共查询到20条相似文献,搜索用时 15 毫秒
1.
G.W. Zamponi 《The Journal of membrane biology》1999,167(2):183-192
Piperidines are a relatively novel class of calcium channel blockers which act at a unique receptor site associated with
the calcium channel α1 subunit. Calcium channel blocking affinities ranging from subnanomolar to several hundred micromolar have been reported in
the literature, suggesting that piperidine block is highly sensitive to the cellular environment experienced by the channel.
Here, I have investigated some of the cytoplasmic determinants of haloperidol block of N-type calcium channels expressed in
human embryonic kidney cells. In perforated patch clamp recordings, haloperidol blocks N-type calcium channels with an inhibition
constant of 120 μM. Upon internal dialysis with chloride containing pipette solution, the blocking affinity increases by 40-fold.
This effect could be attributed in part to the presence of internal chloride ions, as replacement of intracellular chloride
with methanesulfonate reduced haloperidol blocking affinity by almost one order of magnitude. Tonic inhibition of N-type channels
by Gβγ subunits further enhanced the blocking effects of haloperidol, suggesting the possibility of direct effects of Gβγ binding on the local environment of the piperidine receptor site. Overall, depending on the cytoplasmic environment experienced
by the channel, the blocking affinity of N-type calcium channels for haloperidol may vary by more than two orders of magnitude.
Thus, absolute blocking affinities at the piperidine receptor site must be interpreted cautiously and in the context of the
particular experimental setting.
Received: 23 July 1998/Revised: 19 October 1998 相似文献
2.
We have previously shown that epithelial Na+ channels in mouse mandibular gland duct cells are controlled by cytosolic Na+ and Cl−, acting, respectively, via G
o
and G
i
proteins. Since we found no evidence for control of epithelial Na+ channels by extracellular Na+ ([Na+]
o
), our findings conflicted with the long-held belief that Na+ channel activators, such as sulfhydryl reagents, like para-chloromercuriphenylsulfonate (PCMPS), and amiloride analogues,
like benzimidazolylguanidinium (BIG) and 5-N-dimethylamiloride (DMA), induce their effects by blocking an extracellular channel
site which otherwise inhibits channel activity in response to increasing [Na+]
o
. Instead, we now show that PCMPS acts by rendering epithelial Na+ channels refractory to inhibition by activated G proteins, thereby eliminating the inhibitory effects of cytosolic Na+ and Cl− on Na+ channel activity. We also show that BIG, DMA, and amiloride itself, when applied from the cytosolic side of the plasma membrane,
block feedback inhibition of Na+ channels by cytosolic Na+, while leaving inhibition by cytosolic Cl− unaffected. Since the inhibitory effects of BIG and amiloride are overcome by the inclusion of the activated α-subunit of
G
o
in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na+ receptor.
Received: 1 October 1997/Revised: 24 December 1997 相似文献
3.
Characterization of Lonidamine and AF2785 Blockade of the Cyclic AMP-Activated Chloride Current in Rat Epididymal Cells 总被引:1,自引:0,他引:1
It has been shown previously that the antifertility agents Lonidamine and its analogue AF2785, [1-(2,4-dichlorobenzyl)-indazole-3-acrylic
acid] are potent inhibitors of the cAMP-activated chloride channel (CFTR) in rat epididymal cells. In this study, we further
characterized the blocking actions of these two compounds and compared them with the known chloride channel blocker diphenylamine-2-carboxylate
(DPC). Results show that the order of potency in blocking the cAMP-activated current is AF2785 > Lonidamine > DPC. All three
compounds shared similar blocking characteristics. Firstly, their blockade of the current exhibited voltage dependence; all
three agents blocked the current more markedly at negative than at positive membrane potentials. Secondly, they blocked the
channels from the outside of the cell. Thirdly, their blocking efficacies were maximal at low extracellular pH. Lastly, the
time course of the block by AF2785 and DPC appeared to be more rapid than that of Lonidamine. It is hoped that further studies
with other indazole compounds will add knowledge to the physiology and pharmacology of CFTR in the epididymis. Such information
will be of great importance to our quest for novel male contraceptives.
