Hierarchy of Motions and Quasi-Particles in a Simplified Model of Potassium Channel Selectivity Filter

We develop a simplified model of themultiply occupied Kcsa-like selectivityfilter based on the best availablestructural data. The existence of hierarchyof motions in the selectivity filter isshown. Fast fluctuations of the ion-iondistances may be considered adiabaticallydecoupled from the slow diffusive motion ofthe ions' center of masses. The latter canbe considered as a quasi-particle, called aquasi-ion, moving in an effectivepotential. In the Kcsa-like selectivityfilter occupied by three ions the effectivepotential allows free barrier-lessdiffusional motion of the quasi-ions. Theconcept of the quasi-ions performing iontranslocation through the channel may bevital in explaining barrier-less `knock-on' conduction postulated for real channels.     

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals'' access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal''s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal''s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.     

农作物钾离子通道基因的生物信息学分析

以AtAKT1所编码的氨基酸序列为检索序列,通过对GenBank NR数据库以及EMBL、DDBJ、PDB数据库进行检索,发现水稻、小麦、大麦、玉米、烟草等作物中具有cds全长的34个疑似K~+通道基因.其中:水稻7个,玉米6个,烟草4个,马铃薯、胡萝卜、大麦各3个,葡萄、番茄各2个,蚕豆、小麦、油菜、甜瓜等各1个;有25个基因经过功能验证确定为K~+通道基因.利用比较基因组学,将剩余的9个基因确定为K~+通道基因.对34个K~+基因进化关系的分析表明,与AtAKT1的同源性较高的K~+通道基因为马铃薯的SKT1(相似性71%)、番茄的LKT1(相似性71%)、胡萝卜的DKT1(相似性69%)、烟草的NKT1(相似性62%).     

Identification of the Intracellular Na+ Sensor in Slo2.1 Potassium Channels

Slo2 potassium channels have a very low open probability under normal physiological conditions, but are readily activated in response to an elevated [Na⁺]_i (e.g. during ischemia). An intracellular Na⁺ coordination motif (DX(R/K)XXH) was previously identified in Kir3.2, Kir3.4, Kir5.1, and Slo2.2 channel subunits. Based loosely on this sequence, we identified five potential Na⁺ coordination motifs in the C terminus of the Slo2.1 subunit. The Asp residue in each sequence was substituted with Arg, and single mutant channels were heterologously expressed in Xenopus oocytes. The Na⁺ sensitivity of each of the mutant channels was assessed by voltage clamp of oocytes using micropipettes filled with 2 m NaCl. Wild-type channels and four of the mutant Slo2.1 channels were rapidly activated by leakage of NaCl solution into the cytoplasm. D757R Slo2.1 channels were not activated by NaCl, but were activated by the fenamate niflumic acid, confirming their functional expression. In whole cell voltage clamp recordings of HEK293 cells, wild-type but not D757R Slo2.1 channels were activated by a [NaCl]_i of 70 mm. Thus, a single Asp residue can account for the sensitivity of Slo2.1 channels to intracellular Na⁺. In excised inside-out macropatches of HEK293 cells, activation of wild-type Slo2.1 currents by 3 mm niflumic acid was 14-fold greater than activation achieved by increasing [NaCl]_i from 3 to 100 mm. Thus, relative to fenamates, intracellular Na⁺ is a poor activator of Slo2.1.     

Two new species and a new subgenus of toothed Brachyhypopomus electric knifefishes (Gymnotiformes,Hypopomidae) from the central Amazon and considerations pertaining to the evolution of a monophasic electric organ discharge

