A proton pore in a potassium channel voltage sensor reveals a focused electric field

Voltage-dependent potassium channels are essential for the generation of nerve impulses. Voltage sensitivity is conferred by charged residues located mainly in the fourth transmembrane segment (S4) of each of the four identical subunits that make up the channel. These charged segments relocate when the potential difference across the membrane changes, controlling the ability of the pore to conduct ions. In the crystal structure of the Aeropyrum pernix potassium channel KvAP, the S4 and part of the third (S3B) transmembrane alpha-helices are connected by a hairpin turn in an arrangement termed the 'voltage-sensor paddle'. This structure was proposed to move through the lipid bilayer during channel activation, transporting positive charges across a large fraction of the membrane. Here we show that replacing the first S4 arginine by histidine in the Shaker potassium channel creates a proton pore when the cell is hyperpolarized. Formation of this pore does not support the paddle model, as protons would not have access to a lipid-buried histidine. We conclude that, at hyperpolarized potentials, water and protons from the internal and external solutions must be separated by a narrow barrier in the channel protein that focuses the electric field to a small voltage-sensitive region.     

Calcium channel characteristics conferred on the sodium channel by single mutations.

The sodium channel, one of the family of structurally homologous voltage-gated ion channels, differs from other members, such as the calcium and the potassium channels, in its high selectivity for Na+. This selectivity presumably reflects a distinct structure of its ion-conducting pore. We have recently identified two clusters of predominantly negatively charged amino-acid residues, located at equivalent positions in the four internal repeats of the sodium channel as the main determinants of sensitivity to the blockers tetrodotoxin and saxitoxin. All site-directed mutations reducing net negative charge at these positions also caused a marked decrease in single-channel conductance. Thus these two amino-acid clusters probably form part of the extracellular mouth and/or the pore wall of the sodium channel. We report here the effects on ion selectivity of replacing lysine at position 1,422 in repeat III and/or alanine at position 1,714 in repeat IV of rat sodium channel II (ref. 3), each located in one of the two clusters, by glutamic acid, which occurs at the equivalent positions in calcium channels. These amino-acid substitutions, unlike other substitutions in the adjacent regions, alter ion-selection properties of the sodium channel to resemble those of calcium channels. This result indicates that lysine 1,422 and alanine 1,714 are critical in determining the ion selectivity of the sodium channel, suggesting that these residues constitute part of the selectivity filter of the channel.     

Cloning of a probable potassium channel gene from mouse brain

Potassium channels comprise a diverse class of ion channels important for neuronal excitability and plasticity. The recent cloning of the Shaker locus from Drosophila melanogaster has provided a starting point for molecular studies of potassium channels. Predicted Shaker proteins appear to be integral membrane proteins and have a sequence similar to the sequence of the S4 segment of the vertebrate sodium channel, where the S4 segment has been proposed to be the voltage sensor. Expression studies in frog oocytes confirm that Shaker encodes a component of a potassium channel (the A channel) that conducts a fast transient potassium current. Here we report the isolation of complementary DNA clones from the mouse brain, the nucleotide sequences of which predict a protein remarkably similar to the Shaker protein. The strong conservation of the predicted protein sequence in flies and mammals suggests that these mouse clones encode a potassium channel component and that the conserved amino acids may be essential to some aspect of potassium channel function.     

Calcium activation of BK(Ca) potassium channels lacking the calcium bowl and RCK domains

In many physiological systems such as neurotransmitter release, smooth muscle relaxation and frequency tuning of auditory hair cells, large-conductance calcium-activated potassium (BK(Ca)) channels create a connection between calcium signalling pathways and membrane excitability. BK(Ca) channels are activated by voltage and by micromolar concentrations of intracellular calcium. Although it is possible to open BK(Ca) channels in the absence of calcium, calcium binding is essential for their activation under physiological conditions. In the presence of intracellular calcium, BK(Ca) channels open at more negative membrane potentials. Many experiments investigating the molecular mechanism of calcium activation of the BK(Ca) channel have focused on the large intracellular carboxy terminus, and much evidence supports the hypothesis that calcium-binding sites are located in this region of the channel. Here we show that BK(Ca) channels that lack the whole intracellular C terminus retain wild-type calcium sensitivity. These results show that the intracellular C terminus, including the 'calcium bowl' and the RCK domain, is not necessary for the calcium-activated opening of these channels.     

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.     

Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence.

Voltage-dependent potassium, sodium and calcium ion channels may share a common mechanism of activation, in which the conserved S4 sequence acts as the primary voltage sensor. Site-directed mutagenesis of the S4 sequence of the Shaker potassium channel and electrophysiological analysis suggest that voltage-dependent activation involves the S4 sequence but is not solely due to electrostatic interactions.     

Polycystin-L is a calcium-regulated cation channel permeable to calcium ions.

