共查询到20条相似文献,搜索用时 15 毫秒
1.
E. Wolf M. Zahr R. Benz J. F. Imhoff A. Lustig E. Schiltz J. Stahl-Zeng J. Weckesser 《Archives of microbiology》1996,166(3):169-175
Major outer membrane proteins with porin activity were isolated from cell envelopes of the halophilic strains Ectothiorhodospira shaposhnikovii N1 and Ectothiorhodospira vacuolataβ1. The porins were obtained as oligomers. They dissociated into monomers by heat or EDTA treatment. The molecular masses
of the monomers were determined by mass spectrometry to be 39,285 and 37,160 Da for E. shaposhnikovii N1 and E. vacuolataβ1, respectively. Both were shown by analytical ultracentrifugation to be trimers of about 112,000 Da. Circular dichroism
spectra indicated predominantly β-sheet structure. The 18 N-terminal amino acid sequences of the two porins were identical
except for the amino acids in positions 12 and 14. No sequence similarity with the primary structure of known porins was found.
In reconstitution experiments with lipid bilayers, the porins of E. shaposhnikovii N1 and E. vacuolataβ1 formed channels with a single-channel conductance of 1.5 and 0.7 nS, respectively, in 1 M KCl. The single-channel conductance
saturated with increasing salt concentration, indicating a putative binding-site for anions in the channel since both porins
exhibited anion-selectivity. For the porin of E. vacuolataβ1, but not for that of E. shaposhnikovii N1, an influence of detergent concentration on the single-channel conductance was observed.
Received: 3 April 1996 / Accepted: 31 May 1996 相似文献
2.
S.I. Ortiz-Miranda J.A. Lasalde P.A. Pappone M.G. McNamee 《The Journal of membrane biology》1997,158(1):17-30
We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type α418Cys
residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold,
respectively. The mutation of a homologous cysteine in the β subunit (β447Trp) had similar but smaller effects on mean open
time. Coexpression of α418Trp and β447Trp had the largest effect on channel open time, increasing mean open time 58-fold.
No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested.
However, the coexpression of the α418Trp and β447Trp mutated subunits also produced channels with at least two additional
conductance levels. Block by acetylcholine was apparent in the current records from α418Trp mutants. Burst analysis of the
α418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate
and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the
α- and β subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating,
and that mutations in multiple subunits act additively to increase channel open time.
Received: 27 September 1996/Revised: 28 January 1997 相似文献
3.
The gram-positive bacterium Mycobacterium phlei was treated with detergents. Reconstitution experiments using lipid bilayers suggested that the detergent extracts contain
a channel forming protein. The protein was purified to homogeneity by preparative SDS-PAGE and identified as a protein with
an apparent molecular mass of about 135 kDa. The channel-forming unit dissociated into subunits with a molecular mass of about
22 kDa when it was boiled in 80% dimethylsulfoxid (DMSO). The channel has on average a single channel conductance of 4.5 nS
in 1 m KCl and is highly voltage-dependent in an asymmetric fashion when the protein is added to only one side of the membrane.
Zero-current membrane potential measurements with different salts implied that the channel is highly cation-selective because
of negative point charges in or near the channel mouth. Analysis of the single-channel conductance as a function of the hydrated
cation radii using the Renkin correction factor and the effect of the negative point charges on the single-channel conductance
suggest that the diameter of the cell wall channel is about 1.8 to 2.0 nm. The channel properties were compared with those
of other members of the mycolata and suggest that these channels share common features. Southern blots demonstrated that the
chromosome of M. phlei and other mycolata tested contain homologous sequences to mspA (gene of the cell wall porin of Mycobacterium smegmatis).
Received: 22 December 2000/Revised: 10 April 2001 相似文献
4.
Saier MH 《The Journal of membrane biology》2000,175(3):165-180
Channel-forming proteins/peptides fall into over 100 currently recognized families, most of which are restricted to prokaryotes
or eukaryotes, but a few of which are ubiquitous. These proteins fall into three major currently recognized classes: (i) α-helix-type
channels present in bacterial, archaeal and eukaryotic cytoplasmic and organellar membranes, (ii) β-barrel-type porins present
in the outer membranes of Gram-negative bacterial cells, mitochondria and chloroplasts, and (iii) protein/peptide toxins targeted
to the cytoplasmic membranes of cells other than those that synthesize the toxins. High-resolution 3-dimensional structural
data are available for representative proteins/peptides of all three of these channel-forming types. Each type exhibits distinctive
features that distinguish them from the other channel protein types and from carriers. Structural, functional, and evolutionary
aspects of transmembrane channel-formers are discussed.
