated by both calcium and Mg-ATP from the cytoplasmic face, evidently through separate mechanisms. Activation by cytoplasmic calcium takes place within seconds, is reversible, and does not require ATP. Stimulation by Mg-ATP reverses only slowly over greater than 10 min, or not at all. Unexpectedly, a substantial decrease in exchange current occurs during activation by cytoplasmic sodium, which seems to reflect an inactivation process rather than ion concentration changes or a 'first pass' exchange cycle. This apparent inactivation, and the modulations by cytoplasmic calcium and Mg-ATP, are all abolished by brief treatment of the cytoplasmic surface with chymotrypsin, leaving the exchanger in a maintained state of high activity. Therefore, limited proteolysis deregulates Na(+)-Ca2+ exchange and could contribute to the loss of secondary regulation of the exchange in isolated sarcolemmal vesicles.     

Mechanism of ion permeation through calcium channels

Calcium channels carry out vital functions in a wide variety of excitable cells but they also face special challenges. In the medium outside the channel, Ca2+ ions are vastly outnumbered by other ions. Thus, the calcium channel must be extremely selective if it is to allow Ca2+ influx rather than a general cation influx. In fact, calcium channels show a much greater selectivity for Ca2+ than sodium channels do for Na+ despite the high flux that open Ca channels can support. Relatively little is known about the mechanism of ion permeation through Ca channels. Earlier models assumed ion independence or single-ion occupancy. Here we present evidence for a novel hypothesis of ion movement through Ca channels, based on measurements of Ca channel activity at the level of single cells or single channels. Our results indicate that under physiological conditions, the channel is occupied almost continually by one or more Ca2+ ions which, by electrostatic repulsion, guard the channel against permeation by other ions. On the other hand, repulsion between Ca2+ ions allows high throughput rates and tends to prevent saturation with calcium.     

Functional coordination of intraflagellar transport motors

Cilia have diverse roles in motility and sensory reception, and defects in cilia function contribute to ciliary diseases such as Bardet-Biedl syndrome (BBS). Intraflagellar transport (IFT) motors assemble and maintain cilia by transporting ciliary precursors, bound to protein complexes called IFT particles, from the base of the cilium to their site of incorporation at the distal tip. In Caenorhabditis elegans, this is accomplished by two IFT motors, kinesin-II and osmotic avoidance defective (OSM)-3 kinesin, which cooperate to form two sequential anterograde IFT pathways that build distinct parts of cilia. By observing the movement of fluorescent IFT motors and IFT particles along the cilia of numerous ciliary mutants, we identified three genes whose protein products mediate the functional coordination of these motors. The BBS proteins BBS-7 and BBS-8 are required to stabilize complexes of IFT particles containing both of the IFT motors, because IFT particles in bbs-7 and bbs-8 mutants break down into two subcomplexes, IFT-A and IFT-B, which are moved separately by kinesin-II and OSM-3 kinesin, respectively. A conserved ciliary protein, DYF-1, is specifically required for OSM-3 kinesin to dock onto and move IFT particles, because OSM-3 kinesin is inactive and intact IFT particles are moved by kinesin-II alone in dyf-1 mutants. These findings implicate BBS ciliary disease proteins and an OSM-3 kinesin activator in the formation of two IFT pathways that build functional cilia.     

Crystal structure of the sodium-potassium pump

The Na+,K+-ATPase generates electrochemical gradients for sodium and potassium that are vital to animal cells, exchanging three sodium ions for two potassium ions across the plasma membrane during each cycle of ATP hydrolysis. Here we present the X-ray crystal structure at 3.5 A resolution of the pig renal Na+,K+-ATPase with two rubidium ions bound (as potassium congeners) in an occluded state in the transmembrane part of the alpha-subunit. Several of the residues forming the cavity for rubidium/potassium occlusion in the Na+,K+-ATPase are homologous to those binding calcium in the Ca2+-ATPase of sarco(endo)plasmic reticulum. The beta- and gamma-subunits specific to the Na+,K+-ATPase are associated with transmembrane helices alphaM7/alphaM10 and alphaM9, respectively. The gamma-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the alpha-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.     

