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1.
Structural inferences for the native skeletal muscle sodium channel as derived from patterns of endogenous proteolysis 总被引:2,自引:0,他引:2
The alpha subunit (Mr approximately 260,000) of the rat skeletal muscle sodium channel is sensitive to cleavage by endogenous proteases during the isolation of muscle surface membrane. Antisera against synthetic oligopeptides were used to map the resultant fragments in order to identify protease-sensitive regions of the channel's structure in its native membrane environment. Antibodies to the amino terminus labeled major fragments of Mr approximately 130,000 and 90,000 and lesser amounts of other peptides as small as Mr approximately 12,000. Antisera to epitopes within the carboxyl-terminal half of the primary sequence recognized two fragments of Mr approximately 110,000 and 78,000. Individual antisera also selectively labeled smaller polypeptides in the most extensively cleaved preparations. The immunoreactivity patterns of monoclonal antibodies previously raised against the purified channel were then surveyed. The binding sites for one group of monoclonals, including several that recognize subtype-specific epitopes in the channel structure, were localized within a 12-kDa fragment near the amino terminus. The distribution of carbohydrate along the primary structure of the channel was also assessed by quantitating 125I-wheat germ agglutinin and 125I-concanavalin A binding to the proteolytic peptides. Most of the carbohydrate detected by these lectins was located between 22 and 90 kDa from the amino terminus of the protein. No lectin binding was detected to fragments arising from carboxyl-terminal half of the protein. These results were analyzed in terms of current models of sodium channel tertiary structure. In its normal membrane environment, the skeletal muscle sodium channel appears sensitive to cleavage by endogenous proteases in regions predicted to link the four repeat domains on the cytoplasmic side of the membrane while the repeat domains themselves are resistant to proteolysis. 相似文献
2.
Antibodies were raised against three peptides corresponding to the potential protein phosphorylation sites of rat-brain sodium channels by the cAMP-dependent protein kinase (PKA). One of the antibody against sequence (C561-575) reacted to the channel molecule. This immunoreaction occurred in a sequence-specific manner, as it was inhibited by the antigen peptide itself but not inhibited by two other peptides. Although PKA phosphorylates two synthetic peptides, C561-575 and C681-689, of the three, anti-(C561-575) antibody can only inhibit the phosphorylation of peptide (C561-575). PKA catalyzed the incorporation of 3.1-3.5 mol of phosphates into the alpha subunit of the purified sodium channel. The anti-(C561-575) antibody inhibited the channel phosphorylation by 40%. Digestion of the phosphorylated sodium channel with lysyl endoproteinase yielded four major phosphorylated fragments of 3.5, 5.0, 7.0, and 10 kDa. However, similar digestion of the channel that was phosphorylated in the presence of anti-(C561-575) antibody did not yield the phosphorylated fragment of 3.5 kDa and gave the 7.0 kDa fragment in reducing yield. Inspection of these phosphorylated fragments by the predicted sizes of the peptide fragments containing the five potential phosphorylation sites gives a conclusion that anti-(C561-575) antibody inhibits the phosphorylation on Ser-573 completely, and on either Ser-610 or Ser-623 partially, probably due to their proximity orientation in the tertiary structure. 相似文献
3.
Peptide fragments reproducing the sequences of S4 segments extended with L45 linkers from the four homologous domains of
the electric eel sodium channel were chemically synthesized and purified to allow circular dichroism studies in various solvents
and conductance assays in planar lipid bilayers. Repeats III (with proline) and IV (lacking proline) present the lowest and
highest helicities, respectively. The conformational transition (from helix to β-strand) shown to occur on an increase of solvent dielectric constant is broader with repeat III. Analytical ultracentrifugation
(interference fringe pattern) is consistent with a monodispersion of the peptide. In macroscopic conductance experiments,
the proline containing peptides (repeats I, II and especially III) display higher voltage-sensitivities than repeat IV. The
apparent and averaged number of monomers per intramembrane conducting aggregate is 4 – 5. The influence of proline is confirmed
in similar experiments carried out on homologous S4 segments of repeat IV of the human skeletal muscle sodium channel comparing
the wild type and an analogue where the fourth arginine was substituted with a proline. Thus, both conformational switching
and voltage-sensitivity appear correlated to the presence and position of a single proline residue. Since voltage sensors
are likely to experience different polarity environments in the channel open and closed states, our results suggest an alternative
gating mechanism, i. e. a voltage-driven conformational change of S4L45s. The data also implies a plausible functional asymmetry,
namely a “three- or four-stroke” activation sequentially involving the four domains of the sodium channel.
