Role of the Plasmalemma in Controlling the Gravitropic Response of Wheat Coleoptiles

The changes in the specific binding of ³H-IAA to the plasmalemma from segments of wheat (Triticum aestivumL.) coleoptiles and the physiological activity of the IAA–protein complexes thus formed in dependence on the duration of gravitational stimulation (GS) (1 g) were studied. The overall inhibition of the formation of IAA–protein complexes was accompanied by a transverse polarization of their functional activity occurring as early as within two minutes after the onset of GS. The pretreatment of plasmalemmal vesicles with 0.1 M CaCl₂prevented the in vitroIAA–protein complex formation in the plasmalemma. It is suggested that the GS results in an increase in the plasmalemma permeability for Ca²⁺, which reduces the capacity of the plasmalemma to bind IAA at the early stages of the gravitropic response.     

Calcium and photoperiodic flower induction in Pharbitis nil

The relationship between phytochrome-mediated induction of flowering, Ca²⁺ transport and metabolism in Pharbitis nil Chois cv. Violet seedlings has been investigated. Ethyleneglycol-bis-(β-aminoethylether)-N,N,N', N'-tetraacetic acid (EGTA), a specific Ca⁺ chelator, caused a 30–40% inhibition of flowering in Pharbitis subjected to complete photoperiodic induction. It was most effective when applied during the light period preceding along inductive dark period. The agonist of calcium channels. Bay K-8644, did not affect flowering, while Nifedipine, Verapamil and La³⁺ (antagonists of calcium channels) only slightly inhibited this process. A similar small effect has been found when the plants were treated with Li⁺ (inhibitor of the membrane phospholipids pathway), and with chlorpromazine (a camodulin inhibitor). Except for EGTA, the effect of the other substances did not depend on the timing of their application. The results of the present study suggest that the effect of all the substances applied was not specific, and flowering is not directly dependent on transport and intracellular metabolism of Ca²⁺.     

Molecular Basis of Regulating High Voltage-Activated Calcium Channels by S-Nitrosylation

Nitric oxide (NO) is involved in a variety of physiological processes, such as vasoregulation and neurotransmission, and has a complex role in the regulation of pain transduction and synaptic transmission. We have shown previously that NO inhibits high voltage-activated Ca²⁺ channels in primary sensory neurons and excitatory synaptic transmission in the spinal dorsal horn. However, the molecular mechanism involved in this inhibitory action remains unclear. In this study, we investigated the role of S-nitrosylation in the NO regulation of high voltage-activated Ca²⁺ channels. The NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP) rapidly reduced N-type currents when Cav2.2 was coexpressed with the Cavβ1 or Cavβ3 subunits in HEK293 cells. In contrast, SNAP only slightly inhibited P/Q-type and L-type currents reconstituted with various Cavβ subunits. SNAP caused a depolarizing shift in voltage-dependent N-type channel activation, but it had no effect on Cav2.2 protein levels on the membrane surface. The inhibitory effect of SNAP on N-type currents was blocked by the sulfhydryl-specific modifying reagent methanethiosulfonate ethylammonium. Furthermore, the consensus motifs of S-nitrosylation were much more abundant in Cav2.2 than in Cav1.2 and Cav2.1. Site-directed mutagenesis studies showed that Cys-805, Cys-930, and Cys-1045 in the II-III intracellular loop, Cys-1835 and Cys-2145 in the C terminus of Cav2.2, and Cys-346 in the Cavβ3 subunit were nitrosylation sites mediating NO sensitivity of N-type channels. Our findings demonstrate that the consensus motifs of S-nitrosylation in cytoplasmically accessible sites are critically involved in post-translational regulation of N-type Ca²⁺ channels by NO. S-Nitrosylation mediates the feedback regulation of N-type channels by NO.     