Effects of ATP and vanadate on calcium efflux from barnacle muscle fibres

Calcium ions carry the inward current during depolarization of barnacle muscle fibres and are involved in the contraction process. Intracellular ionized calcium ([Ca2+]i) in barnacle muscle, as in other cells, is kept at a very low concentration, against a large electrochemical gradient. This large gradient is maintained by Ca2+ extrusion mechanisms. When [Ca2+]i is below the contraction threshold, Ca2+ efflux from giant barnacle muscle fibres is, largely, both ATP dependent and external Na+ (Na+0) dependent (see also refs 5,6). When [Ca2+]i is raised to the level expected during muscle contraction (2-5 muM), most of the Ca2+ efflux from perfused fibres is Na0 dependent; as in squid axons, this Na+0-dependent Ca2+ efflux is ATP independent. Orthovanadate is an inhibitor of (Na+ + K+) ATPase and the red cell Ca2+-ATpase. We report here that vanadate inhibits ATP-promoted, Na+0-dependent Ca2+ efflux from barnacle muscle fibres perfused with low [Ca2+]i (0.2-0.5 microM), but has little effect on the Na+0-dependent, ATP-independent Ca2+ efflux from fibres with a high [Ca]i (2-5 microM). Nevertheless, ATP depletion or vanadate treatment of high [Ca2+]i fibres causes an approximately 50-fold increase of Ca2+ efflux into Ca2+-containing lithium seawater. These results demonstrate that both vanadate and ATP affect Ca2+ extrusion, including the Na+0-dependent Ca2+ efflux (Na-Ca exchange), in barnacle muscle.     

Effect of ions on the light-sensitive current in retinal rods

The effect of ions on the light-sensitive current of retinal rods was studied by sucking the inner segment into a tightly fitting capillary with the outer segment projecting into a flowing solution. This new method showed that the light-sensitive pathway, in which Na+ is the normal carrier of current, has an ionic selectivity different from that of other known sodium channels. Externàl calcium has a striking effect on the current, which increased about 20-fold when all calcium was removed. Reducing the sodium concentration gradient greatly prolonged the response to a flash of light, as would be expected if internal calcium blocks sodium channels and if light releases calcium which is subsequently extruded by a sodium-calcium exchange mechanism.     

External Na dependence of ouabain-sensitive ATP:ADP exchange initiated by photolysis of intracellular caged-ATP in human red cell ghosts

Coupled active transport of Na+ and K+ across cellular plasma membranes is mediated by (Na+ + K+)-stimulated Mg2+-dependent ATPase. Active cation transport by this Na pump involves a cyclic Na-dependent phosphorylation of the enzyme by intracellular ATP and hydrolytic dephosphorylation of the phosphoenzyme, stimulated by K+ (ref. 1). In human red blood cells, skeletal muscle and squid axons, replacement of extracellular K by Na results in a ouabain-sensitive efflux of Na coupled to an influx of extracellular Na. There is apparently no net Na movement nor net hydrolysis of ATP. The rate of Na:Na exchange is stimulated by increased levels of ADP and exchange transport is not observed in cells totally depleted of intracellular ATP. These characteristics suggest that the biochemical mechanism underlying the Na exchange mode of the Na pump involves phosphorylation of the enzyme by ATP (which requires intracellular Na) followed by its dephosphorylation by ADP. Such a reaction has been observed in partially purified (Na+ + K+) ATPase from a variety of sources and its dependence on Na concentration has been described (although not previously for the red cell enzyme). In the present work, intracellular ATP:ADP exchange reaction was initiated by photoreleased ATP following brief irradiation at 350 nm of ghosts containing caged-ATP. The ouabain-sensitive component of the ensuing ATP:ADP exchange reaction shows a biphasic response to extracellular Na. External Na in the range 0--10 mM has an inhibitory effect whilst increasing concentrations beyond this range stimulate the rate of exchange in a roughly linear fashion up to 100 mM Na. These results represent the first direct demonstration of the sidedness of the effects of Na on this partial sequence in the overall enzyme cycle and bear a qualitative resemblance to the Na effects on the Na-ATPase which occur in the absence of intracellular ADP in human red blood cells.     