Received: 28 October 1997 / Revised version: 4 March 1998 / Accepted: 26 March 1998 相似文献
4.
《Channels (Austin, Tex.)》2013,7(5):281-290
Epithelial Sodium Channel (ENaC) proteolysis at sites in the extracellular loop of the α and γ subunits leads to marked activation. The mechanism of this effect remains debated, as well as the role of the N- and C-terminal fragments of these subunits created by cleavage. We introduced cysteines at sites bracketing upstream and downstream the cleavage regions in α and γ ENaC to examine the role of these fragments in the activated channel. Using thiol modifying reagents, as well as examining the effects of cleavage by exogenous proteases we constructed a functional model that determines the potential interactions of the termini near the cleavage regions. We report that the N-terminal fragments of both α and γ ENaC interact with the channel complex; with interactions between the N-terminal γ and the C-terminal α fragments being the most critical to channel function and activation by exogenous cleavage by subtilisin. Positive charge modification at a.a.135 in the N-terminal fragment of γ exhibited the largest inhibition of channel function. This region was found to interact with the C-terminal α fragment between a.a. 205 and 221; a tract which was previously identified to be the site of subtilisin's action. These data provide the first evidence for the functional channel rearrangement caused by proteolysis of the α and γ subunit and indicate that the untethered N-terminal fragments of these subunits interact with the channel complex. 相似文献
5.
The voltage-sensitive sodium channel from the electroplax of Electrophorus electricus is selectively phosphorylated by the catalytic subunit of cyclic-AMP-dependent protein kinase (protein kinase A) but not by protein kinase C. Under identical limiting conditions, the protein was phosphorylated 20% as rapidly as the synthetic model substrate kemptamide. A maximum of 1.7 +/- 0.6 equiv of phosphate is incorporated per mole. Phosphoamino acid analysis revealed labeled phosphoserine and phosphothreonine at a constant ratio of 3.3:1. Seven distinct phosphopeptides were identified among tryptic fragments prepared from radiolabeled, affinity-purified protein and resolved by HPLC. The three most rapidly labeled fragments were further purified and sequenced. Four phosphorylated amino acids were identified deriving from three consensus phosphorylation sites. These were serine 6, serine 7, and threonine 17 from the amino terminus and a residue within 47 amino acids of the carboxyl terminus, apparently serine 1776. The alpha-subunits of brain sodium channels, like the electroplax protein, are readily phosphorylated by protein kinase A. However, these are also phosphorylated by protein kinase C and exhibit a markedly different pattern of incorporation. Each of three brain alpha-subunits displays an approximately 200 amino acid segment between homologous repeat domains I and II, which is missing from the electroplax and skeletal muscle proteins [Noda et al. (1986) Nature (London) 320, 188; Kayano et al. (1988) FEBS Lett. 228, 1878; Trimmer et al. (1989) Neuron 3, 33]. Most of the phosphorylation of the brain proteins occurs on a cluster of consensus phosphorylation sites located in this segment. This contrasts with the pattern of highly active sites on the amino and carboxyl termini of the electroplax protein. The detection of seven labeled tryptic phosphopeptides compared to the maximal labeling stoichiometry of approximately 2 suggests that many of the acceptor sites on the protein may be blocked by endogenous phosphorylation. 相似文献
6.
J Littlechild A Malcolm K Paterakis I Ackermann J Dijk 《Biochimica et biophysica acta》1987,913(2):245-255
Ribosomal proteins from Escherichia coli have been isolated by a mild purification procedure. Their tertiary structure has been explored by two techniques, proton magnetic resonance and limited proteolysis. A number of proteins when subjected to limited proteolysis produce resistant fragments in good yields. In most cases this does not depend on the specificity of the enzyme used. The proteins S15, S16, S17 and L30 are not degraded at all, whereas a few proteins are very susceptible to proteolysis. 1H-NMR experiments show that the majority of the ribosomal proteins have a uniquely folded tertiary structure. This is particularly pronounced in the four proteins mentioned above which resist proteolysis. In general, a good agreement is observed between the degree of proteolytic resistance and the amount of folding indicated by NMR spectroscopy. Similar studies on a few ribosomal proteins purified under denaturing conditions show that, in contrast, these protein preparations are not structurally homogeneous and that they contain a mixture of denatured and renatured molecules. The results are interpreted in terms of a compactly folded tertiary structure for the four proteinase-resistant proteins while the majority of the other proteins appear to have two domains, one compactly folded and resistant to proteinase and the other flexible and susceptible to proteolysis. A few proteins seem to have a completely flexible structure and can therefore be easily degraded. 相似文献
7.