Polycystic kidney diseases are genetic disorders in which the renal parenchyma is progressively replaced by fluid-filled cysts. Two members of the polycystin family (polycystin-1 and -2) are mutated in autosomal dominant polycystic kidney disease (ADPKD), and polycystin-L is deleted in mice with renal and retinal defects. Polycystins are membrane proteins that share significant sequence homology, especially polycystin-2 and -L (50% identity and 71% similarity). The functions of the polycystins remain unknown. Here we show that polycystin-L is a calcium-modulated nonselective cation channel that is permeable to sodium, potassium and calcium ions. Patch-clamp experiments revealed single-channel activity with a unitary conductance of 137 pS. Channel activity was substantially increased when either the extracellular or intracellular calcium-ion concentration was raised, indicating that polycystin-L may act as a transducer of calcium-mediated signalling in vivo. Its large single-channel conductance and regulation by calcium ions distinguish it from other structurally related cation channels.     

A biological role for prokaryotic ClC chloride channels

An unexpected finding emerging from large-scale genome analyses is that prokaryotes express ion channels belonging to molecular families long studied in neurons. Bacteria and archaea are now known to carry genes for potassium channels of the voltage-gated, inward rectifier and calcium-activated classes, ClC-type chloride channels, an ionotropic glutamate receptor and a sodium channel. For two potassium channels and a chloride channel, these homologues have provided a means to direct structure determination. And yet the purposes of these ion channels in bacteria are unknown. Strong conservation of functionally important sequences from bacteria to vertebrates, and of structure itself, suggests that prokaryotes use ion channels in roles more adaptive than providing high-quality protein to structural biologists. Here we show that Escherichia coli uses chloride channels of the widespread ClC family in the extreme acid resistance response. We propose that the channels function as an electrical shunt for an outwardly directed virtual proton pump that is linked to amino acid decarboxylation.     

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.     

Anion channels activated by adrenaline in cardiac myocytes

In heart cells, the catecholamine-activated cyclic AMP system regulates calcium and potassium channels. We report here a novel class of chloride channels that can be activated by adrenaline in mammalian ventricular cells. Like the agonist-activated Cl- channel currents of airway and colonic epithelial cells, the cardiac Cl(-)-channel current shows outward rectification. But the unit conductance of cardiac Cl- channels is smaller than that of epithelial Cl- channels. The cardiac Cl- channel is functionally voltage-independent, in contrast to the Cl- channel in colonic epithelial cells. This channel could be responsible for the beta-catecholamine-induced increase in cardiac membrane conductance that has been attributed to activation of a Cl- current. Thus, sympathetic control of cardiac electrical activity involves not only the voltage-dependent, excitation-related cation channels, but also anion channels that generate a steady current.     

Endfeet of retinal glial cells have higher densities of ion channels that mediate K+ buffering

A major function of glial cells in the central nervous system is to buffer the extracellular potassium concentration, [K+]o. A local rise in [K+]o causes potassium ions to enter glial cells, which have membranes that are highly permeable to K+; potassium then leaves the glial cells at other locations where [K+]o has not risen. We report here the first study of the individual ion channels mediating potassium buffering by glial cells. The patch-clamp technique was employed to record single channel currents in Müller cells, the radial glia of the vertebrate retina. Those cells have 94% of their potassium conductance in an endfoot apposed to the vitreous humour, causing K+ released from active retinal neurones to be buffered preferentially to the vitreous. Recordings from patches of endfoot and cell body membrane show that a single type of inward-rectifying K+ channel mediates potassium buffering at both cell locations. The non-uniform density of K+ conductance is due to a non-uniform distribution of one type of K+ channel, rather than to the cell expressing high conductance channels at the endfoot and low conductance channels elsewhere on the cell.     

海葵多肽类神经毒素的结构与药理学功能

海葵以其触手刺细胞中的毒液行使捕食和防御功能,其毒液中富含各种多肽类神经毒素,分子量为3￣7kDa之间,分子序列中含多对二硫键以稳定其结构。海葵神经毒素以钠离子通道毒素和钾离子通道毒素为其主要成分,此外还发现有作用于其他离子通道的成分,此外,还有部分海葵毒素目前尚不清楚其分子靶标。不同类型的海葵毒素具有不同的空间结构。海葵毒素多肽的分子多样性使其成为动物毒素研究的一个重要分支,同时海葵多肽毒素对不同离子通道的特异性和高亲和性,使得它们成为神经生理学和药理学研究的一种重要工具。     

Ankyrin and spectrin associate with voltage-dependent sodium channels in brain

The segregation of voltage-dependent sodium channels to specialized regions of the neuron is crucial for propagation of an action potential. Studies of their lateral mobility indicate that sodium channels are freely mobile on the neuronal cell body but are immobile at the axon hillock, presynaptic terminal and at focal points along the axon. To elucidate the mechanisms that regulate sodium channel topography and mobility, we searched for specific proteins from the brain that associate with sodium channels. Here we show that sodium channels labelled with 3H-saxitoxin (STX) are precipitated in the presence of exogenous brain ankyrin by anti-ankyrin antibodies and that 125I-labelled ankyrin binds with high affinity to sodium channels reconstituted into lipid vesicles. The cytoplasmic domain of the erythrocyte anion transporter competes for the latter interaction. Neither the neuronal GABA (gamma-aminobutyric acid) receptor channel complex nor the dihydropyridine (DHP) receptor bind brain ankyrin. The results indicate that brain ankyrin links the voltage-dependent sodium channel to the underlying cytoskeleton and may help to maintain axolemmal membrane heterogeneity and control sodium channel mobility.     

