Modulation of Cav3.2 T-type calcium channel permeability by asparagine-linked glycosylation

Low-voltage-gated T-type calcium channels are expressed throughout the nervous system where they play an essential role in shaping neuronal excitability. Defects in T-type channel expression have been linked to various neuronal disorders including neuropathic pain and epilepsy. Currently, little is known about the cellular mechanisms controlling the expression and function of T-type channels. Asparagine-linked glycosylation has recently emerged as an essential signaling pathway by which the cellular environment can control expression of T-type channels. However, the role of N-glycans in the conducting function of T-type channels remains elusive. In the present study, we used human Ca_v3.2 glycosylation-deficient channels to assess the role of N-glycosylation on the gating of the channel. Patch-clamp recordings of gating currents revealed that N-glycans attached to hCa_v3.2 channels have a minimal effect on the functioning of the channel voltage-sensor. In contrast, N-glycosylation on specific asparagine residues may have an essential role in the conducting function of the channel by enhancing the channel permeability and / or the pore opening of the channel. Our data suggest that modulation of N-linked glycosylation of hCa_v3.2 channels may play an important physiological role, and could also support the alteration of T-type currents observed in disease states.     

A Cav3.2/Stac1 molecular complex controls T-type channel expression at the plasma membrane

Low-voltage-activated T-type calcium channels are essential contributors to neuronal physiology where they play complex yet fundamentally important roles in shaping intrinsic excitability of nerve cells and neurotransmission. Aberrant neuronal excitability caused by alteration of T-type channel expression has been linked to a number of neuronal disorders including epilepsy, sleep disturbance, autism, and painful chronic neuropathy. Hence, there is increased interest in identifying the cellular mechanisms and actors that underlie the trafficking of T-type channels in normal and pathological conditions. In the present study, we assessed the ability of Stac adaptor proteins to associate with and modulate surface expression of T-type channels. We report the existence of a Ca_v3.2/Stac1 molecular complex that relies on the binding of Stac1 to the amino-terminal region of the channel. This interaction potently modulates expression of the channel protein at the cell surface resulting in an increased T-type conductance. Altogether, our data establish Stac1 as an important modulator of T-type channel expression and provide new insights into the molecular mechanisms underlying the trafficking of T-type channels to the plasma membrane.     

Discovery of N-(1-adamantyl)-2-(4-alkylpiperazin-1-yl)acetamide derivatives as T-type calcium channel (Cav3.2) inhibitors

Chemical evolution of a HTS-based fragment hit resulted in the identification of N-(1-adamantyl)-2-[4-(2-tetrahydropyran-4-ylethyl)piperazin-1-yl]acetamide, a novel, selective T-type calcium channel (Ca(v)3.2) inhibitor with in vivo antihypertensive effect in rats.     

Bipartite syntaxin 1A interactions mediate CaV2.2 calcium channel regulation

Functional interactions between syntaxin 1A and Ca_V2 calcium channels are critical for fast neurotransmitter release in the mammalian brain, and coexpression of syntaxin 1A with these channels not only regulates channel availability, but also promotes G-protein inhibition. Both the syntaxin 1A C-terminal H3 domain, and N-terminal Ha domain have been shown to interact with the Ca_V2.2 channel synprint region, suggesting a bipartite model of functional interaction, however the molecular determinants of this interaction have not been closely investigated. We used in vitro binding assays to assess interactions of syntaxin 1A truncation mutants with Ca_V2.2 synprint and Ca_V2.3 II–III linker regions. We identified two distinct interactions between the Ca_V2.2 synprint region and syntaxin 1A: the first between C-terminal H3c domain of syntaxin 1A and residues 822–872 of Ca_V2.2; and the second between the N-terminal 10 residues of the syntaxin 1A Ha region and residues 718–771 of Ca_V2.2. The N-terminal syntaxin 1A fragment also interacted with the Ca_V2.3 II–III linker. We then performed whole cell patch clamp recordings to test the effects of a putative interacting syntaxin 1A N-terminus peptide with Ca_V2.2 and Ca_V2.3 channels in a recombinant expression system. A YFP-tagged peptide corresponding to the N-terminal 10 residues of the syntaxin 1A Ha domain was sufficient to allosterically inhibit both Ca_V2.2 and Ca_V2.3 channel function but had no effect on G-protein mediated inhibition. Our results support a model of bipartite functional interactions between syntaxin 1A and Ca_V2.2 channels and add accuracy to the two putative interacting domains, consistent with previous studies. Furthermore, we highlight the syntaxin 1A N-terminus as the minimal determinant for functional regulation of Ca_V2.2 and Ca_V2.3 channels.     