Identification of an intracellular domain of the sodium channel having multiple cAMP-dependent phosphorylation sites 总被引:9,自引:0,他引:9
Cyclic AMP-dependent protein kinase catalyzes the incorporation of 3-4 mol of phosphate into the alpha subunit of rat brain sodium channels in vitro or in situ. Digestion of phosphorylated sodium channels with CNBr yielded three major phosphorylated fragments of 25, 31, and 33 kDa. These fragments were specifically immunoprecipitated with site-directed antisera establishing their location within an intracellular loop between the first and second homologous domains containing residues 448 to 630 of sodium channel RI or residues 450-639 of sodium channel RII. Five of the seven major tryptic phosphopeptides generated from intact sodium channel alpha subunits were contained in each of the 25-, 31-, and 33-kDa CNBr fragments, indicating that most cAMP-dependent phosphorylation sites are in this domain. Since CNBr digestion of sodium channels which had been metabolically labeled with 32P in intact neurons yielded the same phosphorylated fragments, the phosphorylated region we have identified is the major location of phosphorylation in situ. Only serine residues were phosphorylated by cAMP-dependent protein kinase in vitro, while approximately 16% of the phosphorylation in intact neurons was on threonine residues that must lie outside the domain we have identified. Since this domain is phosphorylated in intact neurons, our results show that it is located on the intracellular side of the plasma membrane. These results are considered with respect to models for the transmembrane orientation of the alpha subunit. 相似文献
8.
O'Reilly AO Khambay BP Williamson MS Field LM Wallace BA Davies TG 《The Biochemical journal》2006,396(2):255-263
A homology model of the housefly voltage-gated sodium channel was developed to predict the location of binding sites for the insecticides fenvalerate, a synthetic pyrethroid, and DDT an early generation organochlorine. The model successfully addresses the state-dependent affinity of pyrethroid insecticides, their mechanism of action and the role of mutations in the channel that are known to confer insecticide resistance. The sodium channel was modelled in an open conformation with the insecticide-binding site located in a hydrophobic cavity delimited by the domain II S4-S5 linker and the IIS5 and IIIS6 helices. The binding cavity is predicted to be accessible to the lipid bilayer and therefore to lipid-soluble insecticides. The binding of insecticides and the consequent formation of binding contacts across different channel elements could stabilize the channel when in an open state, which is consistent with the prolonged sodium tail currents induced by pyrethroids and DDT. In the closed state, the predicted alternative positioning of the domain II S4-S5 linker would result in disruption of pyrethroid-binding contacts, consistent with the observation that pyrethroids have their highest affinity for the open channel. The model also predicts a key role for the IIS5 and IIIS6 helices in insecticide binding. Some of the residues on the helices that form the putative binding contacts are not conserved between arthropod and non-arthropod species, which is consistent with their contribution to insecticide species selectivity. Additional binding contacts on the II S4-S5 linker can explain the higher potency of pyrethroid insecticides compared with DDT. 相似文献
9.
H Nakayama Y Hatanaka E Yoshida K Oka M Takanohashi Y Amano Y Kanaoka 《Biochemical and biophysical research communications》1992,184(2):900-907
Forty three percent of the labeled sites, at least, in the electroplax sodium channel with a photoactivable tetrodotoxin derivative were identified by probing protease-digested labeled fragments with several sequence-directed antibodies. They are located in the loop between segments S5 and S6 of domain IV, as well as the region containing transmembrane segment S6 and adjacent extracellular and cytoplasmic sequences in domain III. No photolabeled fragments were detected in the corresponding region of domain I. These results suggest that C-11 of tetrodotoxin where the photoreactive moiety is attached orients to the region between S5 and S6 in domain III and IV. Probable orientation of the tetrodotoxin molecule in sodium channels is considered by taking together with the recent report of the site-directed mutagenesis. 相似文献
10.
11.
