Identification of an intracellular peptide segment involved in sodium channel inactivation

Antibodies directed against a conserved intracellular segment of the sodium channel alpha subunit slow the inactivation of sodium channels in rat muscle cells. Of four site-directed antibodies tested, only antibodies against the short intracellular segment between homologous transmembrane domains III and IV slowed inactivation, and their effects were blocked by the corresponding peptide antigen. No effects on the voltage dependence of sodium channel activation or of steady-state inactivation were observed, but the rate of onset of the antibody effect and the extent of slowing of inactivation were voltage-dependent. Antibody binding was more rapid at negative potentials, at which sodium channels are not inactivated; antibody-induced slowing of inactivation was greater during depolarizations to more positive membrane potentials. The peptide segment recognized by this antibody appears to participate directly in rapid sodium channel inactivation during large depolarizations and to undergo a conformational change that reduces its accessibility to antibodies as the channel inactivates.     

A targeting motif involved in sodium channel clustering at the axonal initial segment

The sorting of sodium channels to axons and the formation of clusters are of primary importance for neuronal electrogenesis. Here, we showed that the cytoplasmic loop connecting domains II and III of the Nav1 subunit contains a determinant conferring compartmentalization in the axonal initial segment of rat hippocampal neurons. Expression of a soluble Nav1.2II-III linker protein led to the disorganization of endogenous sodium channels. The motif was sufficient to redirect a somatodendritic potassium channel to the axonal initial segment, a process involving association with ankyrin G. Thus, this motif may play a fundamental role in controlling electrical excitability during development and plasticity.     

Molecular basis of gating charge immobilization in Shaker potassium channels

Voltage-dependent ion channels respond to changes in the membrane potential by means of charged voltage sensors intrinsic to the channel protein. Changes in transmembrane potential cause movement of these charged residues, which results in conformational changes in the channel. Movements of the charged sensors can be detected as currents known as gating currents. Measurement of the gating currents of the Drosophila Shaker potassium channel indicates that the charge on the voltage sensor of the channels is progressively immobilized by prolonged depolarizations. The charge is not immobilized in a mutant of the channel that lacks inactivation. These results show that the region of the molecule responsible for inactivation interacts, directly or indirectly, with the voltage sensor to prevent the return of the charge to its original position. The gating transitions between closed states of the channel appear not to be independent, suggesting that the channel subunits interact during activation.     

Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB

Site-directed mutagenesis experiments have suggested a model for the inactivation mechanism of Shaker potassium channels from Drosophila melanogaster. In this model, the first 20 amino acids form a cytoplasmic domain that interacts with the open channel to cause inactivation. The model was tested by the internal application of a synthetic peptide, with the sequence of the first 20 residues of the ShB alternatively spliced variant, to noninactivating mutant channels expressed in Xenopus oocytes. The peptide restored inactivation in a concentration-dependent manner. Like normal inactivation, peptide-induced inactivation was not noticeably voltage-dependent. Trypsin-treated peptide and peptides with sequences derived from the first 20 residues of noninactivating mutants did not restore inactivation. These results support the proposal that inactivation occurs by a cytoplasmic domain that occludes the ion-conducting pore of the channel.     

Inactivation and block of calcium channels by photo-released Ca2+ in dorsal root ganglion neurons

Calcium channels are inactivated by voltage and intracellular calcium. To study the kinetics and the mechanism of calcium-induced inactivation of calcium channels, a "caged" calcium compound, dimethoxy-nitrophen was used to photo-release about 50 microM calcium ion within 0.2 millisecond in dorsal root ganglion neurons. When divalent cations were the charge carriers, intracellular photo-release of calcium inactivated the calcium channel with an invariant rate [time constant (tau) approximately equal to 7 milliseconds]. When the monovalent cation sodium was the charge carrier, photorelease of calcium inside or outside of the cell blocked the channel rapidly (tau approximately equal to 0.4 millisecond), but the block was greater from the external side. Thus the kinetics of calcium-induced calcium channel inactivation depends on the valency of the permeant cation. The data imply that calcium channels exist in either of two conformational states, the calcium- and sodium-permeant forms, or, alternatively, calcium-induced inactivation occurs at a site closely associated with the internal permeating site.     