Vanadate inhibits uncoupled Ca efflux but not Na--Ca exchange in squid axons.

Nerve cells can maintain a very low intracellular calcium concentration ([Ca2+]i) against large Ca2+ electrochemical gradients (see ref. 1 for review). The properties of the calcium efflux from these cells depend on [Ca2+]i (ref. 2), and within the physiological range, most Ca efflux depends on ATP (which stimulates with high affinity) and is insensitive to Na1, Na0 and Ca0 (uncoupled Ca efflux). When the [Ca2+]i is well above the physiological range, Ca efflux becomes only partially dependent on ATP (acting now with low affinity), is inhibited by Nai and is stimulated by Na0 and Ca0 (Na--Ca exchange). Orthovanadate, a powerful inhibitor of the (Na+ + K+)ATPase and the Na pump, also inhibits the Ca-stimulated ATPase activity, which is the enzymatic basis for the uncoupled Ca pump, in human red cells. The experiments reported here show that in squid axons the ATP-dependent uncoupled Ca efflux can be fully and reversibly inhibited by vanadate, whereas concentrations of vanadate 10 times higher have no effect on the Na--Ca exchange. This is another indication that the uncoupled Ca efflux represents an ATP-driven Ca pump, and supports the suggestion that the uncoupled Ca efflux and Na--Ca exchange are mediated by different mechanisms.     

钙离子与钠离子对浆料Zeta电位的影响

用氯化钙和氯化钠调节添加了不同化学助剂的去金属离子浆料电导率,用德国mütek 公司SZP-04型Zeta电位仪检测对比浆料Zeta电位的变化情况,探讨了Na+和Ca2+对浆料Zeta电位的不同影响,并应用Minitab软件对实验测得的结果进行量化分析。结果表明：无机盐离子使浆料Zeta电位绝对值下降,Ca2+对浆料Zeta电位绝对值的降低作用高于Na+。浆料Zeta电位（ζ）随浆料电导率（σ）以及浆料初始Zeta电位值（ζ0）的变化符合数学模型：ζ=-0.388+0988ζ0+0.202lnσ-ζ00.091lnσ（氯化钙调节电导率时）, ζ=-0.806+ 0.960ζ0-0.316lnσ-ζ00.102lnσ（氯化钠调节电导率时）。浆料Zeta电位在等电点附近时,CPAM的助留助滤性能最佳。与Na+相比,Ca2+更容易使造纸系统的Zeta电位值过高甚至达到正值,从而影响浆料的性能。     

Motility and mechanosensitivity of macrocilia in the ctenophore Bero?

Mechanical activation of the microtubule sliding mechanism in cilia and flagella by local passive bending has been postulated to be essential for the initiation and propagation of bending waves along the axoneme. In addition, responsiveness of cilia to hydrodynamic forces imposed externally by their neighbours is thought to be responsible for metachronal coordination of ciliary activity, as well as for synchronal beating of component cilia within compound ciliary organelles. Direct tests of the mechanosensitivity of motile cilia are limited, but generally support these views. It remains problematical, however, whether mechanical interaction between cilia operates continuously during both the effective and recovery phases of the asymmetrical beat cycle. Moreover, the directional sensitivity and temporal responsiveness of motile cilia to mechanical stimuli have been explored in only a few cases. Finally, the continuous nature of the ciliary beat cycle has hindered investigation of the 'switch point hypothesis' in which doublet sliding is assumed to be activated sequentially on the two halves of the axoneme to produce bends in opposite directions. Here we report that macrocilia on the ctenophore Bero? beat discontinuously with separate effective and recovery strokes, resulting in 'split-cycle' waves of metachronal coordination. This new pattern of ciliary beating is used to investigate the motile responses of cilia to controlled mechanical stimuli during each phase of the beat cycle.     