CACNA1H missense mutations associated with amyotrophic lateral sclerosis alter Cav3.2 T-type calcium channel activity and reticular thalamic neuron firing

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. In a recent study by Steinberg and colleagues, 2 recessive missense mutations were identified in the Ca_v3.2 T-type calcium channel gene (CACNA1H), in a family with an affected proband (early onset, long duration ALS) and 2 unaffected parents. We have introduced and functionally characterized these mutations using transiently expressed human Ca_v3.2 channels in tsA-201 cells. Both of these mutations produced mild but significant changes on T-type channel activity that are consistent with a loss of channel function. Computer modeling in thalamic reticular neurons suggested that these mutations result in decreased neuronal excitability of thalamic structures. Taken together, these findings implicate CACNA1H as a susceptibility gene in amyotrophic lateral sclerosis.     

Gating of the expressed Cav3.1 calcium channel

Intramembrane charge movement originating from Cav3.1 (T-type) channel expressed in HEK 293 cells was investigated. Ion current was blocked by 1 mM La3+. Charge movement was detectable for depolarizations above approximately -70 mV and saturated above +60 mV. The voltage dependence of charge movement followed a single Boltzmann function with half-maximal activation voltage +12.9 mV and +12.3 mV and with slopes of 22.4 mV and 18.1 mV for the ON- and OFF-charge movement, respectively. Inactivation of I(Ca) by prolonged depolarization pulse did not immobilize intramembrane charge movement in the Cav3.1 channel.     

调控T型钙通道Cav3.1电压依赖性失活的分子结构域

T型钙通道(Cav3)广泛分布于各类细胞,其显著的电生理学特点是低电位激活和快速的电压依赖性失活.失活在通道的生理功能调节中起十分重要的作用,但具体参与通道失活的分子基础目前并不完全清楚.为明确Cav3.1通道中调控电压依赖性失活的结构域,用Cav1.2通道(无电压依赖性失活)结构域Ⅰ和Ⅱ中的S1~S4、S5~S6区及Ⅰ和Ⅱ间的联系区替换Cav3.1中的相应区域,构建嵌合通道,并在卵母细胞中表达,用电压钳技术分析通道的电生理学特性.结果表明,替换Ⅰ中的S1~S4或S5~S6区可使Cav3.1的失活特性显著改变,但这种改变主要是由激活-失活偶联所致.Ⅱ的替换使通道的失活曲线参数发生显著改变,表明结构域Ⅱ,包括S1~S4和S5~S6均参与Cav3.1失活过程的调控.Ⅰ、Ⅱ间的联系区及Ⅰ中的S5~S6主要调控Cav3.1的失活速率,Ⅰ和Ⅱ中的S1~S4对通道失活速率无影响.综上所述,结构域Ⅱ是调控Cav3.1电压依赖性失活的关键因素,结构域Ⅰ不参与该通道失活过程的调控.Ⅰ、Ⅱ间的联系区及Ⅰ中的S5~S6主要调控Cav3.1通道的失活速率,Ⅰ、Ⅱ中的S1~S4对通道失活速率无影响.     

Endogenous and exogenous hydrogen sulfide facilitates T-type calcium channel currents in Cav3.2-expressing HEK293 cells

Hydrogen sulfide (H₂S), a gasotransmitter, is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE). We have shown that an H₂S donor, NaHS, causes hyperalgesia in rodents, an effect inhibited by knockdown of Ca_v3.2 T-type Ca²⁺ channels (T-channels), and that NaHS facilitates T-channel-dependent currents (T-currents) in NG108-15 cells that naturally express Ca_v3.2. In the present study, we asked if endogenous and exogenous H₂S participates in regulation of the channel functions in Ca_v3.2-transfected HEK293 (Ca_v3.2-HEK293) cells. dl-Propargylglycine (PPG), a CSE inhibitor, significantly decreased T-currents in Ca_v3.2-HEK293 cells, but not in NG108-15 cells. NaHS at 1.5 mM did not affect T-currents in Ca_v3.2-HEK293 cells, but enhanced T-currents in NG108-15 cells. In the presence of PPG, NaHS at 1.5 mM, but not 0.1–0.3 mM, increased T-currents in Ca_v3.2-HEK293 cells. Similarly, Na₂S, another H₂S donor, at 0.1–0.3 mM significantly increased T-currents in the presence, but not absence, of PPG in Ca_v3.2-HEK293 cells. Expression of CSE was detected at protein and mRNA levels in HEK293 cells. Intraplantar administration of Na₂S, like NaHS, caused mechanical hyperalgesia, an effect blocked by NNC 55-0396, a T-channel inhibitor. The in vivo potency of Na₂S was higher than NaHS. These results suggest that the function of Ca_v3.2 T-channels is tonically enhanced by endogenous H₂S synthesized by CSE in Ca_v3.2-HEK293 cells, and that exogenous H₂S is capable of enhancing Ca_v3.2 function when endogenous H₂S production by CSE is inhibited. In addition, Na₂S is considered a more potent H₂S donor than NaHS in vitro as well as in vivo.     

Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Voltage-activated Ca²⁺ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca²⁺ selective filters of all high-voltage-activated Ca²⁺ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca²⁺ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Ca_v3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Ca_v3.2, including relative permeability between Ba²⁺ and Ca²⁺, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd²⁺ blocking sensitivity, and pH effects, in distinctive ways.     

Effect of protein tyrosine kinase inhibitors on the current through the Ca(V)3.1 channel

In the present study, we have investigated the effects of protein tyrosine kinase (PTK) inhibitors on the Ca(V)3.1 calcium channel stably transfected in HEK293 cells using the whole-cell configuration of the patch-clamp technique. We have tested two different tyrosine kinase inhibitors, genistein and tyrphostin AG213, and their inactive analogs, genistin and tyrphostin AG9. Bath application of genistein, but not genistin, decreased the T-type calcium current amplitude in a concentration-dependent manner with an IC(50) of 24.7+/-2.0 microM. This effect of genistein was accompanied by deceleration of channel activation and acceleration of channel inactivation. Intracellular application of neither genistein nor genistin had a significant effect on the calcium current. Extracellular application of 50 microM tyrphostin AG213 and its inactive analogue, tyrphostin AG9, did not affect the current through the Ca(V)3.1 channel. The effect of genistein on the channel was also not affected by the presence of catalytically active PTK, p60(c-src) inside the cell. We have concluded that genistein directly inhibited the channel. This mechanism does not involve a PTK-dependent pathway. The alteration of the channel kinetics by genistein suggests an interaction with the voltage sensor of the channel together with the channel pore occlusion.     

Pore structure influences gating properties of the T-type Ca2+ channel alpha1G

The selectivity filter of all known T-type Ca2+ channels is built by an arrangement of two glutamate and two aspartate residues, each one located in the P-loops of domains I-IV of the alpha1 subunit (EEDD locus). The mutations of the aspartate residues to glutamate induce changes in the conduction properties, enhance Cd2+ and proton affinities, and modify the activation curve of the channel. Here we further analyze the role of the selectivity filter in the gating mechanisms of T-type channels by comparing the kinetic properties of the alpha1G subunit (CaV3.1) to those of pore mutants containing aspartate-to-glutamate substitution in domains III (EEED) or IV (EEDE). The change of the extracellular pH induced similar effects on the activation properties of alpha1G and both pore mutants, indicating that the larger affinity of the mutant channels for protons is not the cause of the gating modifications. Both mutants showed alterations in several gating properties with respect to alpha1G, i.e., faster macroscopic inactivation in the voltage range from -10 to 50 mV, positive voltage shift and decrease in the voltage sensitivity of the time constants of activation and deactivation, decrease of the voltage sensitivity of the steady-state inactivation, and faster recovery from inactivation for long repolarization periods. Kinetic modeling suggests that aspartate-to-glutamate mutations in the EEDD locus of alpha1G modify the movement of the gating charges and alter the rate of several gating transitions. These changes are independent of the alterations of the selectivity properties and channel protonation.     

东北典型旱作农田N2O和CH4排放通量研究

应用封闭式箱法技术测定了玉米、大豆田中N₂O和CH₄全年的通量变化。指出N₂O排放有明显的季节变化和明显的日变化。大量的N₂O排放发生在作物生长季节中。在冰雪溶化期和收割作物后也有一定量的N₂O从土壤中排放。此外,实验结果也指出,玉米和大豆田作为大气CH₄源或汇的作用不明显。     

Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels

Ca_v3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca_v3.2 and modulation by redox agents. His¹⁹¹ is found on the extracellular face of the Ca_v3.2 channel on the IS3-S4 linker and is not conserved in other Ca_v3 channels. Mutation of the corresponding residue in Ca_v3.1 to histidine, Gln¹⁷², significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca_v3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3–S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.     

一株兼性氧化亚氮还原菌的还原N2O能力

【目的】从水稻土中分离筛选出一株兼性氧化亚氮还原菌,并探索其在不同条件下还原N_2O的能力,为减少温室气体N_2O的排放提供重要依据。【方法】通过微生物富集培养分离技术从水稻土中分离得到纯菌;利用nosZ基因和16S rRNA的测序分析鉴定菌株;通过测定菌株在不同条件下N_2O的还原量,分析该菌株还原N_2O的能力及调控因子。【结果】经鉴定,该菌株含有nos Z基因,属于假单胞菌属,在温度30°C、厌氧条件下还原N_2O速率高达0.0219μmol/min以上,改变不同温度和氧气浓度后其能力相对减弱,但仍具备较强的还原N_2O作用。【结论】从水稻土中分离筛选得到的兼性氧化亚氮还原菌为假单胞菌,它在不同环境条件下都具备较强的还原N_2O能力,该菌株可能为减少土壤N_2O排放提供新途径,对保障生态环境安全具有重要的应用价值。     

