Soluble,highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants

Green fluorescent protein (GFP) from Aequorea victoria has rapidly become a standard reporter in many biological systems. However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding mRNA and by protein insolubility. It has been shown that GFP can be expressed in Arabidopsis thaliana after altering the codon usage in the region that is incorrectly spliced, but the fluorescence signal is weak, possibly due to aggregation of the encoded protein. Through site-directed mutagenesis, we have generated a more soluble version of the codon-modified GFP called soluble-modified GFP (smGFP). The excitation and emission spectra for this protein are nearly identical to wild-type GFP. When introduced into A. thaliana, greater fluorescence was observed compared to the codon-modified GFP, implying that smGFP is brighter because more of it is present in a soluble and functional form. Using the smGFP template, two spectral variants were created, a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP). The increased fluorescence output of smGFP will further the use of this reporter in higher plants. In addition, the distinct spectral characters of smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions.     

Tuning magnesium sensitivity of BK channels by mutations

Intracellular Mg(2+) at physiological concentrations activates mSlo1 BK channels by binding to a metal-binding site in the cytosolic domain. Previous studies suggest that residues E374, Q397, and E399 are important in Mg(2+) binding. In the present study, we show that mutations of E374 or E399 to other amino acids, except for Asp, abolish Mg(2+) sensitivity. These results further support that the side chains of E374 and E399 are essential for Mg(2+) coordination. To the contrary, none of the Q397 mutations abolishes Mg(2+) sensitivity, suggesting that its side chain may not coordinate to Mg(2+). However, because Q397 is spatially close to E374 and E399, its mutations affect the Mg(2+) sensitivity of channel gating by either reducing or increasing the Mg(2+) binding affinity. The pattern of mutational effects and the effect of chemical modification of Q397C indicate that Q397 is involved in the Mg(2+)-dependent activation of BK channels and that mutations of Q397 alter Mg(2+) sensitivity by affecting the conformation of the Mg(2+) binding site as well as by electrostatic interactions with the bound Mg(2+) ion.     

Acid sensing ion channels regulate neuronal excitability by inhibiting BK potassium channels

Acid sensing ion channels (ASICs), Ca²⁺ and voltage-activated potassium channels (BK) are widely present throughout the central nervous system. Previous studies have shown that when expressed together in heterologous cells, ASICs inhibit BK channels, and this inhibition is relieved by acidic extracellular pH. We hypothesized that ASIC and BK channels might interact in neurons, and that ASICs may regulate BK channel activity. We found that ASICs inhibited BK currents in cultured wild-type cortical neurons, but not in ASIC1a/2/3 triple knockout neurons. The inhibition in the wild-type was partially relieved by a drop in extracellular pH to 6. To test the consequences of ASIC-BK interaction for neuronal excitability, we compared action potential firing in cultured cortical neurons from wild-type and ASIC1a/2/3 null mice. We found that in the knockout, action potentials were narrow and exhibited increased after-hyperpolarization. Moreover, the excitability of these neurons was significantly increased. These findings are consistent with increased BK channel activity in the neurons from ASIC1a/2/3 null mice. Our data suggest that ASICs can act as endogenous pH-dependent inhibitors of BK channels, and thereby can reduce neuronal excitability.     

Structural and functional analysis of natural capsid variants suggests sialic acid-independent entry of BK polyomavirus

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State-dependent inhibition of BK channels by the opioid agonist loperamide

Large-conductance Ca²⁺-activated K⁺ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K⁺ efflux through intestinal BK channels.     

