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Molecular basis of inward rectification: polyamine interaction sites located by combined channel and ligand mutagenesis 下载免费PDF全文
Kurata HT Phillips LR Rose T Loussouarn G Herlitze S Fritzenschaft H Enkvetchakul D Nichols CG Baukrowitz T 《The Journal of general physiology》2004,124(5):541-554
Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter. 相似文献
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Dev K. Singh Avia Rosenhouse-Dantsker Colin G. Nichols Decha Enkvetchakul Irena Levitan 《The Journal of biological chemistry》2009,284(44):30727-30736
Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by 86Rb+ uptake. Our results show that 86Rb+ flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses 86Rb+ flux by >50%, and activity is completely inhibited at 12–15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated 86Rb+ uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 μg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific.Inwardly rectifying potassium channels (Kir) are known to play critical roles in the regulation of multiple cellular functions including membrane excitability, heart rate, and vascular tone (1–3). Kir channels are classified into seven subfamilies (Kir1–7) identified by distinct biophysical properties and sensitivities to different regulators (2). Our earlier studies have shown that Kir2 channels, one of the major subfamilies of Kir that are responsible for maintaining membrane potential in a variety of cell types, are strongly suppressed by the elevation of membrane cholesterol (4, 5). Cholesterol-induced suppression of Kir2 was first observed in aortic endothelial cells (4), in which resting K+ conductance is dominated by Kir2.1 and Kir2.2 channels (6), and then when channels were heterologously expressed in Chinese hamster ovary cells (5, 7). Furthermore, the same effect was observed ex vivo in endothelial cells and bone marrow-derived progenitor cells isolated from hypercholesterolemic pigs (8, 9).In terms of the mechanism, the first insights came from comparing the effects of cholesterol and of its chiral analogue, epicholesterol. Although the two sterols are known to have almost identical effects on the biophysical properties of the lipid bilayer (10, 11), their impact on Kir activity is completely different; partial substitution of endogenous cholesterol with epicholesterol resulted in significant increase in Kir current in endothelial cells (4). These observations suggest that specific sterol-protein interactions may be involved in the cholesterol sensitivity of Kir2 channels. However, in the complex environment of the plasma membrane, cholesterol may interact not only with the channels themselves but also with other proteins, which in turn may regulate the activity of the channels. In the cellular environment, therefore, it is impossible to discriminate between direct channel-cholesterol interactions and indirect effects. Moreover, it is impossible to define the actual concentrations of cholesterol in any given membrane compartment. To quantitatively test direct cholesterol-protein interactions, it is necessary to examine sensitivity of pure Kir channels to membrane cholesterol in a membrane of defined lipid composition. To date, only the cytoplasmic domains of several mammalian Kir channels have been purified (Kir2.1, Kir3.1, and Kir3.2) (12–15). We therefore concentrate in this study on the effect of cholesterol on two bacterial K+ channels that differ in the level of their homology to mammalian Kir channels, KirBac1.1 and KcsA. KirBac channels have high sequence homology with mammalian Kirs (e.g. 52% homology between KirBac1.1 and Kir2.1; see Fig. 7A) and have now been extensively used as structural models of mammalian Kir channels (3, 16, 17). The sequence similarity between KcsA and mammalian K channels lies mainly in the transmembrane domain (18). The overall sequence homology of KcsA to mammalian Kir channels is relatively low (e.g. 22% homology between KcsA and Kir2.1; see Fig. 7A), with an entirely different cytoplasmic domain structure.Open in a separate windowFIGURE 7.Cholesterol has no effect on KcsA-mediated 86Rb+ uptake. A, time courses of 86Rb+ uptake into liposomes reconstituted with 50 μg of cholesterol/mg of PL and as compared with liposomes containing no cholesterol (control). Both batches of liposomes contained 5 μg of KcsA/mg of PL. Blank liposomes contain no protein. The points represent averages of three independent experiments (means ± S.D.). B, normalized time courses of 86Rb+ uptake in liposomes incorporating 50, 150, and 250 μg of cholesterol/mg of PL. C, maximal uptake of 86Rb+ after 240 s in liposomes containing 10, 25, 50, 100, 150, 200, and 250 μg of cholesterol/mg of PL normalized to control (means ± S.D. of 3–5 independent experiments; *, p < 0.05). DPM, disintegrations per minute.Here we show that, similarly to Kir2 channels, prokaryotic Kir channels incorporated into liposomes are strongly suppressed by an increase in membrane cholesterol. Furthermore, the sensitivity of prokaryotic Kir to cholesterol is stereo-selective to cholesterol optical analogues. In contrast, KscA channels are insensitive to membrane cholesterol. These observations suggest that cholesterol directly suppresses Kir channels. 相似文献