Portability of paddle motif function and pharmacology in voltage sensors

Voltage-sensing domains enable membrane proteins to sense and react to changes in membrane voltage. Although identifiable S1-S4 voltage-sensing domains are found in an array of conventional ion channels and in other membrane proteins that lack pore domains, the extent to which their voltage-sensing mechanisms are conserved is unknown. Here we show that the voltage-sensor paddle, a motif composed of S3b and S4 helices, can drive channel opening with membrane depolarization when transplanted from an archaebacterial voltage-activated potassium channel (KvAP) or voltage-sensing domain proteins (Hv1 and Ci-VSP) into eukaryotic voltage-activated potassium channels. Tarantula toxins that partition into membranes can interact with these paddle motifs at the protein-lipid interface and similarly perturb voltage-sensor activation in both ion channels and proteins with a voltage-sensing domain. Our results show that paddle motifs are modular, that their functions are conserved in voltage sensors, and that they move in the relatively unconstrained environment of the lipid membrane. The widespread targeting of voltage-sensor paddles by toxins demonstrates that this modular structural motif is an important pharmacological target.     

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.     

Noradrenaline contracts arteries by activating voltage-dependent calcium channels

Noradrenaline (NA) regulates arterial smooth muscle tone and hence blood vessel diameter and blood flow. NA apparently increases tone by causing a calcium influx through the cell membrane. Two calcium influx pathways have been proposed: voltage-activated calcium channels and NA-activated calcium-permeable channels that are voltage-insensitive. Although voltage-activated calcium channels have been identified in arterial smooth muscle, voltage-insensitive calcium channels activated by NA have not. We show here that NA contractions of rabbit mesenteric arteries increase with depolarization. The increase parallels the elevation of open-state probability (P0) of single, voltage-dependent calcium channels. The action of noradrenaline can be explained by NA-activating voltage-dependent calcium channels, rather than by opening a second type of channel. We show directly that NA increases the open-state probability of single calcium channels. Thus, in the presence of NA, calcium entry through voltage-dependent calcium channels can regulate smooth muscle tone at physiological membrane potentials. These results may have relevance to pathophysiological conditions such as hypertension.     

Blocker protection in the pore of a voltage-gated K+ channel and its structural implications

The structure of the bacterial potassium channel KcsA has provided a framework for understanding the related voltage-gated potassium channels (Kv channels) that are used for signalling in neurons. Opening and closing of these Kv channels (gating) occurs at the intracellular entrance to the pore, and this is also the site at which many open channel blockers affect Kv channels. To learn more about the sites of blocker binding and about the structure of the open Kv channel, we investigated here the ability of blockers to protect against chemical modification of cysteines introduced at sites in transmembrane segment S6, which contributes to the intracellular entrance. Within the intracellular half of S6 we found an abrupt cessation of protection for both large and small blockers that is inconsistent with the narrow 'inner pore' seen in the KcsA structure. These and other results are most readily explained by supposing that the structure of Kv channels differs from that of the non-voltage-gated bacterial channel by the introduction of a sharp bend in the inner (S6) helices. This bend would occur at a Pro-X-Pro sequence that is highly conserved in Kv channels, near the site of activation gating.     

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.     

蝎昆虫神经毒素及其基因研究进展

蝎昆虫神经毒素是一类对昆虫有专一性麻痹致死作用，而对哺乳动物无害或毒性很小的蛋白。因此分离和表达蝎昆虫神经毒素基因，有利于开发研究新型高效生物杀虫剂，并为把该基因导入植物培育新抗虫品种提供必要的基础。另外，此类蝎昆虫神经毒素还有利于研究电压依从性离子通道结构与功能的相互关系。对蝎昆虫神经毒素的研究已成为国内外生物科学工作者关注的课题之一。本文综述了近几年来蝎昆虫神经毒素及其基因的研究进展概况。     

Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2

Voltage-gated calcium channels (VGCCs) conduct calcium into cells after membrane depolarization and are vital for diverse biological events. They are regulated by various signalling pathways, which has profound functional consequences. The activity of VGCCs decreases with time in whole-cell and inside-out patch-clamp recordings. This rundown reflects persistent intrinsic modulation of VGCCs in intact cells. Although several mechanisms have been reported to contribute to rundown of L-type channels, the mechanism of rundown of other types of VGCC is poorly understood. Here we show that phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), an essential regulator of ion channels and transporters, is crucial for maintaining the activity of P/Q- and N-type channels. Activation of membrane receptors that stimulate hydrolysis of PtdIns(4,5)P2 causes channel inhibition in oocytes and neurons. PtdIns(4,5)P2 also inhibits P/Q-type channels by altering the voltage dependence of channel activation and making the channels more difficult to open. This inhibition is alleviated by phosphorylation by protein kinase A. The dual actions of PtdIns(4,5)P2 and the crosstalk between PtdIns(4,5)P2 and protein kinase A set up a dynamic mechanism through which the activity of VGCCs can be finely tuned by various neurotransmitters, hormones and trophic factors.