Received: 10 September 1999/Revised: 11 February 2000 相似文献
5.
The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm
cis/150 mm
trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much
lower concentrations (1.1–2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to
420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P
Cl/P
NMDG permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current
flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of
the pores formed by 100–500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P
Cl/P
NMDG permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect
midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties.
Received: 23 February 2001/Revised: 15 June 2001 相似文献
6.
Arbing MA Dahan D Boismenu D Mamer OA Hanrahan JW Coulton JW 《The Journal of membrane biology》2000,178(3):185-193
Porin of Haemophilus influenzae type b (341 amino acids; M
r
37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected
to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up
to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and
matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified
lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid
bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel
conductance compared to Hib porin (1.24+/−0.41 vs. 0.85+/−0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin.
The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin
was 50–55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89–91% of the instantaneous current levels.
We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b.
Received: 11 August 2000/Revised: 8 September 2000 相似文献
7.
Detergent-solubilized cell wall extracts of the gram-positive, strictly aerobic bacterium Nocardia asteroides contain channel-forming activity as judged from reconstitution experiments using lipid bilayer membranes. The cell wall porin
was identified as a protein with an apparent molecular mass of about 84 kDa based on SDS-PAGE. The porin was purified to homogeneity
using preparative SDS-PAGE. The 84-kDa protein was no longer observed after heating in SDS buffer. The presumed dissociation
products were not observed on SDS-polyacrylamide gels. The cell wall porin increased the specific conductance of artificial
lipid bilayer membranes from phosphatidylcholine/phosphatidylserine mixtures by the formation of cation-selective channels,
which had an average single-channel conductance of 3.0 nS in 1 M KCl. The single-channel conductance was only moderately dependent
on the bulk aqueous KCl concentration, which indicated negative point charge effects on the channel properties. The analysis
of the concentration dependence of the single-channel conductance using the effect of negative charges on channel conductance
suggested that the diameter of the cell wall channel is about 1.4 nm. Asymmetric addition of the cell wall porin to lipid
bilayer membranes resulted in an asymmetric voltage dependence. The cell wall channel switched into substates, when the cis side of the membrane, the side of the addition of the protein, had negative polarity. Positive potentials at the cis side had no influence on the conductance of the cell wall channel.
Received: 23 September 1998 / Accepted: 9 December 1998 相似文献
8.
We have previously reported that intralobular salivary duct cells contain an amiloride-sensitive Na+ conductance (probably located in the apical membranes). Since the amiloride-sensitive Na+ conductances in other tight epithelia have been reported to be controlled by extracellular (luminal) Na+, we decided to use whole-cell patch clamp techniques to investigate whether the Na+ conductance in salivary duct cells is also regulated by extracellular Na+. Using Na+-free pipette solutions, we observed that the whole-cell Na+ conductance increased when the extracellular Na+ was increased, whereas the whole-cell Na+ permeability, as defined in the Goldman equation, decreased. The dependency of the whole-cell Na+ conductance on extracellular Na+ could be described by the Michaelis-Menten equation with a K
m
of 47.3 mmol/1 and a maximum conductance (G
max) of 2.18 nS. To investigate whether this saturation of the Na+ conductance with increasing extracellular Na+ was due to a reduction in channel activity or to saturation of the single-channel current, we used fluctuation analysis of
the noise generated during the onset of blockade of the Na+ current with 200 μmol/l 6-chloro-3,5-diaminopyrazine-2-carboxamide. Using this technique, we estimated the single channel
conductance to be 4 pS when the channel was bathed symmetrically in 150 mmol/l Na+ solutions. We found that Na+ channel activity, defined as the open probability multiplied by the number of available channels, did not alter with increasing
extracellular Na+. On the other hand, the single-channel current saturated with increasing extracellular Na+ and, consequently, whole-cell Na+ permeability declined. In other words, the decline in Na+ permeability in salivary duct cells with increasing extracellular Na+ concentration is due simply to saturation of the single-channel Na+ conductance rather than to inactivation of channel activity.