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Shizhen Wang Yewande Alimi Ailing Tong Colin G. Nichols Decha Enkvetchakul 《The Journal of biological chemistry》2009,284(5):2854-2860
Potassium channels are tetrameric proteins that mediate
K+-selective transmembrane diffusion. For KcsA, tetramer stability
depends on interactions between permeant ions and the channel pore. We have
examined the role of pore blockers on the tetramer stability of KirBac1.1. In
150 mm KCl, purified KirBac1.1 protein migrates as a monomer
(∼40 kDa) on SDS-PAGE. Addition of Ba2+
(K1/2 ∼ 50 μm) prior to loading results
in an additional tetramer band (∼160 kDa). Mutation A109C, at a residue
located near the expected Ba2+-binding site, decreased tetramer
stabilization by Ba2+ (K1/2 ∼ 300
μm), whereas I131C, located nearby, stabilized tetramers in the
absence of Ba2+. Neither mutation affected Ba2+ block of
channel activity (using 86Rb+ flux assay). In contrast
to Ba2+, Mg2+ had no effect on tetramer stability (even
though Mg2+ was a potent blocker). Many studies have shown
Cd2+ block of K+ channels as a result of cysteine
substitution of cavity-lining M2 (S6) residues, with the implicit
interpretation that coordination of a single ion by cysteine side chains along
the central axis effectively blocks the pore. We examined blocking and
tetramer-stabilizing effects of Cd2+ on KirBac1.1 with cysteine
substitutions in M2. Cd2+ block potency followed an α-helical
pattern consistent with the crystal structure. Significantly, Cd2+
strongly stabilized tetramers of I138C, located in the center of the inner
cavity. This stabilization was additive with the effect of Ba2+,
consistent with both ions simultaneously occupying the channel:
Ba2+ at the selectivity filter entrance and Cd2+
coordinated by I138C side chains in the inner cavity.Potassium channels are expressed in many cell types and are key players in
a wide range of physiological processes. One subset of potassium channels, the
inward-rectifying potassium (Kir) channels, are functionally blocked by
cytosolic cations such as Mg2+ and polyamines and contribute to the
regulation of membrane excitability, cardiac rhythm, vascular tone, insulin
release, and salt flow across epithelia
(1–3).
There are seven subfamilies of eukaryotic Kir channel genes. Among them, Kir1
encodes weak rectifiers, whereas Kir2 and Kir5 encode strong rectifiers; Kir3
encodes G-protein-regulated channels; and Kir6 encodes ATP-sensitive channels
(4). Recently, a related
bacterial family of genes (KirBac) has been identified
(5,
6), and in 2003, the first
member (KirBac1.1) was crystallized
(7), providing a structural
model for eukaryotic channels.The crystal structure of KirBac1.1 revealed a tetrameric pore structure
similar to that seen in KcsA and a novel cytoplasmic domain
(7,
8). The selectivity filter of
both KirBac1.1 and KcsA consists of an extremely conserved pore loop followed
by a central cavity, forming a transmembrane ion-selective permeation pore
(7,
8). The linear arrangement of
five oxygen rings (four from carbonyl oxygens and one from a Thr side chain)
in the selectivity filter coordinates with ions, compensating for the energy
barrier caused by K+ dehydration, thereby facilitating the rapid
diffusion of K+ across the membrane
(8–12).
Two-thirds of the KirBac1.1 amino acid residues constitute the cytosolic
domain that is highly conserved among the Kir subfamilies and form the
cytosolic vestibule
(13–16),
which, together with the transmembrane pore, generates an 88-Å-long ion
conduction pore (7).The prototypic potassium channel KcsA exists very stably as a tetramer,
even in the harsh conditions of SDS-PAGE
(17). In addition to
protein-protein interaction between monomers, protein-lipid and protein-ion
interactions play important roles in stabilizing the KcsA tetramer
(17–20).
The selectivity filter of KcsA, coordinated with K+ ions, can serve
as a bridge between the four monomers to maintain the structure of the
selectivity filter and the tetrameric architecture of the channel as a whole
(11,
21). Blocking ions, such as
Ba2+, also act as strong stabilizers
(17). In the crystal structure
of KcsA, Ba2+ occupies a site equivalent to the S4
K+-binding site within the selectivity filter
(22). Other permeant ions
(Rb+, Cs+, Tl+, and
NH+4) and strong blockers (Sr2+) can also
contribute to the thermostability of the KcsA tetramer in SDS-PAGE
(17). In contrast, impermeant
ions such as Na+ and Li+ or weak blockers such as
Mg2+ tend to destabilize the KcsA tetramer
(17,
19).Like KcsA, KirBac1.1 purified using decylmaltoside or tridecylmaltoside is
active and presumably stable as a tetramer in mild detergent solutions.