State-dependent block of BK channels by synthesized shaker ball peptides

Crystal structures of potassium channels have strongly corroborated an earlier hypothetical picture based on functional studies, in which the channel gate was located on the cytoplasmic side of the pore. However, accessibility studies on several types of ligand-sensitive K(+) channels have suggested that their activation gates may be located near or within the selectivity filter instead. It remains to be determined to what extent the physical location of the gate is conserved across the large K(+) channel family. Direct evidence about the location of the gate in large conductance calcium-activated K(+) (BK) channels, which are gated by both voltage and ligand (calcium), has been scarce. Our earlier kinetic measurements of the block of BK channels by internal quaternary ammonium ions have raised the possibility that they may lack a cytoplasmic gate. We show in this study that a synthesized Shaker ball peptide (ShBP) homologue acts as a state-dependent blocker for BK channels when applied internally, suggesting a widening at the intracellular end of the channel pore upon gating. This is consistent with a gating-related conformational change at the cytoplasmic end of the pore-lining helices, as suggested by previous functional and structural studies on other K(+) channels. Furthermore, our results from two BK channel mutations demonstrate that similar types of interactions between ball peptides and channels are shared by BK and other K(+) channel types.     

State-independent block of BK channels by an intracellular quaternary ammonium

Intracellular blockade by quaternary ammonium (QA) molecules of many potassium channels is state dependent, where the requirement for channel opening is evidenced by a time-dependent component of block in the macroscopic record. Whether this is the case for Ca(2+)- and voltage-activated potassium (BK) channels, however, remains unclear. Previous work (Li, W., and R.W. Aldrich. 2004. J. Gen. Physiol. 124:43-57) tentatively proposed a state-dependent, trapping model, but left open the possibility of state-independent block. Here, we found BK channel blockade by a novel QA derivative, bbTBA, was time dependent, raising the possibility of state-dependent, open channel block. Alternatively, the observed voltage dependence of block could be sufficient to explain time-dependent block. We have used steady-state and kinetic measurements of bbTBA blockade in order to discriminate between these two possibilities. bbTBA did not significantly slow deactivation kinetics at potentials between -200 and -100 mV, suggesting that channels can close unhindered by bound bbTBA. We further find no evidence that bbTBA is trapped inside BK channels after closing. Measurements of steady state fractional block at +40 mV revealed a 1.3-fold change in apparent affinity for a 33-fold change in P(o), in striking contrast to the 31-fold change predicted by state-dependent block. Finally, the appearance of a third kinetic component of bbTBA blockade at high concentrations is incompatible with state-dependent block. Our results suggest that access of intracellular bbTBA to the BK channel cavity is not strictly gated by channel opening and closing, and imply that the permeation gate for BK channels may not be intracellular.     

Generation of functional I-Ed variants from an antigen-presenting macrophage cell line

We report the ability of the BAC1.2 macrophage cell line to present antigen both to populations of antigen-primed lymph node cells and to T cell hybridomas. We also describe the selection of I-Ed variants derived from this cell line. These variants have lost the ability to present antigen to an I-Ed plus ovalbumin-specific T cell line, but retain the ability to stimulate an I-Ad plus ovalbumin-restricted T cell line, and a T cell line reactive with I-Ed alone. These variants should aid in the elucidation of structural elements of the I-E molecule involved in the interaction with helper T cells.     

Closed-channel block of BK potassium channels by bbTBA requires partial activation

Blockade of large-conductance Ca²⁺-activated K⁺ (BK) channels by the bulky quaternary ammonium compound, N-(4-[benzoyl]benzyl)-N,N,N-tributylammonium (bbTBA), exhibits features consistent with blockade of both closed and open states. Here, we examine block of closed BK channels by bbTBA and how it may differ from block of open channels. Although our observations generally confirm earlier results, we describe three observations that are inconsistent with a model in which closed and open channels are equally accessible to blockade by bbTBA. First, block by bbTBA exhibits Ca²⁺-dependent features that are inconsistent with strictly state-independent block. Second, the steady-state voltage dependence of bbTBA block at negative potentials shows that any block of completely closed states either does not occur or is completely voltage independent. Third, determination of the fractional unblock by bbTBA at either low or high Ca²⁺ reveals deviations from a model in which open- and closed-state block is identical. The results support the view that bbTBA blockade of fully closed channels does not occur. We imagine two general types of explanation. First, a stronger voltage dependence of closed-channel block may minimize the contribution of closed-channel block at negative potentials. Second, voltage-dependent conformational changes among closed-channel states may permit block by bbTBA. The analysis supports the latter view, suggesting that bbTBA blockade of fully closed channels does not occur, but the ability of bbTBA to block a closed channel requires movement of one or more voltage sensors. Models in which block is coupled to voltage sensor movement can qualitatively account for (1) the ability of open-channel block to better fit block of conductance–voltage curves at high Ca²⁺; (2) the voltage dependence of fractional availability; and (3) the fractional unblock at different open probabilities. BK channels appear to undergo voltage-dependent conformational changes among closed states that are permissive for bbTBA block.     