Block of N-type Calcium Channels in Chick Sensory Neurons by External Sodium

L-type Ca²⁺ channels select for Ca²⁺ over sodium Na⁺ by an affinity-based mechanism. The prevailing model of Ca²⁺ channel permeation describes a multi-ion pore that requires pore occupancy by at least two Ca²⁺ ions to generate a Ca²⁺ current. At [Ca²⁺] < 1 μM, Ca²⁺ channels conduct Na⁺. Due to the high affinity of the intrapore binding sites for Ca²⁺ relative to Na⁺, addition of μM concentrations of Ca²⁺ block Na⁺ conductance through the channel. There is little information, however, about the potential for interaction between Na⁺ and Ca²⁺ for the second binding site in a Ca²⁺ channel already occupied by one Ca²⁺. The two simplest possibilities, (a) that Na⁺ and Ca²⁺ compete for the second binding site or (b) that full time occupancy by one Ca²⁺ excludes Na⁺ from the pore altogether, would imply considerably different mechanisms of channel permeation. We are studying permeation mechanisms in N-type Ca²⁺ channels. Similar to L-type Ca²⁺ channels, N-type channels conduct Na⁺ well in the absence of external Ca²⁺. Addition of 10 μM Ca²⁺ inhibited Na⁺ conductance by 95%, and addition of 1 mM Mg²⁺ inhibited Na⁺ conductance by 80%. At divalent ion concentrations of 2 mM, 120 mM Na⁺ blocked both Ca²⁺ and Ba²⁺ currents. With 2 mM Ba²⁺, the IC₅₀ for block of Ba²⁺ currents by Na⁺ was 119 mM. External Li⁺ also blocked Ba²⁺ currents in a concentration-dependent manner, with an IC₅₀ of 97 mM. Na⁺ block of Ba²⁺ currents was dependent on [Ba²⁺]; increasing [Ba²⁺] progressively reduced block with an IC₅₀ of 2 mM. External Na⁺ had no effect on voltage-dependent activation or inactivation of the channel. These data suggest that at physiological concentrations, Na⁺ and Ca²⁺ compete for occupancy in a pore already occupied by a single Ca²⁺. Occupancy of the pore by Na⁺ reduced Ca²⁺ channel conductance, such that in physiological solutions, Ca²⁺ channel currents are between 50 and 70% of maximal.     

FK-506结合蛋白对钙释放通道的调控

细胞内自由钙作为一种重要的细胞信使广泛地参与细胞生理功能调控.胞内钙库(内质网系和肌浆网系)对调节细胞内自由钙水平起着重要的作用.钙库膜上的钙释放通道(ryanodine受体和三磷酸肌醇受体)受许多因素调控,其中之一就是新近研究得相当多的FK506结合蛋白.免疫抑制剂FK506能特异地结合钙库上一种分子质量为12 ku左右的蛋白,这种FK506结合蛋白与钙释放通道形成一种紧密连接的复合体,在正常生理情况下对钙释放通道起着十分重要的调控作用.     

Coriaria Lactone Increased the Intracellular Level of Calcium through the Voltage-gated Calcium Channels in Rat Hippocampal Neurons

To investigate the effects of Coriaria Lactone (CL), an epileptogenic substance, on intracellular levels of calcium ([Ca²⁺]_i) and physiological properties of voltage-gated calcium channels (VGCCs). Ratiometric calcium imaging using Fura Red and whole-cell voltage patch-clamp technique were explored on freshly isolated rat hippocampal neurons exposed to CL. Coriaria Lactone increased [Ca²⁺]_i from 118 ± 21 to 440 ± 35 nM; VGCCs and calcium influx through NMDA receptor served as the main routes of entry. Coriaria Lactone could enhance both Low voltage activated (LVA) and High voltage activated calcium currents in a concentration-dependent way, and its effect on LVA current was more potent (about 60%). The increased calcium currents were accompanied by the shift of voltage-dependent steady-state inactivation to more positive potentials. These effects of CL, especially its impact on LVA current, could activate different calcium-dependent signaling pathways, and influence cellular excitable properties as well, which might play an important role in CL’s epileptogenic process. Q. Zhang and X. Lai contributed equally to this article.     

Distribution of Membrane-bound Calcium and Activited Calmodulin in Isolated Zygotes and Young Embryos of Triticum aestivum

The method of non-enzymatic, manual microdissection was established to isolate zygotes and young embryos in Triticum aestivum L. The distribution of membrane-bound calcium and activated calmodulin in the isolated zygotes and young embryos was visualized by chlorotetracycline (CTC) and fluphenanize (FPZ) fluorescence probe respectively. The CTC fluorescence was polar distributed in the zygote protoplast. The distribution of the CFC and FPZ fluorescence from twocelled embryos to multicellular embryos was observed. In the young pear-shaped embryos the CTC and FPZ fluorescence of the embryos was slightly higher than that of the suspensor. In a pear-shaped embryo beginning with differentiation the CTC fluorescence was restricted to several-layer of cells between embryo and suspensor and the several ventral cells of the embryo. In the embryos with newly differentiated plumule the basal part of the embryo possessed a higher CTC fluorescence, while the FPZ fluorescence was only distributed in the basal part. It indicated that the distribution of CTC and FPZ fluorescence was in coincidence with the sites that plumule and radicle were beginning to differentiate. The technique of isolated zygotes and the possible function of calcium and calmodulin during embryo development are discussed.     