Received: 8 February 2000/Revised: 25 September 2000 相似文献
4.
Veratridine, an alkaloid isolated from the rhizome of V. album, binds and slows the inactivation of the brain sodium channels. The synthetic pentapeptide KIFMK causes a voltage- and use-dependent open-channel block of the RIIA (rat brain type IIA) sodium channel (Eaholtz, Scheuer
& Catterall, 1994). Our studies on the RIIA sodium channel expressed in CHO cells reveal that the fraction of veratridine
modified sodium channels decreases linearly with increasing KIFMK concentration. However, the time constant for dissociation of veratridine from the channel remains unchanged in the presence
of a high concentration of KIFMK, as opposed to that in the presence of QX314 where the dissociation appears to be more complex. These data are consistent
with mutually exclusive binding of the open channel blocking peptide and veratridine to the brain sodium channel.
Received: 19 November 1996/Revised: 31 July 1997 相似文献
5.
Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K+ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root
hair cells. K+ channel blockers are potent tools for studying the physiological functions and structural properties of K+ channels. In the present study the structural and biophysical mechanisms of Cs+ and TEA+ block of a cloned Arabidopsis inward-rectifying K+ channel (KAT1) were analyzed. Effects of the channel blockers Cs+ and TEA+ were characterized both extracellularly and intracellularly. Both external Cs+ and TEA+ block KAT1 currents. A mutant of KAT1 (``m2KAT1'; H267T, E269V) was produced by site-directed mutagenesis of two amino acid
residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs+ and TEA+ than the wild-type K+ channel. Internal TEA+ and Cs+ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence
and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced
mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K+ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested
for outward-rectifying K+ channels.
Received: 31 July 1995/Revised: 5 January 1996 相似文献
6.
The effect of ethanol on maxi Ca2+-activated K+ channels (BK channels) in GH3 pituitary tumor cells was investigated using single-channel recordings and focusing on intracellular
signal transduction. In outside-out patches, ethanol caused a transient concentration-dependent increase of BK-channel activity.
30 mm (1.4‰) ethanol significantly increased mean channel open time and channel open probability by 26.3 ± 9% and 78.8 ± 10%, respectively;
single-channel current amplitude was not affected by ethanol. The augmenting effect of ethanol was blocked in the presence
of protein kinase C (PKC) inhibitors staurosporine, bisindolylmaleimide, and PKC (19–31) pseudosubstrate inhibitor as well
as by AMP-PNP (5′-adenylylimidodiphosphate), a nonhydrolyzable ATP-analogue, but not by the phospholipase C blocker U-73122.
Phosphatase inhibitors microcystin-LR and okadaic acid promoted the ethanol effect. The blocking effect was released at higher
concentrations of ethanol (100 mm) suggesting a second site of action or a competition between blockers and ethanol. Our results suggest that the effect of
ethanol on BK-channels is mediated by PKC stimulation and phosphorylation of the channels which increases channel activity
and hence may influence action potentials duration and hormone secretion.
Received: 24 July 1996/Revised: 27 December 1996 相似文献
7.
We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH2-terminal domain of the Shaker K+ channel with purified, ryanodine-modified, cardiac Ca2+-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a
holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a
single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec−1μm
−1 for ShB and 2000 ± 200 sec−1μm
−1 for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied
potential with the bulk of the voltage-dependence residing in Koff. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As
is the case with inactivation and block of K+ channels, this is mediated by a large increase in Kon. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites
which will contribute to the ion handling properties of this channel.
Received: 15 December 1997/Revised: 13 March 1998 相似文献
8.
Lanthanide ions such as La3+ are frequently used as blockers to test the involvement of calcium channels in plant and animal signal transduction pathways.