Received: 27 July 1995/Revised: 7 December 1995 相似文献
9.
O.V. Krasilnikov M.-F.P. Capistrano L.N. Yuldasheva R.A. Nogueira 《The Journal of membrane biology》1997,156(2):157-172
Replacement of an amino acid residue at position 130 -Gly by Cys- in the primary structure of Staphylococcus aureus alpha-toxin
decreases the single-channel conductance induced by the toxin in planar lipid bilayers. Concomitantly, the pH value at which
the channel becomes unable to discriminate between Cl− and K+ ions is also decreased. By contrast, the pH dependence of the efficiency of the mutant toxin to form ion channels in lipid
bilayers was unchanged (maximum efficiency at pH 5.5–6.0). The asymmetry and nonlinearity of the current-voltage characteristics
of the channel were increased by the point mutation but the diameter of the water pore induced by the mutant toxin, evaluated
in lipid bilayers and in erythrocyte membranes, was found to be indistinguishable from that formed by wild-type toxin and
equal to 2.4–2.6 nm.
Alterations at the ``trans mouth' were found to be responsible for all observed changes of the channel properties. This mouth is situated close to
the surface of the second leaflet of a bilayer lipid membrane. The data obtained allows us to propose that the region around
residue 130 in fact determines the main features of the ST-channel and takes part in the formation of the trans entrance of the channel.
Received: 8 September 1995/Revised: 20 November 1996 相似文献
10.
GABAA channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline
and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches).
The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application
of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay
that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased
with GABA concentration from less than 10 pS (0.5 μm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC
50 value of 33 μm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of
1 μm diazepam, the GABA EC
50 decreased to 0.2 μm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA
antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 μm GABA the bicuculline IC
50 was 19 μm. The effect of GABA on channel conductance shows that the role of the ligand in GABAA receptor channel function is more complex than previously thought.
Received: 23 October 2000/Revised: 27 February 2001 相似文献
11.
The rate-limiting step for the maternofetal exchange of low molecular-weight solutes in humans is constituted by transport
across a single epithelial layer (syncytiotrophoblast) of the placenta. Other than the well-established presence of a large-conductance,
multisubstate Cl− channel, the ionic channels occurring in this syncytial tissue are, for the most part, unknown. We have found that fusion
of apical plasma membrane-enriched vesicle fractions with planar lipid bilayers leads, mainly (96% of 353 reconstitutions),
to the reconstitution of nonselective cation channels. Here we describe the properties of this novel placental conductance
at the single-channel level. The channel has a large (>200 pS) and variable conductance, is cation selective (P
Cl
/P
K
≅ 0.024), is reversibly inhibited (presumably blocked) by submillimolar La3+, has very unstable kinetics, and displays a large number (>10) of current sublevels with a ``promiscuous' connectivity pattern.
The occurrence of both ``staircaselike' and ``all-or-nothing' transitions between the minimum and maximum current levels
was intriguing, particularly considering the large number of conductance levels spanned at a time during the concerted current
steps. Single-channel data simulated according to a multistate linear reaction scheme, with rate constants that can vary spontaneously
in time, reproduce many aspects of the recorded subconductance behavior. The channel's sensitivity to lanthanides is reminiscent
of stretch-sensitive channels which, in turn, suggests a physiological role for this ion channel as a mechanotransducer during
syncytiotrophoblast-volume regulation.
Received: 30 August 1999/Revised: 12 November 1999 相似文献
12.
Nickel ions have been reported to exhibit differential effects on distinct subtypes of voltage-activated calcium channels.