Calmodulin binding is dispensable for Rem-mediated Ca2+ channel inhibition

GTPases of the Ras-related RGK family are negative regulators of high voltage-activated (HVA) Ca²⁺ channel activity. In this study, we examined the role of calmodulin (CaM) association in Rem-mediated Ca²⁺ channel inhibition. We found that the Rem/CaM interaction is Ca²⁺-dependent, and that truncation of the Rem C-terminus before position 277 prevents CaM binding. Serial mutagenesis of the Rem C-terminus between residues 265 and 276 to alanine generated two mutants (Rem^L271A and Rem^L274A) that displayed reduced CaM binding, and a subset of these mutants displayed significantly lower cell periphery localization than Rem^WT. However, reductions in CaM association or membrane trafficking did not affect function, as all Rem mutants could completely inhibit Ca²⁺ channels. The Rem^1–275 truncation mutant partially inhibited Ca²⁺ channel activity despite its inability to bind CaM. Taken together, these studies indicate that CaM association is not essential for either Rem-mediated Ca²⁺ channel inhibition or plasma membrane localization. Jonathan Satin is an established investigator of the American Heart Association.     

Discovery and evaluation of Cav3.2-selective T-type calcium channel blockers

We identified and characterized a series of pyrrole amides as potent, selective Ca_v3.2-blockers. This series culminated with the identification of pyrrole amides 13b and 26d, with excellent potencies and/or selectivities toward the Ca_v3.1- and Ca_v3.3-channels. These compounds display poor physicochemical and DMPK properties, making their use difficult for in vivo applications. Nevertheless, they are well-suited for in vitro studies.     

Analysis of the T-type calcium channel in embryonic chick ventricular myocytes

Summary T-type calcium channels (I _T channels) were studied in cell-attached patch electrode recordings from the ventricular cell membrane of 14-day embryonic chick heart. All experiments were performed in the absence of Ca²⁺ with Na⁺ (120mm) as the charge carrier.I _T channels were distinguished from L-type calcium channels (I _L) by their more negative activation and inactivation potential ranges; their smaller unitary slope conductance (26 pS), and their insensitivity to isoproterenol or D600. Inactivation kinetics were voltage dependent. The time constant of inactivation was 37 msec when the membrane potential was depolarized 40 mV from rest (R+40 mV), and 20 msec atR+60 mV. The frequency histogram of channel open times ₀ was fit by a single-exponential curve while that of closed times _c was biexponeintial. _o was the same atR+40 mV andR+60 mV whereas _c was shortened atR+60 mV. The open-state probability (P _o) increased with depolarization: 0.35 atR+40 mV, 0.8 atR+60 mV and 0.88 atR+80 mV. This increase inP _o at depolarized potentials could be accounted for by the decrease in _c.     

Gating charges per channel of CaV2.2 channels are modified by G protein activation in rat sympathetic neurons

It has been suggested that voltage-dependent G protein modulation of Ca_V2.2 channels is carried out at closed states of the channel. Our purpose was to estimate the number of gating charges of Ca_V2.2 channel in control and G protein-modulated conditions. By using a Cole-Moore protocol we observed a significant delay in Ca_V2.2 channel activation according to a transit of the channel through a series of closed states before channel opening. If G protein voltage-dependent modulation were carried out at these closed states, then we would have expected a greater Cole-Moore lag in the presence of a neurotransmitter. This prediction was confirmed for noradrenaline, while no change was observed in the presence of angiotensin II, a voltage-insensitive G protein modulator. We used the limiting slope method for calculation of the gating charge per channel. Effective charge z was 6.32 ± 0.65 for Ca_V2.2 channels in unregulated conditions, while GTPγS reduced elementary charge by ∼4 e₀. Accordingly, increased concentration of noradrenaline induced a gradual decrease on z, indicating that this decrement was due to a G protein voltage-sensitive modulation. This paper shows for the first time a significant and reversible decrease in charge transfer of Ca_V2.2 channels under G protein modulation, which might depend on the activated G protein inhibitory pathway.     

Structural hybridization of pyrrolidine-based T-type calcium channel inhibitors and exploration of their analgesic effects in a neuropathic pain model

Highly effective and safe drugs for the treatment of neuropathic pain are urgently required and it was shown that blocking T-type calcium channels can be a promising strategy for drug development for neuropathic pain. We have developed pyrrolidine-based T-type calcium channel inhibitors by structural hybridization and subsequent assessment of in vitro activities against Ca_v3.1 and Ca_v3.2 channels. Profiling of in vitro ADME properties of compounds was also carried out. The representative compound 17h showed comparable in vivo efficacy to gabapentin in the SNL model, which indicates T-type calcium channel inhibitors can be developed as effective therapeutics for neuropathic pain.