A positive feedback mechanism governs the polarity and motion of motile cilia

Ciliated epithelia produce fluid flow in many organ systems, ranging from the respiratory tract where it clears mucus to the ventricles of the brain where it transports cerebrospinal fluid. Human diseases that disable ciliary flow, such as primary ciliary dyskinesia, can compromise organ function or the ability to resist pathogens, resulting in recurring respiratory infections, otitis, hydrocephaly and infertility. To create a ciliary flow, the cilia within each cell need to be polarized coordinately along the planar axis of the epithelium, but how polarity is established in any ciliated epithelia is not known. Here we analyse the developmental mechanisms that polarize cilia, using the ciliated cells in the developing Xenopus larval skin as a model system. We show that cilia acquire polarity through a sequence of events, beginning with a polar bias set by tissue patterning, followed by a refinement phase. Our results indicate that during refinement, fluid flow is both necessary and sufficient in determining cilia polarity. These findings reveal a novel mechanism in which tissue patterning coupled with fluid flow act in a positive feedback loop to direct the planar polarity of cilia.     

Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium

Chloride (Cl-) secretion by the airway epithelium regulates, in part, the quantity and composition of the respiratory tract fluid, thereby facilitating mucociliary clearance. The rate of Cl- secretion is controlled by apical membrane Cl- channels. Apical Cl- channels are opened and Cl- secretion is stimulated by a variety of hormones and neurotransmitters that increase intracellular levels of cyclic AMP (cAMP). In cystic fibrosis (CF), a common lethal genetic disease of Caucasians, airway, sweat-gland duct, secretory-coil and possibly other epithelia are anion impermeable. This abnormality may explain several of the clinical manifestations of the disease. The Cl- impermeability in CF-airway epithelia has been localized to the apical cell membrane, where regulation of Cl- channels is abnormal: hormonal secretagogues stimulate cAMP accumulation appropriately but Cl- channels fail to open. Here we report that the purified catalytic subunit of cAMP-dependent protein kinase plus ATP opens Cl- channels in excised, cell-free patches of membrane from normal cells, but fails to open Cl- channels in CF cells. These results indicate that in normal cells, the cAMP-dependent protein kinase phosphorylates the Cl- channel or an associated regulatory protein, causing the channel to open. The failure of CF Cl- channels to open suggests a defect either in the channel or in such an associated regulatory protein.     

带相反电荷无机纳米片胶体稳定存在的溶剂

为了探讨采用不同溶剂所获得的带正、负电荷无机纳米片均能以稳定胶体状态存在的溶剂,用熔融盐法和均匀沉淀法分别合成带负电及正电层板的层状化合物birnessite型锰酸钠和CoAl层状双金属氢氧化物LDH,并对它们进行离子交换、剥离而制得在四甲基胺离子水溶液和甲酰胺中稳定存在的MnO2和CoAl-LDH纳米片.研究发现,甲酰胺可作为此2种纳米片胶体稳定存在的溶剂.     

Vertebrate Smoothened functions at the primary cilium

The unanticipated involvement of several intraflagellar transport proteins in the mammalian Hedgehog (Hh) pathway has hinted at a functional connection between cilia and Hh signal transduction. Here we show that mammalian Smoothened (Smo), a seven-transmembrane protein essential for Hh signalling, is expressed on the primary cilium. This ciliary expression is regulated by Hh pathway activity; Sonic hedgehog or activating mutations in Smo promote ciliary localization, whereas the Smo antagonist cyclopamine inhibits ciliary localization. The translocation of Smo to primary cilia depends upon a conserved hydrophobic and basic residue sequence homologous to a domain previously shown to be required for the ciliary localization of seven-transmembrane proteins in Caenorhabditis elegans. Mutation of this domain not only prevents ciliary localization but also eliminates Smo activity both in cultured cells and in zebrafish embryos. Thus, Hh-dependent translocation to cilia is essential for Smo activity, suggesting that Smo acts at the primary cilium.     