To more precisely determine the effects of nickel, we have investigated the action of nickel on four classes of cloned neuronal
calcium channels (α1A, α1B, α1C, and α1E) transiently expressed in Xenopus oocytes. Nickel caused two major effects: (i) block detected as a reduction of the maximum slope conductance and (ii) a shift
in the current-voltage relation towards more depolarized potentials which was paralleled by a decrease in the slope of the
activation-curve. Block followed 1:1 kinetics and was most pronounced for α1C, followed by α1E > α1A > α1B channels. In contrast, the change in activation-gating was most dramatic with α1E, with the remaining channel subtypes significantly less affected. The current-voltage shift was well described by a simple
model in which nickel binding to a saturable site resulted in altered gating behavior. The affinity for both the blocking
site and the putative gating site were reduced with increasing concentration of external permeant ion. Replacement of barium
with calcium reduced both the degree of nickel block and the maximal effect on gating for α1A channels, but increased the nickel blocking affinity for α1E channels. The coexpression of Ca channel β subunits was found to differentially influence nickel effects on α1A, as coexpression with β2a or with β4 resulted in larger current-voltage shifts than those observed in the presence of β1b, while elimination of the β subunit almost completely abolished the gating shifts. In contrast, block was similar for the
three β subunits tested, while complete removal of the β subunit resulted in an increase in blocking affinity. Our data suggest
that the effect of nickel on calcium channels is complex, cannot be described by a single site of action, and differs qualitatively
and quantitatively among individual subtypes and subunit combinations.
Received: 12 October 1995/Revised: 17 January 1996 相似文献
13.
14.
J.-P. Bénitah J.R. Balser E. Marban G.F. Tomaselli 《The Journal of membrane biology》1997,155(2):121-131
Extracellular acidosis affects both permeation and gating of the expressed rat skeletal muscle Na+ channel (μ1). Reduction of the extracellular pH produced a progressive decrease in the maximal whole-cell conductance and
a depolarizing shift in the whole-cell current-voltage relationship. A smaller depolarizing shift in the steady-state inactivation
curve was observed. The pK of the reduction of maximal conductance was 6.1 over the pH range studied. An upper limit estimate
of the pK of the shift of the half-activation voltage was 6.1. The relative reduction in the maximal whole-cell conductance
did not change with higher [Na+]
o
. The conductance of single fenvalerate-modified Na+ channels was reduced by extracellular protons. Although the single-channel conductance increased with higher [Na+]
o
, the maximal conductances at pH 7.6, 7.0 and 6.0 did not converge at [Na+]
o
up to 280 mm, inconsistent with a simple electrostatic effect. A model incorporating both Na+ and H+ binding in the pore and cation binding to a Gouy-Chapman surface charge provided a robust fit to the single-channel conductance
data with an estimated surface charge density of 1e−/439?2. Neither surface charge nor proton block alone suffices to explain the effects of extracellular acidosis on Na+ channel permeation; both effects play major roles in mediating the response to extracellular pH.
Received: 14 May 1996/Revised: 19 September 1996 相似文献
15.
A K+ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid
bilayers. This channel was identified as an ATP-sensitive K+ channel (KATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na2. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0
mV and with a 5/250 KCl gradient, the main conductance of the KATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil
stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels.
In general, KATP channels were characterized by brief openings followed by long closings (open probability, P
o
≈ 0.02); only occasionally (3 out of 12 experiments) did the KATP channels have a high open probability (P
o
≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl2 to the internal side of the channel.
Received: 7 November 1995/Revised: 10 June 1996 相似文献
16.
In the first part of this study, photofrin II sensitized membrane modifications of OK-cells were investigated at the level
of macroscopic membrane currents. In this second part, the inside-out configuration of the patch-clamp technique is applied
to analyze the phenomena at the microscopic level. It is shown that the characteristic single channel fluctuations of the
electric current disappear after the start of illumination of membrane patches in the presence of photofrin II. This holds
for all three types of ion channels investigated: the large-conductance Ca2+-dependent K+ channel (maxi-KCa), a K+ channel of small conductance (sK), and a stretch-activated nonselective cation channel (SA-cat). Part of the experiments
show a transient activation of the channels (indicated by an increase of the probability in the open-channel state) before
the channels are converted into a closed nonconductive state. Inactivation of all three channel types proceeds by a continuous
reduction of their open probability, while the single channel conductance values are not affected. The process of photodynamically
induced channel inactivation is followed by a pronounced increase of the leak conductance of the plasma membrane. The latter
process — after light-induced initiation — is found to continue in the dark. The ionic pathways underlying the leak conductance
also allow permeation of Ca2+ ions. The resulting Ca2+-flux may contribute to the photodynamically induced increase of the intracellular Ca2+ concentration observed in various cell lines.