For example, the large rise in cytoplasmic Ca2+ concentration triggered by cold shock in Arabidopsis seedlings is effectively blocked by 10 mm La3+ and we show here that the simultaneous large membrane depolarization is similarly blocked. However, a pharmacological tool
is only as useful as it is selective and the specificity of La3+ for calcium channels was brought into question by our finding that it also blocked a blue light (BL)-induced depolarization
that results from anion channel activation and believed not to involve calcium channels. This unexpected inhibitory effect
of La3+ on the BL-induced depolarization is explained by our finding that 10 mm La3+ directly and completely blocked the BL-activated anion channel when applied to excised patches. We have investigated the
ability of La3+ to block noncalcium channels in Arabidopsis. In addition to the BL-activated anion channel, 10 mm La3+ blocked a cation channel and a stretch-activated channel in patches of plasma membrane excised from hypocotyl cells. In root
cells, 10 mm La3+ inhibited the activity of an outward-rectifying potassium channel at the whole cell and single-channel level by 47% and 58%,
respectively. We conclude that La3+ is a nonspecific blocker of multiple ionic conductances in Arabidopsis and may disrupt signal transduction processes independently of any effect on Ca2+ channels.
Received: 28 July 1997/Revised: 13 November 1997 相似文献
9.
O.V. Krasilnikov J.B. Da Cruz L.N. Yuldasheva W.A. Varanda R.A. Nogueira 《The Journal of membrane biology》1998,161(1):83-92
This paper describes a new approach to evaluate the inner structure (including a main constriction and its localization)
of the water lumen of an ion channel. The method is based on the determination of channel filling by different nonelectrolyte
molecules through each side of an ion channel. The method has two characteristic features that make its use attractive: (i)
the possibility to ascertain the existence, localization and size of a narrow part inside an ion channel water lumen and (ii)
the chances to determine the maximal size of both entrances of an ion channel and to obtain additional information about the
geometry of its water lumen at the same time. Determinations were made on colicin Ia ion channels inserted into planar lipid
bilayers. This channel was chosen because there is an apparent contradiction between its low single channel conductance and
the large diameter of its water lumen. Our results show that the water lumen of the colicin Ia channel has a funnel-like structure
with a small trans-entrance, with a diameter of about 1.0 nm, and a large cis-entrance, with a diameter of approximately 1.8 nm. A constriction with a diameter of approximately 0.7 nm is shown to be
located close to the trans-entrance of the channel. The method can also be applied to patch clamp studies of single ion channels.
Received: 20 February 1997/Revised: 19 August 1997 相似文献
10.
The putative role(s) of a mechanically gated (MG) cation channel in Xenopus oocyte growth, maturation, fertilization and embryogenesis has been examined. Using a pharmacological approach, we have tested
the effects of the MG channel blockers, gadolinium, gentamicin and amiloride on the above developmental events. Our results
indicate that oocyte maturation, fertilization and early embryogenesis (up to the free-swimming stage 45) can proceed normally
in the presence of concentrations of agents that either completely abolish (i.e., ≥10 μm Gd3+) or partially block (i.e., 1 mm gentamicin) single MG channel activity as measured by patch-clamp recording. However, we also find that higher concentrations
of Gd3+ (≥50 μm) can lead to an increased percentage (>20%) of axis-perturbed embryos compared with control (<1%) and that amiloride (0.5
mm) reduces the success of fertilization (from 100% to <50%) and increases mortality (by ∼75%) in developing embryos. Furthermore,
we find that all three agents inhibit oocyte growth in vitro. However, their order of effectiveness (amiloride > gentamicin
> Gd3+) is opposite to their order for blocking MG channels (Gd3+≫ gentamicin > amiloride). These discrepancies indicated that the drugs effects occur by mechanisms other than, or in addition
to, MG channel block. Our results provide no compelling evidence for the idea that MG channel activity is critical for development
in Xenopus. This could mean that there are other mechanisms in the oocyte that can compensate when MG channel activity is blocked or
that the protein that forms the channel can undergo additional interactions that result in a function insensitive to MG channel
blockers.
Received: 27 March 1998/Revised: 10 June 1998 相似文献
11.
