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1.
A chloride-permeable channel from Phaseolus vulgaris roots incorporated into planar lipid bilayers 总被引:1,自引:0,他引:1
Balleza D Quinto C Sánchez F Gómez-Lagunas F 《Biochemical and biophysical research communications》2003,307(1):114-118
Ion channels are key participants in physiological processes of plant cells. Here, we report the first characterization of a high conductance, Cl(-)-permeable channel, present in enriched fractions of plasma membranes of bean root cells. The Cl(-) channel was incorporated into planar lipid bilayers and its activity was recorded under voltage clamp conditions. The channel is voltage-dependent, excludes the passage of cations (K(+), Na(+), and Ca(2+)), and is inhibited by micromolar concentrations of Zn(2+). The Cl(-) conductance here characterized represents a previously undescribed channel of plant cells. 相似文献
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Gómez-Lagunas F Batista CV Olamendi-Portugal T Ramírez-Domínguez ME Possani LD 《The Journal of general physiology》2004,123(3):265-279
The Shaker B K(+) conductance (G(K)) collapses when the channels are closed (deactivated) in Na(+) solutions that lack K(+) ions. Also, it is known that external TEA (TEA(o)) impedes the collapse of G(K), and that channel block by TEA(o) and scorpion toxins are two mutually exclusive events. Therefore, we tested the ability of scorpion toxins to inhibit the collapse of G(K) in 0 K(+). We have found that these toxins are not uniform regarding the capacity to protect G(K). Those toxins, whose binding to the channels is destabilized by external K(+), are also effective inhibitors of the collapse of G(K). In addition to K(+), other externally added cations also destabilize toxin block, with an effectiveness that does not match the selectivity sequence of K(+) channels. The inhibition of the drop of G(K) follows a saturation relationship with [toxin], which is fitted well by the Michaelis-Menten equation, with an apparent Kd bigger than that of block of the K(+) current. However, another plausible model is also presented and compared with the Michaelis-Menten model. The observations suggest that those toxins that protect G(K) in 0 K(+) do so by interacting either with the most external K(+) binding site of the selectivity filter (suggesting that the K(+) occupancy of only that site of the pore may be enough to preserve G(K)) or with sites capable of binding K(+) located in the outer vestibule of the pore, above the selectivity filter. 相似文献
4.
Side chain flexibility and coupling between the S4‐S5 linker and the TRP domain in thermo‐sensitive TRP channels: Insights from protein modeling
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The transient receptor potential (TRP) superfamily is subdivided into several subfamilies on the basis of sequence similarity, which is highly heterogeneous but shows a molecular architecture that resembles the one present in members of the Kv channel superfamily. Because of this diversity, they produce a large variety of channels with different gating and permeability properties. Elucidation of these particular features necessarily requires comparative studies based on structural and functional data. The present study aims to compilate, analyze, and determine, in a coherent way, the relationship between intrinsic side‐chain flexibility and the allosteric coupling in members of the TRPV, TRPM, and TRPC families. Based on the recently determined structures of TRPV1 and TRPV2, we have generated protein models for single subunits of TRPV5, TRPM8, and TRPC5 channels. With these models, we focused our attention on the apparently crucial role of the GP dipeptide at the center of the S4‐S5 linker and discussed its role in the interaction with the TRP domain, specifically with the highly‐conserved Trp during this coupling. Our analysis suggests an important role of the S4‐S5L flexibility in the thermosensitivity, where heat‐activated channels possess rigid S4‐S5 linkers, whereas cold‐activated channels have flexible ones. Finally, we also present evidence of the key interaction between the conserved Trp residue of the TRP box and of several residues in the S4‐S5L, importantly the central Pro. Proteins 2017; 85:630–646. © 2016 Wiley Periodicals, Inc. 相似文献
5.
Mechanosensitive (MS) channels play a major role in protecting bacterial cells against hypo-osmotic shock. To understand their
function, it is important to identify the conserved motifs using sequence analysis methods. In this study, the sequence conservation
was investigated by an in silico analysis to generate sequence logos. We have identified new conserved motifs in the domains
TM1, TM2 and the cytoplasmic helix from 231 homologs of MS channel of large conductance (MscL). In addition, we have identified
new motifs for the TM3 and the cytoplasmic carboxy-terminal domain from 309 homologs of MS channel of small conductance (MscS).
We found that the conservation in MscL homologs is high for TM1 and TM2 in the three domains of life. The conservation in
MscS homologs is high only for TM3 in Bacteria and Archaea.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
6.