Protective Effect of Retinal Ischemia by Blockers of Voltage-dependent Calcium Channels and Intracellular Calcium Stores

In the present study, the neuroprotective effect of blockers of voltage-dependent calcium channels (VDCC) and intracellular calcium stores on retinal ischemic damage induced by oxygen deprivation-low glucose insult (ODLG) was investigated. Retinal damage induced by ODLG was dependent on the calcium concentration in the perfusion medium. When incubated in medium containing 2.4 mM CaCl₂, cell death in ischemic retinal slices treated with blockers of VDCC, ω-conotoxin GVIA (1.0 μM), ω-conotoxin MVIIC (100 nM) and nifedipine (1.0 μM), was reduced to 62 ± 2.3, 46 ± 4.3 and 47 ± 3.9%, respectively. In the presence of blockers of intracellular calcium stores, dantrolene (100 μM) and 2-APB (100 μM), the cell death was reduced to 46 ± 3.2 and 55 ± 2.9%, respectively. Tetrodotoxin (1.0 μM), reducing the extent of the membrane depolarization reduces the magnitude of calcium influx trough VDCC causing a reduction of the cell death to 55 ± 4.3. Lactate dehydrogenase content of untreated ischemic retinal slices was reduced by 37% and treatment of ischemic slices with BAPTA-AM (100 μM) or 2-APB (100 μM) abolished the leakage of LDH. Dantrolene (100 μM) and nifedipine (1.0 μM) partially blocked the induced reduction on the LDH content of retinal ischemic slices. Histological analysis of retinal ischemic slices showed 40% reduction of ganglion cells that was prevented by BAPTA-AM or dantrolene. 2-APB partially blocked this reduction whilst nifedipine had no effect, p > 0.95. Conclusion Blockers of VDCC and intracellular calcium-sensitive receptors exert neuroprotective effect on retinal ischemia.     

Mechanisms of Calmodulin Regulation of Different Isoforms of Kv7.4 K+ Channels

Calmodulin (CaM), a Ca²⁺-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca²⁺-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca²⁺-independent manner but that only the long isoform (hKv7.4a) is regulated by Ca²⁺/CaM. Ca²⁺/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca²⁺/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca²⁺ on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca²⁺/CaM regulation of the Kv7.4 channel under physiological and pathological conditions.     

内皮细胞电压门控钙离子通道及其功能研究进展

内皮细胞（endothelial cell,EC）作为不可兴奋细胞,早前通常被认为缺乏功能性电压门控钙离子通道（voltagegated calcium channel,VGCC）,如人脐静脉内皮细胞、牛肺动脉内皮细胞、牛主动脉内皮细胞等。随着膜片钳技术、荧光显微技术、聚合酶链式反应（PCR）技术的发展,越来越多的VGCC在各种内皮细胞中被发现,如人主动脉内皮细胞、大鼠主动脉内皮细胞、大鼠肺微血管内皮细胞等。目前对于VGCC存在与否主要有3种检测方法:利用膜片钳技术对离子通道电流的检测、利用荧光显微技术对胞内钙离子浓度变化的检测、利用PCR技术对离子通道基因或蛋白质表达的检测。内皮细胞不单单是血液和其他相邻组织细胞及基质蛋白间的物理屏障,更重要的是通过细胞膜上VGCC的开放和关闭对细胞和血管组织的生理变化产生显著的影响。一方面,VGCC对胞内钙离子浓度变化的影响,控制着一氧化氮（NO）等血管舒张因子的释放,调节血管张力的平衡。另一方面,作为钙离子内流重要途经的VGCC,经过Ras和MEK通路的诱导、磷酸化PI3K和Akt通路,影响内皮细胞迁移和增殖。此外,部分生理现象,如血管内压力产生...     