Block of K+ channels can be influenced by the ability of charged residues on the protein surface to accumulate cationic blocking ions
to concentrations greater than those in bulk solution. We examined the ionic strength dependence of extracellular block of
Shaker K+ channels by tetraethylammonium ions (TEA+) and by a trivalent quaternary ammonium ion, gallamine3+. Wild-type and mutant channels were expressed in Xenopus oocytes and currents recorded with the cut-open oocyte technique. Channel block by both compounds was substantially increased
when the bathing electrolyte ionic strength was lowered, but with a much larger effect for trivalent gallamine. These data
were quantitatively well described by a simple electrostatic model, accounting for accumulation of blocking ions near the
pore of the channel by surface charges. The surface charge density of the wild-type channel consistent with the results was
−0.1 e nm−2. Shaker channels with T449Y mutations have an increased affinity for both TEA and gallamine but the ionic strength dependence of
block was described with the same surface charge density as wild-type channels. Much of the increased sensitivity of Shaker K+ channels to gallamine may be due to a larger local accumulation of the trivalent ion. The negative charge at position 431
contributes to the sensitivity of channels to TEA (MacKinnon & Yellen, 1990). A charge reversal mutation at this location
had little effect on the ionic strength dependence of quaternary ammonium ion block, suggesting that the charge on this amino
acid may directly affect binding affinity but not local ion accumulation.
Received: 7 December 2000/Revised: 27 April 2001 相似文献
12.
The Ach-evoked Ca2+-activated K+ Current in Mouse Mandibular Secretory Cells. Single Channel Studies
Although acetylcholine (ACh) is able to activate voltage- and Ca2+-sensitive K+ (BK) channels in mouse mandibular secretory cells, our recent whole cell studies have suggested that these channels, like
those in sheep parotid secretory cells, do not contribute appreciably to the conductance that carries the ACh-evoked whole
cell K+ current. In the present study, we have used cell-attached patch clamp methods to identify and characterize the K+ channel type responsible for carrying the bulk of this current. When the cells were bathed in a NaCl-rich solution the predominant
channel type activated by ACh (1 μmol/l or 50 nmol/l) had a conductance only of 40 pS; it was not blocked by TEA but it was
sensitive to quinine and it conducted Rb+ to an appreciable extent. BK channels, which could be seen in some but not all patches from resting cells, also showed increased
activity when ACh was added to the bath, but they were much less conspicuous during ACh stimulation than the 40-pS channels.
When the cells were bathed in a KCl-rich rather than a NaCl-rich solution, a small-conductance K+ channel, sensitive to quinine but not to TEA, was still the most conspicuous channel to be activated by ACh although its
conductance was reduced to 25 pS. Our studies confirm that the ACh-evoked whole-cell K+ current is not carried substantially by BK channels and show that it is carried by a small-conductance K+ channel with quite different properties.
Received: 28 September 1995/Revised: 26 December 1995 相似文献
13.
P. K. Kienker X. -Q. Qiu S. L. Slatin A. Finkelstein K. S. Jakes 《The Journal of membrane biology》1997,157(1):27-37
Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of
target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic
segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment
forms an α-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with
the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have
been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed
its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin
added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected
channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first
opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic
hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens.
We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative
study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared
with previous results for wild-type colicin Ia.
Received: 12 November 1996/Revised: 23 January 1997 相似文献
14.
We have investigated the effects of extracellular and intracellular pH on single channel and macroscopic (macropatches) currents
through volume-regulated anion channels (VRAC) in endothelial cells.
Protonation of extracellular binding sites with an apparent pK of 4.6 increased voltage independent of the single-channel
amplitude. Cytosolic acidification had a dual effect on VRAC currents: on the one hand, it increased single channel conductance
by ∼20% due to protonation of a group with an apparent pK of 6.5 and a Hill coefficient of 2. On the other hand, it reduced
channel activity due to protonation of a group with an apparent pK of 6.3 and a Hill coefficient of 2.1. This dual effect
enhances the macroscopic current at a slightly acidic pH but inhibits it at more acidic pH. Cytosolic alkalization also reduced
channel activity with a pK of 8.4 and a Hill coefficient of 1.9, but apparently did not affect single-channel conductance.