A cyclic nucleotide-gated conductance in olfactory receptor cilia

Olfactory transduction is thought to be initiated by the binding of odorants to specific receptor proteins in the cilia of olfactory receptor cells. The mechanism by which odorant binding could initiate membrane depolarization is unknown, but the recent discovery of an odorant-stimulated adenylate cyclase in purified olfactory cilia suggests that cyclic AMP may serve as an intracellular messenger for olfactory transduction. If so, then there might be a conductance in the ciliary plasma membrane which is controlled by cAMP. Here we report that excised patches of ciliary plasma membrane, obtained from dissociated receptor cells, contain a conductance which is gated directly by cAMP. This conductance resembles the cyclic GMP-gated conductance that mediates phototransduction in rod and cone outer segments, but differs in that it is activated by both cAMP and cGMP. Our data provide a mechanistic basis by which an odorant-stimulated increase in cyclic nucleotide concentration could lead to an increase in membrane conductance and therefore, to membrane depolarization. These data suggest a remarkable similarity between the mechanisms of olfactory and visual transduction and indicate considerable conservation of sensory transduction mechanisms.     

Correlation between the ATPase and microtubule translocating activities of sea urchin egg kinesin

Coupling between ATP hydrolysis and microtubule movement was demonstrated several years ago in flagellar axonemes and subsequent studies suggest that the relevant microtubule motor, dynein, uses ATP to drive microtubule sliding by a cross-bridge mechanism analogous to that of myosin in muscles. Kinesin, a microtubule-based motility protein which may participate in organelle transport and mitosis, binds microtubules in a nucleotide-sensitive manner, and requires hydrolysable nucleotides to translocate microtubules over a glass surface. Recently, neuronal kinesin was shown to possess microtubule-activated ATPase activity although coupling between ATP hydrolysis and motility was not demonstrated. Here we report that sea urchin egg kinesin, prepared either with or without a 5'-adenylyl imidodiphosphate(AMPPNP)-induced microtubule binding step, also possesses significant microtubule-activated ATPase activity when Mg-ATP is used as a substrate. This ATPase activity is inhibited in a dose-dependent manner by addition of Mg-free ATP, by chelation of Mg2+ with EDTA, by addition of Na3VO4, or by addition of AMPPNP with or without Mg2+. Addition of these same reagents also inhibits the microtubule-translocating activities of sea urchin egg kinesin in a dose-dependent manner, supporting the hypothesis that kinesin-driven motility is coupled to the microtubule-activated Mg2+-ATPase activity.     

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.     

脱果胶试剂在全棉秆化机浆漂白中的应用

通过原子吸收分光光度计(FAAS)对原料中金属离子含量进行定量分析.以果胶酸钙为模型物,考察氢氧化钠、草酸钠等脱果胶试剂对棉秆皮部的果胶脱除及化学成分的影响,进而探讨改进全棉秆化机浆成浆性能的方法.实验结果表明:各脱果胶试剂与果胶酸钙模型物反应速率的关系为2 2 43 4Na C O NaOHNa POv>v>v;用3%草酸钠进行皮部预处理,棉秆皮部果胶脱除率可达到53.72%.对全棉秆化机浆漂白结果显示,当温度90,℃、草酸钠用量3%的预处理条件下,以6.5%NaOH和11%H2O2制得全棉秆化机浆白度可以达到76.2%,尘埃度可降至50.10,mm2/m2.     

Ca2+/calmodulin binds to and modulates P/Q-type calcium channels.

Neurotransmitter release at many central synapses is initiated by an influx of calcium ions through P/Q-type calcium channels, which are densely localized in nerve terminals. Because neurotransmitter release is proportional to the fourth power of calcium concentration, regulation of its entry can profoundly influence neurotransmission. N- and P/Q-type calcium channels are inhibited by G proteins, and recent evidence indicates feedback regulation of P/Q-type channels by calcium. Although calcium-dependent inactivation of L-type channels is well documented, little is known about how calcium modulates P/Q-type channels. Here we report a calcium-dependent interaction between calmodulin and a novel site in the carboxy-terminal domain of the alpha1A subunit of P/Q-type channels. In the presence of low concentrations of intracellular calcium chelators, calcium influx through P/Q-type channels enhances channel inactivation, increases recovery from inactivation and produces a long-lasting facilitation of the calcium current. These effects are prevented by overexpression of a calmodulin-binding inhibitor peptide and by deletion of the calmodulin-binding domain. Our results reveal an unexpected association of Ca2+/calmodulin with P/Q-type calcium channels that may contribute to calcium-dependent synaptic plasticity.