Epithelial Sodium Channel (ENaC) proteolysis at sites in the extracellular loop of the α and γ subunits leads to marked activation. The mechanism of this effect remains debated, as well as the role of the N- and C-terminal fragments of these subunits created by cleavage. We introduced cysteines at sites bracketing upstream and downstream the cleavage regions in α and γ ENaC to examine the role of these fragments in the activated channel. Using thiol modifying reagents, as well as examining the effects of cleavage by exogenous proteases we constructed a functional model that determines the potential interactions of the termini near the cleavage regions. We report that the N-terminal fragments of both α and γ ENaC interact with the channel complex; with interactions between the N-terminal γ and the C-terminal α fragments being the most critical to channel function and activation by exogenous cleavage by subtilisin. Positive charge modification at a.a.135 in the N-terminal fragment of γ exhibited the largest inhibition of channel function. This region was found to interact with the C-terminal α fragment between a.a. 205 and 221; a tract which was previously identified to be the site of subtilisin''s action. These data provide the first evidence for the functional channel rearrangement caused by proteolysis of the α and γ subunit and indicate that the untethered N-terminal fragments of these subunits interact with the channel complex. 相似文献
12.
Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation 总被引:7,自引:0,他引:7
The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation. 相似文献
13.
MiRP1 (encoded by the KCNE2 gene) is one of a family of five single transmembrane domain voltage-gated potassium (Kv) channel ancillary subunits currently under intense scrutiny to establish their position in channel complexes and elucidate alpha subunit contact points, but its structure is unknown. MiRP1 mutations are associated with inherited and acquired cardiac arrhythmia. Here, synthetic peptides corresponding to human MiRP1 (full-length and separate domains) were structurally analyzed using FTIR and CD spectroscopy. The N-terminal (extracellular) domain was soluble and predominantly non-ordered in aqueous media, but predominantly alpha-helical in L-alpha-lysophosphatidylcholine (LPC) micelles. The MiRP1 transmembrane domain was predominantly a mixture of alpha-helix and non-ordered structure in LPC micelles, with a minor contribution from non-aggregated beta-strand. The intracellular C-terminal domain was insoluble in aqueous solution; reconstitution into non-aqueous environments resulted in solubility and adoption of increasing amounts of alpha-helix, with the solvent order sodium dodecyl sulphate < dimyristoyl L-alpha-phosphatidylcholine (DMPC) < LPC < trifluoroethanol. Correlation of secondary structure changes with lipid transition temperature during heating suggested that the MiRP1 C-terminus incorporates into DMPC bilayers. Full-length MiRP1 was soluble in SDS micelles and calculated to contain 34% alpha-helix, 23% beta-strand and 43% non-ordered structure in this environment, as determined by CD spectroscopy. Thus, MiRP1 is highly dependent upon hydrophobic interaction via lipid and/or protein contacts for adoption of ordered structure without nonspecific aggregation, consistent with a role as a membrane-spanning subunit within Kv channel complexes. These data will provide a structural framework for ongoing mutagenesis-based in situ structure-function studies of MiRP1 and its relatives. 相似文献
14.
G C Chen S Zhu D A Hardman J W Schilling K Lau J P Kane 《The Journal of biological chemistry》1989,264(24):14369-14375
The structural domains of human apolipoprotein B-100 in low density lipoproteins (LDL) and the conformational changes of B-100 that accompany the conversion of very low density lipoproteins (VLDL) to LDL were investigated by limited proteolysis with 12 endoproteases of various specificities, and their cleavage sites were determined. In B-100 of LDL, we identified two peptide regions that are highly susceptible to proteolytic cleavage. One region encompassed about 40 amino acids (residues 1280-1320, designated as the NH2-terminal region) and the other about 100 amino acids (residues 3180-3280, designated as the COOH-terminal region). IN LDL, the cleavage sites in both susceptible regions of B-100 were readily accessible to limited proteolysis; but in VLDL, only sites in the COOH-terminal region were readily accessible. Moreover, B-100 in VLDL appeared less degraded than B-100 in LDL by all enzymes used. Reduction of disulfide bonds of B-100 in both LDL and VLDL before digestion by Staphylococcus aureus V8 protease and clostripain exposed additional cleavage sites and increased the rate of B-100 degradation, suggesting that disulfide bonds probably exert conformational constraints. These results indicate the presence of three principal structural domains in B-100 of LDL that are relatively resistant to limited proteolysis. These three domains are connected by the two susceptible peptide regions. Our results also demonstrate differential accessibility of cleavage sites in B-100 of LDL and VLDL to limited proteolysis. This differential accessibility suggests that substantial changes in the conformation or environment of B-100 accompany the conversion of VLDL to LDL. 相似文献
15.