Gating of Single N-type Calcium Channels Recorded from Bullfrog Sympathetic Neurons

For many neurons, N-type calcium channels provide the primary pathway for calcium influx during an action potential. We investigated the gating properties of single N-type calcium channels using the cell-attached patch technique. With 100 mM Ba²⁺ in the pipet, mean N-channel open probability (P _o, measured over 100 ms) increased with depolarization, but the range at a single voltage was large (e.g., P _o at +40 mV ranged from 0.1 to 0.8). The open dwell time histograms were generally well fit by a single exponential with mean open time (τ_o) increasing from 0.7 ms at +10 mV to 3.1 ms at +40 mV. Shut time histograms were well fit by two exponentials. The brief shut time component (τ_sh1 = 0.3 ms) did not vary with the test potential, while the longer shut time component (τ_sh2) decreased with voltage from 18.9 ms at +10 mV to 2.3 ms at +40 mV. Although N-channel P _o during individual sweeps at +40 mV was often high (∼0.8), mean P _o was reduced by null sweeps, low P _o gating, inactivation, and slow activation. The variability in mean P _o across patches resulted from differences in the frequency these different gating processes were expressed by the channels. Runs analysis showed that null sweeps tended to be clustered in most patches, but that inactivating and slowly activating sweeps were generally distributed randomly. Low P _o gating (P _o = 0.2, τ_o = 1 ms at +40 mV) could be sustained for ∼1 min in some patches. The clustering of null sweeps and sweeps with low P _o gating is consistent with the idea that they result from different modes of N-channel gating. While P _o of the main N-channel gating state is high, the net P _o is reduced to a maximum value of close to 0.5 by other gating processes.     

Functional Aspects of Calcium Channels of Splanchnic Neurons and Chromaffin Cells of the Rat Adrenal Medulla

Effects of the inorganic calcium channel blockers zinc, manganese, cadmium, and nickel on secretion of catecholamines from the perfused adrenal gland of the rat were investigated. Secretion of catecholamines evoked by splanchnic nerve stimulation (1 and 10 Hz) was not affected by nickel (100 microM), partially blocked (50%) by cadmium (100 microM), and almost completely blocked (90%) by zinc (1 mM) or manganese (2 mM). A combination of nickel and cadmium inhibited nerve stimulation-evoked secretion by 80-90%. Catecholamine secretion evoked by direct stimulation of chromaffin cells by acetylcholine (50 micrograms), nicotine (5 microM), muscarine (50 micrograms), and K+ (17.5 mM) was not blocked by either cadmium, nickel, or their combination. However, zinc and manganese almost abolished nicotine- and K(+)-evoked secretion of catecholamines. None of the above agents had any effect on the secretion evoked by muscarine. Acetylcholine-evoked secretion of catecholamines was only partially reduced (50%) by zinc and manganese. We draw the following conclusions from the above findings: (a) cadmium plus nickel selectively blocks the calcium channels of splanchnic neurons but has no effect on calcium channels of the chromaffin cells; (b) zinc and manganese do not discriminate between calcium channels of neurons and calcium channels of chromaffin cells; (c) partial inhibition of acetylcholine-evoked secretion by inorganic calcium channel blockers is consistent with the idea that activation of nicotinic receptors increases Ca2+ influx, and activation of muscarinic receptors mobilizes intracellularly bound Ca2+, which is not affected by calcium channel blockers.     

Response of carbonic anhydrase to polyethylene glycol-mediated water stress in wheat

Carbonic anhydrase (CA) activity in wheat leaves changed upon leaf dehydration: it decreased at mild stress (relative water content, RWC, 81 %), but increased at severe water stress (RWC 74 %). Phosphoenopyruvate carboxylase activity was not significantly affected by these stresses. This revised version was published online in June 2006 with corrections to the Cover Date.     

Selective Dephosphorylation of the Subunits of Skeletal Muscle Calcium Channels by Purified Phosphoprotein Phosphatases

Abstract: Multiple sites on the α1 and β subunits of purified skeletal muscle calcium channels are phosphorylated by cyclic AMP-dependent protein kinase, resulting in three different tryptic phosphopeptides derived from each subunit. Phosphoprotein phosphatases dephosphorylated these sites selectively. Phosphoprotein phosphatase 1 (PP1) and phosphoprotein phosphatase 2A (PP2A) dephosphorylated both α1 and β subunits at similar rates, whereas calcineurin dephosphorylated β subunits preferentially. PP1 dephosphorylated phosphopeptides 1 and 2 of the α1 subunit more rapidly than phosphopeptide 3. In contrast, PP2A dephosphorylated phosphopeptide 3 of the α1 subunit preferentially. All three phosphoprotein phosphatases preferentially dephosphorylated phosphopeptide 1 of the β subunit and dephosphorylated phosphopeptides 2 and 3 more slowly. Mn²⁺ increased the rate and extent of dephosphorylation of all sites by calcineurin so that >80% dephosphorylation of both α1 and β sub-units was obtained. The results demonstrate selective dephosphorylation of different phosphorylation sites on the α1 and β subunits of skeletal muscle calcium channels by the three principal serine/threonine phosphoprotein phosphatases.     