Ca2+-activated K channels in parotid acinar cells: The functional basis for the hyperpolarized activation of BK channels

Fluid secretion relies on a close interplay between Ca²⁺-activated Cl and K channels. Salivary acinar cells contain both large conductance, BK, and intermediate conductance, IK1, K channels. Physiological fluid secretion occurs with only modest (<500 nM) increases in intracellular Ca²⁺ levels but BK channels in many cell types and in heterologous expression systems require very high concentrations for significant activation. We report here our efforts to understand this apparent contradiction. We determined the Ca²⁺ dependence of IK1 and BK channels in mouse parotid acinar cells. IK1 channels activated with an apparent Ca²⁺ affinity of about 350 nM and a hill coefficient near 3. Native parotid BK channels activated at similar Ca²⁺ levels unlike the BK channels in other cell types. Since the parotid BK channel is encoded by an uncommon splice variant, we examined this clone in a heterologous expression system. In contrast to the native parotid channel, activation of this expressed “parslo” channel required very high levels of Ca²⁺. In order to understand the functional basis for the special properties of the native channels, we analyzed the parotid BK channel in the context of the horrigan-Aldrich model of BK channel gating. We found that the shifted activation of parotid BK channels resulted from a hyperpolarizing shift of the voltage dependence of voltage sensor activation and channel opening and included a large change in the coupling of these two processes.Key words: ion channels, Ca²⁺-activated K channels, maxi-K channels, IK1 channels     

Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism

Paxilline, a tremorogenic fungal alkaloid, potently inhibits large conductance Ca²⁺- and voltage-activated K⁺ (BK)-type channels, but little is known about the mechanism underlying this inhibition. Here we show that inhibition is inversely dependent on BK channel open probability (Po), and is fully relieved by conditions that increase Po, even in the constant presence of paxilline. Manipulations that shift BK gating to more negative potentials reduce inhibition by paxilline in accordance with the increase in channel Po. Measurements of Po times the number of channels at negative potentials support the idea that paxilline increases occupancy of closed states, effectively reducing the closed–open equilibrium constant, L(0). Gating current measurements exclude an effect of paxilline on voltage sensors. Steady-state inhibition by multiple paxilline concentrations was determined for four distinct equilibration conditions, each with a distinct Po. The IC₅₀ for paxilline shifted from around 10 nM when channels were largely closed to near 10 µM as maximal Po was approached. Model-dependent analysis suggests a mechanism of inhibition in which binding of a single paxilline molecule allosterically alters the intrinsic L(0) favoring occupancy of closed states, with affinity for the closed conformation being >500-fold greater than affinity for the open conformation. The rate of inhibition of closed channels was linear up through 2 µM paxilline, with a slope of 2 × 10⁶ M⁻¹s⁻¹. Paxilline inhibition was hindered by either the bulky cytosolic blocker, bbTBA, or by concentrations of cytosolic sucrose that hinder ion permeation. However, paxilline does not hinder MTSET modification of the inner cavity residue, A313C. We conclude that paxilline binds more tightly to the closed conformation, favoring occupancy of closed-channel conformations, and propose that it binds to a superficial position near the entrance to the central cavity, but does not hinder access of smaller molecules to this cavity.     