Domain structure of the HSC70 cochaperone, HIP. 总被引:1,自引:0,他引:1
Marion Velten Nathalie Gomez-Vrielynck Alain Chaffotte Moncef M Ladjimi 《The Journal of biological chemistry》2002,277(1):259-266
The domain structure of the HSC70-interacting protein (HIP), a 43-kDa cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor, has been probed by limited proteolysis and biophysical and biochemical approaches. HIP proteolysis by thrombin and chymotrypsin generates essentially two fragments, an NH2-terminal fragment of 25 kDa (N25) and a COOH-terminal fragment of 18 kDa (C18) that appear to be well folded and stable as indicated by circular dichroism and recombinant expression in Escherichia coli. NH2-terminal amino acid sequencing of the respective fragments indicates that both proteases cleave HIP within a predicted alpha-helix following the tetratricopeptide repeat (TPR) region, despite their different specificities and the presence of several potential cleavage sites scattered throughout the sequence, thus suggesting that this region is particularly accessible and may constitute a linker between two structural domains. After size exclusion chromatography, N25 and C18 elute as two distinct and homogeneous species having a Stokes radius of 49 and 24 A, respectively. Equilibrium sedimentation and sedimentation velocity indicate that N25 is a stable dimer, whereas C18 is monomeric in solution, with sedimentation coefficients of 3.2 and 2.3 S and f/f(o) values of 1.5 and 1.1 for N25 and C18, respectively, indicating that the N25 is elongated whereas C18 is globular in shape. Both domains are able to bind to the ATPase domain of HSC70 and inhibit rhodanese aggregation. Moreover, their effects appear to be additive when used in combination, suggesting a cooperation of these domains in the full-length protein not only for HSC70 binding but also for chaperone activity. Altogether, these results indicate that HIP is made of two structural and functional domains, an NH2-terminal 25-kDa domain, responsible for the dimerization and the overall asymmetry of the molecule, and a COOH-terminal 18-kDa globular domain, both involved in HSC70 and unfolded protein binding. 相似文献
16.
The proline-activating activity of the multienzyme gramicidin S synthetase 2 can be recovered on a 115-kDa tryptic fragment 总被引:1,自引:0,他引:1
The multienzyme gramicidin S synthetase 2 was treated with trypsin to obtain fragments capable of activating proline. Three different active fragments were detected. The course of proteolysis was simulated by using a concentration range of trypsin; the cleavage pattern indicated that one of the fragments was particularly stable. This fragment was purified and shown to have a molecular mass of 115 kDa. It was compared chromatographically, by SDS/PAGE, and enzymatically to a Pro-activating fragment produced by a gramicidin-S-negative mutant. It can be concluded that the proteolytic fragment represents a structure which is contained on a continuous part of the polypeptide chain of gramicidin S synthetase 2 and has a relatively compact structure. This provides evidence that the multienzyme gramicidin S synthetase 2 is, at least in part, constructed from functional domains. An approach towards extending these studies to other parts of the gramicidin S synthetase 2 molecule has also been devised. This work complements recombinant DNA studies in the area, providing stable functional fragments. 相似文献
17.
The changes in the main storage protein of seeds of buckwheat ( Fagopyrum esculentum Moench cv. Shatilovskaya 5), 13S globulin, were studied during seed germination. During the first three days of germination the 13S globulin is subjected to a limited proteolysis, which consists in the splitting of some of its subunits into large polypeptide fragments. Insignificant changes in the sedimentation coefficient of the 13S globulin during the first days of germination as well as immunochemical data, indicate that the limited proteolysis of the 13S globulin does not cause any major changes in its structure.
Dormant buckwheat seeds contain a proteolytic enzyme (a metalloproteinase), which can cause limited proteolysis of 13S globulin. The proteinase hydrolyzed some subunits of the 13S globulin to high molecular weight fragments. In the presence of sodium dodecylsulphate the electrophoretic pattern of 13S globulin, isolated from 3-day-old buckwheat seedlings, was almost identical to that of 13S globulin from dormant seeds hydrolyzed with metalloproteinase. It is suggested that the proteolysis of 13S globulin observed in vitro may also take place in vivo in the course of seed germination. 相似文献
Dormant buckwheat seeds contain a proteolytic enzyme (a metalloproteinase), which can cause limited proteolysis of 13S globulin. The proteinase hydrolyzed some subunits of the 13S globulin to high molecular weight fragments. In the presence of sodium dodecylsulphate the electrophoretic pattern of 13S globulin, isolated from 3-day-old buckwheat seedlings, was almost identical to that of 13S globulin from dormant seeds hydrolyzed with metalloproteinase. It is suggested that the proteolysis of 13S globulin observed in vitro may also take place in vivo in the course of seed germination. 相似文献
18.