We describe two new, closely related species of toothed Brachyhypopomus (Hypopomidae: Gymnotiformes: Teleostei) from the central Amazon basin and create a new subgenus for them. Odontohypopomus, new subgenus of Brachyhypopomus, is diagnosed by (1) small teeth present on premaxillae; (2) medialmost two branchiostegal rays thin with blades oriented more vertically than remaining three rays; (3) background color in life (and to lesser extent in preservation) distinctly yellowish with head and sides peppered with small, widely spaced, very dark brown stellate chromatophores that greatly contrast with light background coloration; (4) a dark blotch or bar of subcutaneous pigment below the eye; (5) electric organ discharge waveform of very long duration (head-positive phase approx. 2 milliseconds or longer, head-negative phase shorter or absent) and slow pulse repetition rate (3–16 Hz). The type species of the new subgenus, Brachyhypopomus (Odontohypopomus) walteri sp. n., is diagnosed by the following additional character states: (1) subcutaneous dark pigment at base of orbit particularly prominent, (2) body semi-translucent and nearly bright yellow background coloration in life, (3) a biphasic electric organ discharge (EOD) waveform of very long duration (between 3.5 and 4 milliseconds at 25° C) with head-positive first phase significantly longer than second head-negative phase in both sexes. Brachyhypopomus (Odontohypopomus) bennetti sp. n. is diagnosed by two character states in addition to those used to diagnose the subgenus Odontohypopomus: (1) a deep electric organ, visible as large semi-transparent area, occupying approximately 14–17% body depth directly posterior to the abdominal cavity in combination with a short, but deep, caudal filament, and (2) a monophasic, head-positive EOD waveform, approximately 2.1 milliseconds in duration in both sexes. These are the only described rhamphichthyoid gymnotiforms with oral teeth, and Brachyhypopomus bennetti is the first Brachyhypopomus reported to have a monophasic (head-positive) EOD waveform. Unlike biphasic species, the waveform of its EOD is largely unaffected by tail damage from predators. Such injuries are common among specimens in our collections. This species’ preference for floating meadow habitat along the major channels of the Amazon River basin may put it at particularly high risk of predation and “tail grazing.”     

Role of the S3-S4 Linker in Shaker Potassium Channel Activation

Structural models of voltage-gated channels suggest that flexibility of the S3-S4 linker region may beimportant in allowing the S4 region to undergo large conformational changes in its putative voltage-sensing function. We report here the initial characterization of 18 mutations in the S3-S4 linker of the Shaker channel, including deletions, insertions, charge changes, substitution of prolines, and chimeras replacing the 25-residue Shakerlinker with 7- or 9-residue sequences from Shab, Shaw, or Shal. As measured in Xenopus oocytes with a two-microelectrode voltage clamp, each mutant construct yielded robust currents. Changes in the voltage dependence of activation were small, with activation voltage shifts of 13 mV or less. Substitution of linkers from the slowly activatingShab and Shaw channels resulted in a three- to fourfold slowing of activation and deactivation. It is concluded thatthe S3-S4 linker is unlikely to participate in a large conformational change during channel activation. The linker,which in some channel subfamilies has highly conserved sequences, may however be a determinant of activationkinetics in potassium channels, as previously has been suggested in the case of calcium channels.     

High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer

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Competing Lipid-Protein and Protein-Protein Interactions Determine Clustering and Gating Patterns in the Potassium Channel from Streptomyces lividans (KcsA)

There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp⁶⁷–Glu⁷¹–Asp⁸⁰ inactivation triad within the channel structure and its bearing on the selectivity filter.     

Biomimetic Sensors: Active Electrolocation of Weakly Electric Fish as a Model for Active Sensing in Technical Systems

Instead of vision, many nocturnal animals use alternative senses for navigation and object detection in their dark environment. For this purpose, weakly electric mormyrid fish employ active electrolocation, during which they discharge a specialized electric organ in their tail which discharges electrical pulses. Each discharge builds up an electrical field around the fish, which is sensed by cutaneous electroreceptor organs that are distributed over most of the body surface of the fish. Nearby objects distort this electrical field and cause a local alteration in current flow in those electroreceptors that are closest to the object. By constantly monitoring responses of its electroreceptor organs, a fish can detect, localize, and identify environmental objects.Inspired by the remarkable capabilities of weakly electric fish in detecting and recognizing objects, we designed technical sensor systems that can solve similar problems of remote object sensing. We applied the principles of active electrolocation to technical systems by building devices that produce electrical current pulses in a conducting medium (water or ionized gases) and simultaneously sense local current density. Depending on the specific task a sensor was designed for devices could (i) detect an object, (ii) localize it in space, (iii) determine its distance, and (iv) measure properties such as material properties, thickness, or material faults. Our systems proved to be relatively insensitive to environmental disturbances such as heat, pressure, or turbidity. They have a wide range of applications including material identification, quality control, non-contact distance measurements, medical applications and many more. Despite their astonishing capacities, our sensors still lag far behind what electric fish are able to achieve during active electrolocation. The understanding of the neural principles governing electric fish sensory physiology and the corresponding optimization of our sensors to solve certain technical tasks therefore remain ongoing goals of our research.     

Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers

Summary A Ca-activated, K-selective channel from plasma membrane of rat skeletal muscle was studied in artificial lipid bilayers formed from either phosphatidylethanolamine (PE) or phosphatidylserine (PS). In PE, the single-channel conductance exhibited a complex dependence on symmetrical K⁺ concentration that could not be described by simple Michaelis-Menten saturation. At low K⁺ concentrations the channel conductance was higher in PS membranes, but approached the same conductance observed in PE above 0.4m KCl. At the same Ca²⁺ concentration and voltage, the probability of channel opening was significantly greater in PS than PE. The differences in the conduction and gating, observed in the two lipids, can be explained by the negative surface charge of PS compared to the neutral PE membrane. Model calculations of the expected concentrations of K⁺ and Ca²⁺ at various distances from a PS membrane surface, using Gouy-Chapman-Stern theory, suggest that the K⁺-conduction and Ca²⁺-activation sites sense a similar fraction of the surface potential, equivalent to the local electrostatic potential at a distance of 9 Å from the surface.     

Src Kinase Is the Connecting Player between Protein Kinase A (PKA) Activation and Hyperpolarization through SLO3 Potassium Channel Regulation in Mouse Sperm

Plasma membrane hyperpolarization is crucial for mammalian sperm to acquire acrosomal responsiveness during capacitation. Among the signaling events leading to mammalian sperm capacitation, the immediate activation of protein kinase A plays a pivotal role, promoting the subsequent stimulation of protein tyrosine phosphorylation that associates with fertilizing capacity. We have shown previously that mice deficient in the tyrosine kinase cSrc are infertile and exhibit improper cauda epididymis development. It is therefore not clear whether lack of sperm functionality is due to problems in epididymal maturation or to the absence of cSrc in sperm. To further address this problem, we investigated the kinetics of cSrc activation using anti-Tyr(P)-416-cSrc antibodies that only recognize active cSrc. Our results provide evidence that cSrc is activated downstream of PKA and that inhibition of its activity blocks the capacitation-induced hyperpolarization of the sperm plasma membrane without blocking the increase in tyrosine phosphorylation that accompanies capacitation. In addition, we show that cSrc inhibition also blocks the agonist-induced acrosome reaction and that this inhibition is overcome by pharmacological hyperpolarization. Considering that capacitation-induced hyperpolarization is mediated by SLO3, we evaluated the action of cSrc inhibitors on the heterologously expressed SLO3 channel. Our results indicate that, similar to SLO1 K⁺ channels, cSrc blockers significantly decreased SLO3-mediated currents. Together, these results are consistent with findings showing that hyperpolarization of the sperm plasma membrane is necessary and sufficient to prepare the sperm for the acrosome reaction and suggest that changes in sperm membrane potential are mediated by cSrc activation.     

Reception and learning of electric fields in bees

Honeybees, like other insects, accumulate electric charge in flight, and when their body parts are moved or rubbed together. We report that bees emit constant and modulated electric fields when flying, landing, walking and during the waggle dance. The electric fields emitted by dancing bees consist of low- and high-frequency components. Both components induce passive antennal movements in stationary bees according to Coulomb''s law. Bees learn both the constant and the modulated electric field components in the context of appetitive proboscis extension response conditioning. Using this paradigm, we identify mechanoreceptors in both joints of the antennae as sensors. Other mechanoreceptors on the bee body are potentially involved but are less sensitive. Using laser vibrometry, we show that the electrically charged flagellum is moved by constant and modulated electric fields and more strongly so if sound and electric fields interact. Recordings from axons of the Johnston organ document its sensitivity to electric field stimuli. Our analyses identify electric fields emanating from the surface charge of bees as stimuli for mechanoreceptors, and as biologically relevant stimuli, which may play a role in social communication.     

Functional Consequences of a Decreased Potassium Affinity in a Potassium Channel Pore : Ion Interactions and C-Type Inactivation

Ions bound near the external mouth of the potassium channel pore impede the C-type inactivation conformational change (Lopez-Barneo, J., T. Hoshi, S. Heinemann, and R. Aldrich. 1993. Receptors Channels. 1:61– 71; Baukrowitz, T., and G. Yellen. 1995. Neuron. 15:951–960). In this study, we present evidence that the occupancy of the C-type inactivation modulatory site by permeant ions is not solely dependent on its intrinsic affinity, but is also a function of the relative affinities of the neighboring sites in the potassium channel pore. The A463C mutation in the S6 region of Shaker decreases the affinity of an internal ion binding site in the pore (Ogielska, E.M., and R.W. Aldrich, 1998). However, we have found that this mutation also decreases the C-type inactivation rate of the channel. Our studies indicate that the C-type inactivation effects observed with substitutions at position A463 most likely result from changes in the pore occupancy of the channel, rather than a change in the C-type inactivation conformational change. We have found that a decrease in the potassium affinity of the internal ion binding site in the pore results in lowered (electrostatic) interactions among ions in the pore and as a result prolongs the time an ion remains bound at the external C-type inactivation site. We also present evidence that the C-type inactivation constriction is quite local and does not involve a general collapse of the selectivity filter. Our data indicate that in A463C potassium can bind within the selectivity filter without interfering with the process of C-type inactivation.     