Unique inner pore properties of BK channels revealed by quaternary ammonium block

Potassium channels have a very wide distribution of single-channel conductance, with BK type Ca(2+)-activated K(+) channels having by far the largest. Even though crystallographic views of K(+) channel pores have become available, the structural basis underlying BK channels' large conductance has not been completely understood. In this study we use intracellularly applied quaternary ammonium compounds to probe the pore of BK channels. We show that molecules as large as decyltriethylammonium (C(10)) and tetrabutylammonium (TBA) have much faster block and unblock rates in BK channels when compared with any other tested K(+) channel types. Additionally, our results suggest that at repolarization large QA molecules may be trapped inside blocked BK channels without slowing the overall process of deactivation. Based on these findings we propose that BK channels may differ from other K(+) channels in its geometrical design at the inner mouth, with an enlarged cavity and inner pore providing less spatially restricted access to the cytoplasmic solution. These features could potentially contribute to the large conductance of BK channels.     

Martentoxin:一种大电导钙激活钾离子通道的独有配体(英文)

大电导钙激活钾离子(BK)通道广泛分布于可兴奋细胞与非兴奋细胞中,行使着一系列重要的生理功能。以源于蝎粗毒的高亲和性毒素作为研究工具,使BK通道的药理学和结构性质正逐步被揭示。Martentoxin是一种分离提取自东亚短钳蝎(Buthus martensi Karsch)粗毒的短链多肽,由37个氨基酸残基构成。研究表明,其对BK通道的特异性远高于其它各类型的电压门控钾通道(Kv)。迄今为止,由于用以探明BK通道亚型结构与功能及相关病理的特异性药物工具仍然稀缺,因此阐明martentoxin与BK通道间的相互作用模式就显得至关重要了。鉴于此原因,本综述将针对martentoxin的药理性质和其与BK通道相互作用的分子机制做进一步阐明。     

GFP在绿色荧光裸鼠不同组织中的表达及分析

目的研究外源绿色荧光蛋白（green fluorescent protein,简称GFP）基因在BALB/c绿色荧光裸鼠主要器官组织中的表达及其差异。方法小动物成像系统和RT-PCR方法检测GFP的组织分布以及荧光表达水平情况。结果经活体荧光影像系统观察及PCR方法检测发现GFP可以在裸鼠多个器官组织中表达,其中在胰腺、心脏、全脑、皮肤、睾丸中表达量较高。结论外源绿色荧光蛋白可以在模型动物体内成功表达且稳定遗传,其中在胰腺组织中高表达。     

Mechanism of beta4 subunit modulation of BK channels

Large-conductance (BK-type) Ca(2+)-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca(2+). BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (beta1-beta4). Biophysical characterization has shown that the beta4 subunit confers properties of the so-called "type II" BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the beta4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca(2+) sensitivity. Specifically, channel activity at low Ca(2+) is inhibited, while at high Ca(2+), activity is enhanced. The goal of this study is to understand the mechanism underlying beta4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that beta4's most profound effect is a decrease in P(o) (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, beta4 promotes channel opening by increasing voltage dependence of P(o)-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of beta4 on BK channels. beta4 reduces channel opening by decreasing the intrinsic gating equilibrium (L(0)), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, beta4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vh(o)) to more negative membrane potentials. The consequence is that beta4 causes a net positive shift of the G-V relationship (relative to alpha subunit alone) at low calcium. At higher calcium, the contribution by Vh(o) and an increase in allosteric coupling to Ca(2+) binding (C) promotes a negative G-V shift of alpha+beta4 channels as compared to alpha subunits alone. This manner of modulation predicts that type II BK channels are downregulated by beta4 at resting voltages through effects on L(0). However, beta4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.     

Regulation of endothelial BK channels by heme oxygenase-derived carbon monoxide and caveolin-1