Genomic organization and deduced amino acid sequence of a putative sodium channel gene in Drosophila

The deduced amino acid sequence of a Drosophila gene isolated with a vertebrate sodium channel complementary DNA probe revealed an organization virtually identical to the vertebrate sodium channel protein; four homologous domains containing all putative membrane-spanning regions are repeated in tandem with connecting linkers of various sizes. All areas of the protein presumed to be critical for channel function show high evolutionary conservation. These include those proposed to function in voltage-sensitive gating, inactivation, and ion selectivity. All 24 putative gating charges of the vertebrate protein are in identical positions in the Drosophila gene. Ten introns interrupt the coding regions of the four homology units; introns with positions conserved among homology units bracket a region hypothesized to be the selectivity filter for the channel. The Drosophila gene maps to the right arm of the second chromosome in region 60D-E. This position does not coincide with any known mutations that confer behavioral phenotypes, but is close to the seizure locus (60A-B), which has been hypothesized to code for a voltage-sensitive sodium channel.     

Functional properties of rat brain sodium channels expressed in a somatic cell line

Transfection of Chinese hamster ovary cells with complementary DNA encoding the RIIA sodium channel alpha subunit from rat brain led to expression of functional sodium channels with the rapid, voltage-dependent activation and inactivation characteristic of sodium channels in brain neurons. The sodium currents mediated by these transfected channels were inhibited by tetrodotoxin, persistently activated by veratridine, and prolonged by Leiurus alpha-scorpion toxin, indicating that neurotoxin receptor sites 1 through 3 were present in functional form. The RIIA sodium channel alpha subunit cDNA alone is sufficient for stable expression of functional sodium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Biophysical and molecular mechanisms of Shaker potassium channel inactivation

The potassium channels encoded by the Drosophila Shaker gene activate and inactivate rapidly when the membrane potential becomes more positive. Site-directed mutagenesis and single-channel patch-clamp recording were used to explore the molecular transitions that underlie inactivation in Shaker potassium channels expressed in Xenopus oocytes. A region near the amino terminus with an important role in inactivation has now been identified. The results suggest a model where this region forms a cytoplasmic domain that interacts with the open channel to cause inactivation.     

Alignment of rat cardionatrin sequences with the preprocardionatrin sequence from complementary DNA

Mammalian atria contain peptides that promote the excretion of salt and water from the kidney. When rat atrial tissue is extracted under conditions known to inhibit proteolysis, four natriuretic peptides, cardionatrins I to IV, are consistently isolated. These peptides derive from a common precursor, preprocardionatrin, of 152 amino acids, whose sequence was determined by DNA sequencing of a complementary DNA clone. Amino acid sequencing located the start points of cardionatrins I, III, and IV in the overall sequence. Cardionatrin IV most closely resembles procardionatrin because it begins immediately after the signal sequence at residue 25. Cardionatrin III begins at residue 73, and cardionatrin I, sequenced previously, begins at residue 123. Compositional analysis indicated that each of these cardionatrins extends up to tyrosine at position 150 but lacks the terminal two arginine residues.     

Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids

Voltage-gated potassium (Kv) channels control action potential repolarization, interspike membrane potential, and action potential frequency in excitable cells. It is thought that the combinatorial association between distinct alpha and beta subunits determines whether Kv channels function as non-inactivating delayed rectifiers or as rapidly inactivating A-type channels. We show that membrane lipids can convert A-type channels into delayed rectifiers and vice versa. Phosphoinositides remove N-type inactivation from A-type channels by immobilizing the inactivation domains. Conversely, arachidonic acid and its amide anandamide endow delayed rectifiers with rapid voltage-dependent inactivation. The bidirectional control of Kv channel gating by lipids may provide a mechanism for the dynamic regulation of electrical signaling in the nervous system.     

普洱茶中氨基酸与贮期、级别及品质关系的研究

以不同储藏时期、不同级别的普洱茶为材料,对氨基酸总量及组分含量进行理化分析.结果表明,普洱茶贮藏过程中随着贮藏时间的加长,普洱茶中游离氨基酸总量明显降低,其中丝氨酸、甘氨酸、丙氨酸、异亮氨酸、亮氨酸、组氨酸已基本消减,仅检出精氨酸、脯氨酸、赖氨酸、苯丙氨酸、酪氨酸、缬氨酸、胱氨酸,其含量变化无明显规律性;从7个级别标准样中检出15种氨基酸,未检出苏氨酸、组氨酸、色氨酸,其中氨基酸含量最高的组分是精氨酸,其次是天冬氨酸、赖氨酸、苯丙氨酸、酪氨酸、谷氨酸.茶氨酸在普洱茶中的含量与其它茶类不同,其含量仅占普洱茶中游离氨基酸总量的30％左右.与其它茶类中氨基酸含量相比,普洱茶具有氨基酸种类齐全,营养配比全面、均衡的特点,在保健方面有特殊的意义.     