The role of hydrophobic residues in voltage sensors S4 of voltage-sensitive ion channels is less documented than that of charged residues. We performed alanine-substitution of branched-sidechain residues contiguous to the third, fourth and fifth positively charged residues in S4s of the first three domains of the sodium channel expressed in HEK cells. These locations were selected because they are close to the arginines and lysines important in gating. Mutations in the first two domains (DIS4 and DIIS4) altered steady-state activation curves. In DIIIS4, the mutation L1131A next to the third arginine greatly slowed inactivation in a manner similar to that for substitutions of charged residues in DIVS4, whereas the mutation L1137A next to the fifth arginine preserved wild-type behaviour. Homology models of domain III, based on the structure of a crystallized mammalian potassium channel, shows that L1131 is located at the interface between S3 and S4 helices, whereas L1137, on the opposite side of S4, does not interact with the voltage sensor. The two mutated residues are closer to each other in domains I and II than in domain III, as may be corroborated by their different electrophysiological effects. 相似文献
19.
The role of hydrophobic residues in voltage sensors S4 of voltage-sensitive ion channels is less documented than that of charged residues. We performed alanine-substitution of branched-sidechain residues contiguous to the third, fourth and fifth positively charged residues in S4s of the first three domains of the sodium channel expressed in HEK cells. These locations were selected because they are close to the arginines and lysines important in gating. Mutations in the first two domains (DIS4 and DIIS4) altered steady-state activation curves. In DIIIS4, the mutation L1131A next to the third arginine greatly slowed inactivation in a manner similar to that for substitutions of charged residues in DIVS4, whereas the mutation L1137A next to the fifth arginine preserved wild-type behaviour. Homology models of domain III, based on the structure of a crystallized mammalian potassium channel, shows that L1131 is located at the interface between S3 and S4 helices, whereas L1137, on the opposite side of S4, does not interact with the voltage sensor. The two mutated residues are closer to each other in domains I and II than in domain III, as may be corroborated by their different electrophysiological effects. 相似文献
20.
Structural determinants of the closed KCa3.1 channel pore in relation to channel gating: results from a substituted cysteine accessibility analysis
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Klein H Garneau L Banderali U Simoes M Parent L Sauvé R 《The Journal of general physiology》2007,129(4):299-315
In this work we address the question of the KCa3.1 channel pore structure in the closed configuration in relation to the contribution of the C-terminal end of the S6 segments to the Ca(2+)-dependent gating process. Our results based on SCAM (substituted cysteine accessibility method) experiments first demonstrate that the S6 transmembrane segment of the open KCa3.1 channel contains two distinct functional domains delimited by V282 with MTSEA and MTSET binding leading to a total channel inhibition at positions V275, T278, and V282 and to a steep channel activation at positions A283 and A286. The rates of modification by MTSEA (diameter 4.6 A) of the 275C (central cavity) and 286C residues (S6 C-terminal end) for the closed channel configuration were found to differ by less than sevenfold, whereas experiments performed with the larger MTSET reagent (diameter 5.8 A) resulted in modification rates 10(3)-10(4) faster for cysteines at 286 compared with 275. Consistent with these results, the modification rates of the cavity lining 275C residue by MTSEA, Et-Hg(+), and Ag(+) appeared poorly state dependent, whereas modification rates by MTSET were 10(3) faster for the open than the closed configuration. A SCAM analysis of the channel inner vestibule in the closed state revealed in addition that cysteine residues at 286 were accessible to MTS reagents as large as MTS-PtrEA, a result supported by the observation that binding of MTSET to cysteines at positions 283 or 286 could neither sterically nor electrostatically block the access of MTSEA to the closed channel cavity (275C). It follows that the closed KCa3.1 structure can hardly be accountable by an inverted teepee-like structure as described for KcsA, but is better represented by a narrow passage centered at V282 (equivalent to V474 in Shaker) connecting the channel central cavity to the cytosolic medium. This passage would not be however restrictive to the diffusion of small reagents such as MTSEA, Et-Hg(+), and Ag(+), arguing against the C-terminal end of S6 forming an obstructive barrier to the diffusion of K(+) ions for the closed channel configuration. 相似文献