Received: 26 May 1998/Revised: 8 September 1998 相似文献
17.
Structure-Function Relationship of the Ion Channel Formed by Diphtheria Toxin in Vero Cell Membranes
Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed
by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane
of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming
transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel,
and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin,
Δg
m
, was initially determined. DT-AB induced a ∼10-fold lower Δg
m
than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Δg
m
, whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Δg
m
. Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches.
No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B
D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability
and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is
reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode.
Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane
domain (T-domain) strongly affect the overall channel activity expressed as Δg
m
and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero
cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel.
Received: 20 June 1996/Revised: 8 November 1996 相似文献
18.
Membrane potential and whole-cell current were studied in rat pancreatic β-cells using the `perforated patch' technique and
cell volume measured by a video-imaging method. Exposure of β-cells to the α-ketoaldehyde methylglyoxal (1 mm) resulted in depolarization and electrical activity. In cells voltage-clamped at −70 mV, this effect was accompanied by the
development of inward current noise. In voltage-pulse experiments, methylglyoxal activated an outwardly rectifying conductance
which was virtually identical to the volume-sensitive anion conductance previously described in these cells. Two inhibitors
of this conductance, 4,4′-dithiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid
(NPPB), also inhibited the depolarization and inward current evoked by methylglyoxal. Methylglyoxal increased β-cell volume
to a relative value of 1.33 after 10 min with a gradual return towards basal levels following withdrawal of the α-ketoaldehyde.
None of the effects of methylglyoxal was observed in response to t-butylglyoxal which, unlike methylglyoxal, is a poor substrate for the glyoxalase pathway. Methylglyoxal had no apparent effect
on β-cell K+ channel activity. It is suggested that the metabolism of methylglyoxal to d-lactate causes β-cell swelling and activation of the volume-sensitive anion channel, leading to depolarization. These findings
could be relevant to the stimulatory action of d-glucose, the metabolism of which generates significant quantities of l-lactate.
Received: 15 May 1998/Revised: 25 September 1998 相似文献
19.
Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive,Glutamate-Gated Chloride Channels 总被引:4,自引:0,他引:4
Demetrios K. Vassilatis Keith O. Elliston Philip S. Paress Michel Hamelin Joseph P. Arena James M. Schaeffer Lex H.T. Van der Ploeg Doris F. Cully 《Journal of molecular evolution》1997,44(5):501-508
Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin
class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as
well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of
related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined
the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these
channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic
analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several
intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position.
Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily
and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties
similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride
channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination
with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel
family that may represent genes orthologous to the vertebrate glycine channels.
Received: 30 September 1996 / Accepted: 15 November 1996 相似文献
20.
A number of peptide toxins derived from marine snails and various spiders have been shown to potently inhibit voltage-dependent
calcium channels. Here, we describe the effect of calcicludine, a 60 amino-acid peptide isolated from the venom of the green
mamba (Dendroaspis angusticeps), on transiently expressed high voltage-activated calcium channels. Upon application of calcicludine, L-type (α1
C
) calcium channels underwent a rapid, irreversible decrease in peak current amplitude with no change in current kinetics,
or any apparent voltage-dependence. However, even at saturating toxin concentrations, block was always incomplete with a maximum
inhibition of 58%, indicating either partial pore block, or an effect on channel gating. Block nonetheless was of high affinity
with an IC50 value of 88 nm. Three other types of high voltage activated channels tested (α1
A
, α1
B
, and α1
E
) exhibited a diametrically different response to calcicludine. First, the maximal inhibition observed was around 10%, furthermore,
the voltage-dependence of channel activation was shifted slightly towards more negative potentials. Thus, at relatively hyperpolarized
test potentials, calcicludine actually upregulated current activity of (N-type) α1
B
channels by as much as 50%. Finally, the use of several chimeric channels combining the major transmembrane domains of α1
C
and α1
E
revealed that calcicludine block of L-type calcium channels involves interactions with multiple structural domains. Overall,
calcicludine is a potent and selective inhibitor of neuronal L-type channels with a unique mode of action.
Received: 22 September 1999/Revised: 1 December 1999 相似文献