蒙顶山名茶游离氨基酸总量及组分的测定分析

【目的】探讨蒙顶山名茶"鲜香"与"鲜味"特征的产生机制,提供蒙顶山名茶品质评价的理论依据。【方法】试验通过全量法和杯茶法制备茶汤试液,分别采用水合茚三酮比色法和L-8900型全自动氨基酸分析仪测定了4种蒙顶山名茶中的游离氨基酸总量和组分含量。【结果】4种蒙顶山名茶的游离氨基酸总量较高,分别为蒙顶黄芽(5.28±0.57)%,蒙顶石花(5.59±0.51)%,蒙顶甘露(4.65±0.82)%,蒙顶毛峰(5.34±0.96)%;在杯茶法1次冲泡5min条件下,蒙顶山名茶游离氨基酸的浸出率与浸出浓度亦较高,浸出率为55.24%～76.14%,浸出浓度为58.73～78.24mg/100mL;蒙顶山名茶游离氨基酸组分含量的特征为茶氨酸、天冬氨酸、谷氨酸、精氨酸和丝氨酸的含量在1 000mg/kg以上,色氨酸、半光氨酸、组氨酸和络氨酸组分含量为100～500mg/kg。【结论】蒙顶山名茶高氨基酸总量、丰富的组分含量及独特配比是其茶香"鲜香高长"、茶味"鲜浓爽口"的重要物质基础。     

Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor

The inhibition by charybdotoxin of A-type potassium channels expressed in Xenopus oocytes was studied for several splicing variants of the Drosophila Shaker gene and for several site-directed mutants of this channel. Charybdotoxin blocking affinity is lowered by a factor of 3.5 upon replacing glutamate-422 with glutamine, and by a factor of about 12 upon substituting lysine in this position. Replacement of glutamate-422 by aspartate had no effect on toxin affinity. Thus, the glutamate residue at position 422 of this potassium channel is near or in the externally facing mouth of the potassium conduction pathway, and the positively charged toxin is electrostatically focused toward its blocking site by the negative potential set up by glutamate-422.     

Coding channels in the taste system of the rat

Basic taste qualities are thought to be perceived independently, yet discrete neural coding channels have not been demonstrated in the central nervous system. The response profiles of taste cells in the nucleus tractus solitarius (NTS) of the rat were categorized into four groups, and the effects of amiloride, a passive sodium channel blocker, on each were determined. NTS neurons that responded specifically to sodium chloride (NaCl) or to NaCl and sugars were suppressed by amiloride; those broadly sensitive to salts, acids, and bitter stimuli were unaffected. Moreover, the response profile evoked by NaCl lost its distinctiveness after treatment with amiloride, becoming similar to those evoked by acids and quinine. Receptors that respond to sodium must relay their information through independent coding channels to identifiable subgroups of NTS neurons, the activity of which is responsible for the perception of saltiness.     

Identification and Functional Analysis on Abiotic Stress Response of Soybean Cl~- Channel Gene GmCLCnt

The Cl- homeostasis was known as the major mechanism of soybean to achieve NaCl tolerance, but studies on the role of chloride channel under abiotic stress were relatively few. We cloned a putative CLC-type chloride channel gene GmCLCnt from soybean via RACE and it was predicted to encode a protein of 783 amino acids with 9 possible transmembrane domains and 2 tandem CBS domains. Real-time RT-PCR analysis showed that the GmCLCnt gene was expressed in all tissues of soybean but enriched in leaves and its expression was induced by NaCl, polyethylene glycol (PEG), coldness and ABA treatments. The Arabidopsis seedlings overexpressing GmCLCnt were more tolerant to higher concentration of NaCl than those of wild type. The results suggested that the GmCLCnt may be a CLC-type chloride channel and play an important role in salt tolerance.     