偏头痛发病机制中ASICs通道及P/Q钙通道交互影响的电生理研究

目的:ASICs通道及P/Q钙通道均参与偏头痛发生,分析ASICs通道及P/Q钙通道的电生理相互作用,评价二者的在偏头痛发生中的交互影响。方法:健康SPF级野生型C57BL/6鼠婴,分离培养双侧三叉神经节神经元,采用全细胞膜片钳技术记录三叉神经节神经元的钙电流变化及动作电位变化。结果:酸性外液及阿米洛利对钙通道无直接影响,酸性外液及P/Q通道阻断剂Aga-IVA均增加三叉神经元兴奋性(P0.05),而阿米洛利可阻断这种增加效应(P0.05)。结论:阿米洛利能够抑制Aga-IVA对三叉神经节神经元兴奋性的增加,可能与其阻断ASICs通道有关,提示ASICs通道可能为P/Q通道突变引发偏头痛的下游机制之一。     

Water Channels Are Involved in Stomatal Oscillations Encoded by Parameter-Specific Cytosolic Calcium Oscillations

Earlier studies have shown that various stimuli can induce specific cytosolic calcium （[Ca^2＋]cyt） oscillations in guard cells and various oscillations in stomatal apertures. Exactly how [Ca^2＋]cyt oscillation signaling functions in stomatal oscillation is not known. In the present study, the epidermis of broad bean （Vicia faba L.） was used and a rapid ion-exchange treatment with two shifting buffers differing in K^＋ and Ca^2＋ concentrations was applied. The treatment for fivetransients at a 10-min transient period induced clear and regular stomatal oscillation. However, for other transient numbers and periods, the treatments induced some Irregular oscillations or even no obvious oscillations in stomatal aperture. The results indicate that stomatal oscillation Is encoded by parameter-specific [Ca^2＋]cyt oscillation： the parameters of [Ca^2＋]cyt oscillation affected the occurrence rate and the parameters of stomatal oscillation. The water channel inhibitor HgCl2 completely Inhibited stomatal oscillation and the inhibitory effect could be partially reversed by β-mercaptoethanol （an agent capable of reversing water channel inhibition by HgCl2）. Other Inhibitory treatments against Ion transport （i.e. the application of LaCIs, EGTA, or tetraethylammonlum chloride （TEACI）） weakly impaired stomatal oscillation when the compounds were added after rapid ion-exchange treatment. If these compounds were added before rapid-ion exchange treatment, the inhibitory effect was much more apparent （except In the case of TEACI）. The results of the present study suggest that water channels are involved In stomatal oscillation as a downstream element of [Ca^2＋]cyt oscillation signaling.     

Unstructural Biology of TRP Ion Channels: The Role of Intrinsically Disordered Regions in Channel Function and Regulation

The first genuine high-resolution single particle cryo-electron microscopy structure of a membrane protein determined was a transient receptor potential (TRP) ion channel, TRPV1, in 2013. This methodical breakthrough opened up a whole new world for structural biology and ion channel aficionados alike. TRP channels capture the imagination due to the sheer endless number of tasks they carry out in all aspects of animal physiology. To date, structures of at least one representative member of each of the six mammalian TRP channel subfamilies as well as of a few non-mammalian families have been determined. These structures were instrumental for a better understanding of TRP channel function and regulation. However, all of the TRP channel structures solved so far are incomplete since they miss important information about highly flexible regions found mostly in the channel N- and C-termini. These intrinsically disordered regions (IDRs) can represent between a quarter to almost half of the entire protein sequence and act as important recruitment hubs for lipids and regulatory proteins. Here, we analyze the currently available TRP channel structures with regard to the extent of these “missing” regions and compare these findings to disorder predictions. We discuss select examples of intra- and intermolecular crosstalk of TRP channel IDRs with proteins and lipids as well as the effect of splicing and post-translational modifications, to illuminate their importance for channel function and to complement the prevalently discussed structural biology of these versatile and fascinating proteins with their equally relevant ’unstructural’ biology.     

Role of abscisic acid in regulation of wheat seedling growth under salinity stress

Growth of wheat seedlings (Triticum aestivum L. cv. Mehran-89), in hydroponic culture, was affected by abscisic acid (ABA). Using salinity stress and exogenous ABA application (10-6 M) to enhance endogenous ABA level, the growth of roots was more suppressed than the growth of shoots. On the other hand, norflurazon, which inhibits ABA biosynthesis, reduced only the growth of shoots. This revised version was published online in July 2006 with corrections to the Cover Date.