Pentameric Assembly of Potassium Channel Tetramerization Domain-Containing Protein 5

We report the X-ray crystal structure of human potassium channel tetramerization domain-containing protein 5 (KCTD5), the first member of the family to be so characterized. Four findings were unexpected. First, the structure reveals assemblies of five subunits while tetramers were anticipated; pentameric stoichiometry is observed also in solution by scanning transmission electron microscopy mass analysis and analytical ultracentrifugation. Second, the same BTB (bric-a-brac, tramtrack, broad complex) domain surface mediates the assembly of five KCTD5 and four voltage-gated K ⁺ (Kv) channel subunits; four amino acid differences appear crucial. Third, KCTD5 complexes have well-defined N- and C-terminal modules separated by a flexible linker that swivels by ∼ 30^o; the C-module shows a new fold and is required to bind Golgi reassembly stacking protein 55 with ∼ 1 μM affinity, as judged by surface plasmon resonance and ultracentrifugation. Fourth, despite the homology reflected in its name, KCTD5 does not impact the operation of Kv4.2, Kv3.4, Kv2.1, or Kv1.2 channels.     

Elevated Expression of the G-Protein-Activated Inwardly Rectifying Potassium Channel 2 (GIRK2) in Cerebellar Unipolar Brush Cells of a Down Syndrome Mouse Model

SUMMARY 1. Down syndrome (DS) arises from the presence of three copies of chromosome (Chr.) 21. Fine motor learning deficits found in DS from childhood to adulthood result from expression of extra genes on Chr. 21, however, it remains unclear which if any of these genes are the specific causes of the cognitive and motor dysfunction. DS cerebellum displays morphological abnormalities that likely contribute to the DS motor phenotype.2. The G-protein-activated inwardly rectifying potassium channel subunit 2 (GIRK2) is expressed in cerebellum and can shunt dendritic conductance and attenuate postsynaptic potentials. We have used an interbreeding approach to cross a genetic mouse model of DS (Ts65Dn) with Girk2 knockout mice and examined its relative expression level by quantitative real-time RT-PCR, Western blotting and immunohistochemistry.3. We report here for the first time that GIRK2 is expressed in unipolar brush cells, which are excitatory interneurons of the vestibulocerebellum and dorsal cochlear nucleus. Analysis of disomic-Ts65Dn/Girk2^(+/+/−) and heterozygous-Diploid/Girk2^(+/−) mice shows that GIRK2 expression in Ts65Dn lobule X follows gene dosage. The lobule X of Ts65Dn mice contain greater numbers of unipolar brush cells co-expressing GIRK2 and calretinin than the control mouse groups.4. These results demonstrate that gene triplication can impact specific cell types in the cerebellum. We hypothesize that GIRK2 overexpression will adversely affect cerebellar circuitry in Ts65Dn vestibulocerebellum and dorsal cochlear nucleus due to GIRK2 shunting properties and its effects on resting membrane potential.While Dr. Julius Axelrod’s impact on the development of Neuroscience was significant, one of his major contributions was made indirectly through the people close to him that he influenced. Being a Section Chief and colleague to Julie in the Laboratory of Clinical Science at the National Institute of Mental Health was one of the great honors of my life. It was always a joy observing humility, friendliness and concern of all problems big or small. At laboratory seminars it was a pleasure to watch Julie’s ideas and intuitions that often generated a tremendous amount of good science. He taught all of us how to be curious, incisive and imaginative, and above all to “keep it simple.” His delight in science was contagious. DMJ     

Differentiation of morphology, genetics and electric signals in a region of sympatry between sister species of African electric fish (Mormyridae)