Identification and Functional Analysis on Abiotic Stress Response of SoybeanCl-Channel Gene GmCLCnt

The Cl^- homeostasis was known as the major mechanism of soybean to achieve NaCl tolerance, but studies on the role of chloride channel under abiotic stress were relatively few. We cloned a putative CLC-type chloride channel gene GmCLCnt from soybean via RACE and it was predicted to encode a protein of 783 amino acids with 9 possible transmembrane domains and 2 tandem CBS domains. Real-time RT-PCR analysis showed that the GmCLCnt gene was expressed in all tissues of soybean but enriched in leaves and its expression was induced by NaCl, polyethylene glycol （PEG）, coldness and ABA treatments. The Arabidopsis seedlings overexpressing GmCLCnt were more tolerant to higher concentration of NaCl than those of wild type. The results suggested that the GmCLCnt may be a CLC-type chloride channel and play an important role in salt tolerance.     

Exchange of conduction pathways between two related K+ channels

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.     

Calcium and sodium channels in spontaneously contracting vascular muscle cells

Electrophysiological recordings of inward currents from whole cells showed that vascular muscle cells have one type of sodium channel and two types of calcium channels. One of the calcium channels, the transient calcium channel, was activated by small depolarizations but then rapidly inactivated. It was equally permeable to calcium and barium and was blocked by cadmium, but not by tetrodotoxin. The other type, the sustained calcium channel, was activated by larger depolarizations, but inactivated very little; it was more permeable to barium than calcium. The sustained calcium channel was more sensitive to block by cadmium than the transient channel, but also was not blocked by tetrodotoxin. The sodium channel inactivated 15 times more rapidly than the transient calcium channel and at more negative voltages. This sodium channel, which is unusual because it is only blocked by a very high (60 microM) tetrodotoxin concentration but not by cadmium, is the first to be characterized in vascular muscle, and together with the two calcium channels, provides a basis for different patterns of excitation in vascular muscles.     

野生刺儿菜和刻叶刺儿菜中氨基酸与微量元素含量的比较分析

［目的］为科学地开发利用刻叶刺儿菜的食用及药用价值奠定基础。［方法］ 采用氨基酸自动分析仪和原子吸收分光光度计测定分析野生刺儿菜和刻叶刺儿菜中氨基酸与微量元素的含量。［结果］ 天门冬氨酸、苏氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸、缬氨酸、蛋氨酸、亮氨酸、异亮氨酸、酪氨酸、苯丙氨酸、赖氨酸、组氨酸、精氨酸和脯氨酸在刺儿菜中的含量分别为0.17%、0.09%、0.08%、0.19%、0.10%、0.10%、0.09%、0.01%、0.16%、0.07%、0.06%、0.10%、0.13%、0.04%、0.09%和0.07%,在刻叶刺儿菜中的含量分别为0.17%、0.08%、0.08%、0.18%、0.09%、0.10%、0.09%、0.01%、0.15%、0.07%、0.06%、0.10%、0.13%、0.04%、0.09%和0.06%。钾、钠、钙、镁、铜、锌、铁和锰在刺儿菜中的含量分别为15.376、0.509、142.265、10.701、0.016、0.071、0.271和0.045 mg/g,在刻叶刺儿菜中的含量分别为16.110、1.141、144.834、9.305、0.020、0.053、0.294和0.050 mg/g。［结论］ 刺儿菜和刻叶刺儿菜都含有丰富的氨基酸和人体所需的多种微量元素。     

Voltage sensor of Kv1.2: structural basis of electromechanical coupling

Voltage-dependent ion channels contain voltage sensors that allow them to switch between nonconductive and conductive states over the narrow range of a few hundredths of a volt. We investigated the mechanism by which these channels sense cell membrane voltage by determining the x-ray crystal structure of a mammalian Shaker family potassium ion (K+) channel. The voltage-dependent K+ channel Kv1.2 grew three-dimensional crystals, with an internal arrangement that left the voltage sensors in an apparently native conformation, allowing us to reach three important conclusions. First, the voltage sensors are essentially independent domains inside the membrane. Second, they perform mechanical work on the pore through the S4-S5 linker helices, which are positioned to constrict or dilate the S6 inner helices of the pore. Third, in the open conformation, two of the four conserved Arg residues on S4 are on a lipid-facing surface and two are buried in the voltage sensor. The structure offers a simple picture of how membrane voltage influences the open probability of the channel.