Froylan Vargas 《Journal of neurochemistry》1990,54(3):809-814
3'-Phosphoadenosine 5'-phospho[35S]sulfate [( 35S]PAPS) specific binding properties of rat brain tissue were studied. [35S]PAPS specific binding was optimal at pH 5.8 in either Tris-maleate or potassium phosphate buffers. Association was maximal at low temperature, reaching equilibrium in 20 min. Dissociation was rapid, with a dissociation time of 80 s. Scatchard analysis of [35S]PAPS specific binding was consistent with a single site having a KD of 0.46 +/- 0.06 microM and a Bmax of 20.8 +/- 2.0 pmol/mg of protein. Low concentrations of Triton X-100 (0.025%) were effective in increasing the number of binding sites to a Bmax of 44.5 +/- 4.6 pmol/mg of protein without affecting the affinity. [35S]PAPS specific binding was enriched in crude synaptic membranes (P2) and microsomes (P3). Regional distribution of [35S]PAPS specific binding was quite homogeneous in all brain structures studied. The pharmacological profile of [35S]PAPS specific binding in rat brain microsomes was consistent with a membrane protein having a high selectivity for the 3'-O-phosphoryl group substitution on the ribose moiety. Thus, 3'-phosphoadenosine 5'-phosphate was more potent than 2'-phosphoadenosine 5'-phosphate in competing for [35S]PAPS specific binding. Adenosine 5'-phosphosulfate was a good inhibitor of [35S]PAPS specific binding. ATP and ADP were also good displacers. Dipyridamole, a highly selective marker for adenosine uptake sites, was ineffective. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid, the chloride transporter inhibitor, showed an IC50 of 36 +/- 5.1 microM for inhibition of [35S]PAPS specific binding. 2,6-Dichloro-4-nitrophenol had a low selectivity in competing for the [35S]PAPS binding site.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
Batista CV D'Suze G Gómez-Lagunas F Zamudio FZ Encarnación S Sevcik C Possani LD 《Proteomics》2006,6(12):3718-3727
The Venezuelan scorpion Tityus discrepans is known to cause human fatalities. We describe the first complete proteomic analysis of its venom. By HPLC 58 different fractions were obtained and 205 different components were identified by MS analysis. Components having molecular masses from 272 to 57 908 amu were found. Forty homogeneous components had their N-terminal amino acid sequence determined by Edman degradation, from which two new peptides named TdK2 and TdK3 (meaning T. discrepans (Td) K(+) channel toxins 2 and 3) were fully characterized. The first contains 34 amino acid residues with a molecular mass of 3451 amu, and the second has 36 amino acids with 3832 amu. Both peptides are tightly bound by three disulfide bridges. TdK2 was shown to block reversibly the Shaker B K(+)-channel expressed heterologously in Sf9 cells. The systematic number assigned to TdK2 is alpha-KTx-18.2 and that of TdK3 is alpha-KTx-18.3. Comparative analysis of the amino acid sequences found suggests that this venom contains peptides highly similar to those that block K(+) channels, as well as those that modify the gating mechanisms of Na(+) channels, found in other scorpions. Additionally, peptides similar to defensins were also identified. 相似文献
8.
Saturable specific binding of glycine to synaptosomal membranes from plexiform layers of the retina has been described, which seems to correspond to the modulatory site on NMDA-receptors (26). Spermine inhibited specific [3H]glycine binding to membranes from synaptosomal fractions from the outer (P1) and the inner (P2) plexiform layers of 1–3 day-old chick retinas in a dose-dependent manner with an IC50 = 35 M for the P1 fraction and 32 M for the P2 fraction. Kinetic experiments and non-linear regression analysis of [3H]glycine-specific binding showed a Kd ~ 100–150 nM in both fractions, and a higher Bmax (4.11 ± 0.47 pmol/mg protein) for the inner plexiform layer compared to the outer plexiform layer (Bmax = 2.76 ± 0.25 pmol/mg protein). Strychnine-insensitive [3H]glycine binding was inhibited by 100 M spermine, due to a reduction in Bmax (P1 = 0.84 ± 0.16 pmol/mg protein; P2 = 0.81 ± 0.16 pmol/mg protein) without affecting the Kd. Association and dissociation constants in the absence and presence of 50 M spermine remained unchanged. Results demonstrate the presence of a single modulatory site for spermine on NMDA receptors, in both synaptic layers of the chick retina. 相似文献
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Batista CV Gómez-Lagunas F Rodríguez de la Vega RC Hajdu P Panyi G Gáspár R Possani LD 《Biochimica et biophysica acta》2002,1601(2):123-131
Two novel toxic peptides (Tc30 and Tc32) were isolated and characterized from the venom of the Brazilian scorpion Tityus cambridgei. The first have 37 and the second 35 amino acid residues, with molecular masses of 3,871.8 and 3,521.5, respectively. Both contain three disulfide bridges but share only 27% identity. They are relatively potent inhibitors of K(+)-currents in human T lymphocytes with K(d) values of 10 nM for Tc32 and 16 nM for Tc30, but they are less potent or quite poor blockers of Shaker B K(+)-channels, with respective K(d) values of 74 nM and 4.7 microM. Tc30 has a lysine in position 27 and a tyrosine at position 36 identical to those of charybdotoxin. These two positions conform the dyad considered essential for activity. On the contrary, Tc32 has a serine in the position equivalent to lysine 27 of charybdotoxin and does not contain any aromatic amino acid. Due to its unique primary sequence and to its distinctive preference for K(+)-channels of T lymphocytes, it was classified as the first example of a new subfamily of K(+)-channel-specific peptides (alpha-KT x 18.1). Tc30 is a member of the Tityus toxin II-9 subfamily and was given the number alpha-KT x 4.4. 相似文献
10.
Potassium channels allow the selective flux of K+ excluding the smaller, and more abundant in the extracellular solution, Na+ ions. Here we show that Shab is a typical K+ channel that excludes Na+ under bi-ionic, Nao/Ki or Nao/Rbi, conditions. However, when internal K+ is replaced by Cs+ (Nao/Csi), stable inward Na+ and outward Cs+ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca2+ ions, and compare the effect that internal K+ replacement exerts on both Ca2+ and TEA block. Our observations indicate that Ca2+ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na+ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca2+ is probably coordinated by main chain carbonyls of the pore´s first K+-binding site. 相似文献