Mormyrid fishes produce and sense weak electric organ discharges (EODs) for object detection and communication, and they have been increasingly recognized as useful model organisms for studying signal evolution and speciation. EOD waveform variation can provide important clues to sympatric species boundaries between otherwise similar or morphologically cryptic forms. Endemic to the watersheds of Gabon (Central Africa), Ivindomyrus marchei and Ivindomyrus opdenboschi are morphologically similar to one another. Using morphometric, electrophysiological and molecular characters [cytochrome b sequences and amplified fragment length polymorphism (AFLP) genotypes], we investigated to what extent these nominal mormyrid species have diverged into biological species. Our sampling covered the known distribution of each species with a focus on the Ivindo River, where the two taxa co-occur. An overall pattern of congruence among datasets suggests that I. opdenboschi and I. marchei are mostly distinct. Electric signal analysis showed that EODs of I. opdenboschi tend to have a smaller initial head-positive peak than those of I. marchei, and they often possess a small third waveform peak that is typically absent in EODs of I. marchei. Analysis of sympatric I. opdenboschi and I. marchei populations revealed slight, but significant, genetic partitioning between populations based on AFLP data (F(ST) approximately 0.04). Taken separately, however, none of the characters we evaluated allowed us to discriminate two completely distinct or monophyletic groups. Lack of robust separation on the basis of any single character set may be a consequence of incomplete lineage sorting due to recent ancestry and/or introgressive hybridization. Incongruence between genetic datasets in one individual, which exhibited a mitochondrial haplotype characteristic of I. marchei but nevertheless fell within a genetic cluster of I. opdenboschi based on AFLP genotypes, suggests that a low level of recent hybridization may also be contributing to patterns of character variation in sympatry. Nevertheless, despite less than perfect separability based on any one dataset and inconclusive evidence for complete reproductive isolation between them in the Ivindo River, we find sufficient evidence to support the existence of two distinctive species, I. opdenboschi and I. marchei, even if not 'biological species' in the Mayrian sense.     

Glycosylation and Cell Surface Expression of Kv1.2 Potassium Channel are Regulated by Determinants in the Pore Region

Voltage-gated K⁺ channels contain six membrane spanning segments and a pore-forming domain. We used site-directed mutation to examine the role of specific amino acids in the extracellular region of the pore in Kv1.2. When expressed in CHO cells, a K⁺ current was not observed for mutants S356A, S360A, T383A and T384A. However, coexpression of the Kvβ2 subunit and the S360A mutant resulted in a robust peak current. Immunocytochemistry for Kv1.2 showed staining throughout the cytoplasm in cells coexpressing the β2 and S360A, whereas only the perinuclear region was stained in cells expressing the S360A mutant. Western blotting revealed that the major immunoreactive protein in wild-type- and mutant-expressing cells is 60-kDa, but 87-kDa bands were also detected in cells expressing wild-type Kv1.2 and cells coexpressing β2and S360A. These results suggest that amino acids in the pore region help regulate ion permeability or cellular trafficking by affecting glycosylation of Kv1.2.     

Electric organ discharge frequency and plasma sex steroid levels during gonadal recrudescence in a natural population of the weakly electric fish Sternopygus macrurus

1. Sternopygus macrurus were collected in Venezuela during the period of gonadal recrudescence in early or late dry season. Electric organ discharge (EOD) frequencies were recorded, blood samples were taken for analysis of steroid titers, and gonads were taken for determination of reproductive condition. 2. Mean EOD frequencies were significantly lower in males than in females in all samples. EOD frequency was inversely correlated with body length in males in late, but not early, dry season, and these parameters were never correlated in females. 3. Plasma levels of testosterone (T) and 11-ketotestosterone (11-KT), but not estradiol-17 beta (E2), were inversely correlated with EOD frequency in males. No 11-KT was observed in plasma of females, and plasma levels of T and E2 in females were comparable to those of males. Neither T nor E2 were correlated with EOD frequency in females. 4. Testes collected in late dry season were more mature than those from early dry season; androgen levels and EOD frequency were correlated with testicular maturity. Ovaries collected in early dry season were immature, while those from late dry season were more mature. There was no relationship between EOD frequency and stage of ovarian development. 5. These results suggest that plasma androgens modulate EOD frequency in males during the reproductive season and that plasma E2 has little relationship to EOD frequency in either sex.     

Tyrosine and Serine Phosphorylation of Dystrophin and the 58-kDa Protein in the Postsynaptic Membrane of Torpedo Electric Organ

Abstract: Dystrophin associates with a 58-kDa and an 87-kDa protein in the postsynaptic membrane of the Torpedo electric organ. We have previously shown that the 87-kDa protein is a major phosphotyrosine-containing protein in these membranes. Immunoprecipitation of the 87-kDa protein from phosphorylated postsynaptic membranes results in coimmunoprecipitation of additional phosphoproteins. These phosphoproteins are identified as dystrophin and the 58-kDa protein. Monoclonal antibodies to dystrophin and the 58-kDa protein immunoprecipitate phosphorylated forms of these proteins from postsynaptic membranes phosphorylated in vitro. Phosphoamino acid analysis reveals that dystrophin and the 58-kDa protein are phosphorylated on serine and tyrosine residues. In addition, both dystrophin and the 58-kDa protein are shown to be phosphorylated on tyrosine residues in vivo. These results suggest that the synaptic function of dystrophin and its associated proteins, the 58-kDa and 87-kDa proteins, may be modulated by tyrosine and serine protein Phosphorylation.