A novel vasodilatory influence of endothelial cell (EC) large-conductance Ca(2+)-activated K(+) (BK) channels is present after in vivo exposure to chronic hypoxia (CH) and may exist in other pathological states. However, the mechanism of channel activation that results in altered vasoreactivity is unknown. Previously, we demonstrated that inhibition of either BK channels or heme oxygenase (HO) restores vasoconstrictor reactivity after CH. Additionally, administration of the scaffolding domain of caveolin (Cav)-1 inhibits EC BK activity and restores vasoconstrictor reactivity in this setting. These results led us to hypothesize that CH exposure results in a loss in Cav-1 inhibition of EC BK channels, resulting in their activation by HO-derived carbon monoxide (CO). Experiments were conducted on freshly dispersed aortic ECs from control and CH-exposed (barometric pressure: 380 mmHg for 48 h) rats. In electrophysiology experiments, outward currents were greater in cells from CH rats as well as from cells from control rats treated with the cholesterol-depleting agent methyl-β-cyclodextrin. These enhanced currents were returned to control by HO inhibition. Channel activity could be restored by the CO donor CO-releasing molecule (CORM)-2 during HO inhibition. Administration of the Cav-1 scaffolding domain eliminated BK currents in cells from CH rats, and current was not restored by the addition of CORM-2. Colocalization experiments in ECs from control and CH rats demonstrated an association between HO-2, Cav-1, and BK. We conclude that EC BK channel activity is HO dependent in the absence of the inhibitory effect of the Cav-1 scaffolding domain.     

Intra- and intersubunit cooperativity in activation of BK channels by Ca2+

The activation of BK channels by Ca(2+) is highly cooperative, with small changes in intracellular Ca(2+) concentration having large effects on open probability (Po). Here we examine the mechanism of cooperative activation of BK channels by Ca(2+). Each of the four subunits of BK channels has a large intracellular COOH terminus with two different high-affinity Ca(2+) sensors: an RCK1 sensor (D362/D367) located on the RCK1 (regulator of conductance of K(+)) domain and a Ca-bowl sensor located on or after the RCK2 domain. To determine interactions among these Ca(2+) sensors, we examine channels with eight different configurations of functional high-affinity Ca(2+) sensors on the four subunits. We find that the RCK1 sensor and Ca bowl contribute about equally to Ca(2+) activation of the channel when there is only one high-affinity Ca(2+) sensor per subunit. We also find that an RCK1 sensor and a Ca bowl on the same subunit are much more effective in increasing Po than when they are on different subunits, indicating positive intrasubunit cooperativity. If it is assumed that BK channels have a gating ring similar to MthK channels with alternating RCK1 and RCK2 domains and that the Ca(2+) sensors act at the flexible (rather than fixed) interfaces between RCK domains, then a comparison of the distribution of Ca(2+) sensors with the observed responses suggest that the interface between RCK1 and RCK2 domains on the same subunit is flexible. On this basis, intrasubunit cooperativity arises because two high-affinity Ca(2+) sensors acting across a flexible interface are more effective in opening the channel than when acting at separate interfaces. An allosteric model incorporating intrasubunit cooperativity nested within intersubunit cooperativity could approximate the Po vs. Ca(2+) response for eight possible subunit configurations of the high-affinity Ca(2+) sensors as well as for three additional configurations from a previous study.     

Monitoring membrane binding and insertion of peptides by two-color fluorescent label

Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide-membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.     

Steady-state and closed-state inactivation properties of inactivating BK channels

Calcium-dependent potassium (BK-type) Ca2+ and voltage-dependent K+ channels in chromaffin cells exhibit an inactivation that probably arises from coassembly of Slo1 alpha subunits with auxiliary beta subunits. One goal of this work was to determine whether the Ca2+ dependence of inactivation arises from any mechanism other than coupling of inactivation to the Ca2+ dependence of activation. Steady-state inactivation and the onset of inactivation were studied in inside-out patches and whole-cell recordings from rat adrenal chromaffin cells with parallel experiments on inactivating BK channels resulting from cloned alpha + beta2 subunits. In both cases, steady-state inactivation was shifted to more negative potentials by increases in submembrane [Ca2+] from 1 to 60 microM. At 10 and 60 microM Ca2+, the maximal channel availability at negative potentials was similar despite a shift in the voltage of half availability, suggesting there is no strictly Ca2+-dependent inactivation. In contrast, in the absence of Ca2+, depolarization to potentials positive to +20 mV induces channel inactivation. Thus, voltage-dependent, but not solely Ca2+-dependent, kinetic steps are required for inactivation to occur. Finally, under some conditions, BK channels are shown to inactivate as readily from closed states as from open states, indicative that a key conformational change required for inactivation precedes channel opening.