However, in SDS-PAGE, KirBac1.1 migrates exclusively as a monomer
(23). Because KcsA and
KirBac1.1 are structurally similar in the transmembrane region of the pore, we
hypothesized that permeant and blocking ions would also affect KirBac1.1
tetramer stability in SDS-PAGE. In the present work, the effects of blocking
ions such as Ba2+ and Mg2+ on KirBac1.1 tetramer
stability were examined to provide insight to the physical nature of their
interaction with KirBac1.1, particularly in the selectivity filter and TM2
cavity. The data reveal important differences in the nature of the interaction
of Mg2+ and Ba2+ with the channel as well as provide
previously unavailable evidence for the nature of Cd2+ coordination
within the channel. 相似文献
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Michaela Mühlberg Da’san M.M. Jaradat Rolf Kleineweischede Ilona Papp Decha Dechtrirat Silvia Muth Malgorzata Broncel Christian P.R. Hackenberger 《Bioorganic & medicinal chemistry》2010,18(11):3679-3686
The traceless Staudinger ligation has recently found various applications in the field of peptide synthesis and modification, including immobilization and cyclization strategies. In this report, we utilize the traceless Staudinger ligation in the formation of amide bonds, which allows the acquisition of acylated aminosugars and peptides as well as the cyclization of peptides. A key element in these synthetic procedures is the use of a borane-protected phosphinomethanethiol, which is demonstrated to be prone towards oxidation in its unprotected form, during the synthesis of phosphinothioesters. In combination with acidic and basic deprotection strategies for the borane-protected phosphinothioesters, amide bonds can be formed in the presence of azides in moderate to good overall yields. 相似文献
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Kristian Krag Rasmus Lundegaard Joachim Offenberg Mogens Gissel Nielsen Decha Wiwatwittaya 《Journal of Asia》2010,13(2):97-100
Oecophylla ants are utilized for biological control in fruit plantations in Australia and Asia. In Asia, queen larvae and alates are sold on commercial markets for human and animal consumption. This double utilization has induced an increasing interest in the domestication of these ants, but attempts to rear live colonies have been hindered partly by the length of time it takes from the founding of a colony until it can be utilized commercially. Early growth of a colony may be increased if ants from other colonies are adopted. The present experiments show that Oecophylla smaragdina larvae transplanted from other colonies are readily tolerated by non-nestmate workers and are reared to imagos. These results are fundamental for the future domestication of Oecophylla and elucidate the need for further studies of chemical nestmate recognition. 相似文献
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Many eukaryotic channels, transporters and receptors are activated by phosphatidyl inositol bisphosphate (PIP(2)) in the membrane, and every member of the eukaryotic inward rectifier potassium (Kir) channel family requires membrane PIP(2) for activity. In contrast, a bacterial homolog (KirBac1.1) is specifically inhibited by PIP(2). We speculate that a key evolutionary adaptation in eukaryotic channels is the insertion of additional linkers between transmembrane and cytoplasmic domains, revealed by new crystal structures, that convert PIP(2) inhibition to activation. Such an adaptation may reflect a novel evolutionary drive to protein structure, and that was necessary to permit channel function within the highly negatively charged membranes that evolved in the eukaryotic lineage. 相似文献
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Akinori Yamada Tetsushi Inoue Decha Wiwatwitaya Moriya Ohkuma Toshiaki Kudo Atsuko Sugimoto 《Ecosystems》2006,9(1):75-83
Nitrogen (N) fixed by termites was evaluated as a N input to decomposition processes in two tropical forests, a dry deciduous
forest (DDF) and the neighboring dry evergreen forest (DEF), Thailand. A diverse group of termite species were assayed by
acetylene reduction method and only the wood/litter-feeding termites were found to fix N. More intensive samplings of two
abundant species, Microcerotermes crassus and Globitermes sulphureus, were done across several seasons, suggesting N fixation rates of 0.21 and 0.28 kg ha−1 y−1 by termites in the DDF and DEF, respectively. Also, estimates of asymbiotic N fixation rates were 0.75 and 3.95 kg ha−1 y−1. N fixed by termites and by asymbiotic fixers is directly supplied to decomposers breaking down dead plant material and could
be a major source of their N. N fixed by termites was 7–22% of that fixed by termites and asymbiotic fixers. Although N fixed
by termites is a small input compared to other inputs, this N is likely important for decomposition processes. 相似文献