These data show that VRAC channels are maintained in an active state in a narrow pH range around the normal physiological
pH and shut down outside this range. They also show that HEPES-buffered pipette solutions do not effectively buffer pH in
the vicinity of the VRAC channels.
Received: 31 January 2000/Revised: 21 April 2000 相似文献
15.
Plasma membrane anion channels are thought to play important roles in osmoregulation and signal transduction in higher plant
cells. Knowledge of their pharmacology and regulation is of importance to unravel their physiological functions. In this study,
we explore the pharmacological properties and the nucleotide regulation of the voltage-dependent anion channel of Arabidopsis hypocotyls. The pharmacological profile of this channel is characterized by a low sensitivity to most anion channel blockers.
It is inhibited by niflumic acid with an IC50 of 80 μm, but poorly sensitive to IAA-94 and NPPB and insensitive to 9-AC and DIDS. Nucleotides alter the amplitude, the kinetics
and the voltage-dependence of the channel. The main effect of nucleotides is a shift of the voltage-dependent gate of the
channel toward depolarized potentials leading to a strong reduction of the current amplitude. This regulation does not require
ATP hydrolysis as nonhydrolyzable ATP analogues—AMPPNP and ATPγS—also regulate the anion current. This suggests that a nucleotide
binding site is involved in the regulation. The study of the properties of this putative nucleotide binding site reveals that
(i) ATP regulates the channel with an EC50 of 0.7 mm, (ii) adenyl nucleotides modulate the channel with the following order of effectiveness: ATP > ADP ≫ AMP, and (iii) thiophosphate
nucleotide analogues are the most potent agonists with EC50 in the range of 80 μm. The hypothesis that this regulation may couple the electrical properties of the membrane with the metabolic status of the
cell is discussed.
Received: 26 December 1996/Revised: 21 March 1997 相似文献
16.
We investigated the properties of calcium-activated chloride channels in inside-out membrane patches from the dendritic knobs
of acutely dissociated rat olfactory receptor neurons. Patches typically contained large calcium-activated currents, with
total conductances in the range 30–75 nS. The dose response curve for calcium exhibited an EC50 of about 26 μm. In symmetrical NaCl solutions, the current-voltage relationship reversed at 0 mV and was linear between −80 and +70 mV.
When the intracellular NaCl concentration was progressively reduced from 150 to 25 mM, the reversal potential changed in a manner consistent with a chloride-selective conductance. Indeed, modeling these data
with the Goldman-Hodgkin-Katz equation revealed a PNa/PCl of 0.034. The halide permeability sequence was PCl > PF > PI > PBr indicating that permeation through the channel was dominated by ion binding sites with a high field strength. The channels
were also permeable to the large organic anions, SCN−, acetate−, and gluconate−, with the permeability sequence PCl > PSCN > Pacetate > Pgluconate. Significant permeation to gluconate ions suggested that the channel pore had a minimum diameter of at least 5.8 \A.
Received: 16 April 1997/Revised: 3 October 1997 相似文献
17.
Harper AA Catacuzzeno L Trequattrini C Petris A Franciolini F 《The Journal of membrane biology》2001,179(2):103-111
The effects of verapamil on the large conductance Ca-activated K (BK) channel from rat aortic smooth muscle cells were examined
at the single channel level. Micromolar concentrations of verapamil produced a reversible flickering block of the BK channel
activity. Kinetic analysis showed that verapamil decreased markedly the time constants of the open states, without any significant
change in the time constants of the closed states. The appearance of an additional closed state — specifically, a nonconducting,
open-blocked state — was also observed, whose time constant would reflect the mean residence time of verapamil on the channel.
These observations are indicative of a state-dependent, open-channel block mechanism. Dedicated kinetic (group) analysis confirmed
the state-dependent block exerted by verapamil. D600 (gallopamil), the methoxy derivative of verapamil, was also tested and
found to exert a similar type of block, but with a higher affinity than verapamil. The permanently charged and membrane impermeant
verapamil analogue D890 was used to address other important features of verapamil block, such as the sidedness of action and
the location of the binding site on the channel protein. D890 induced a flickering block of BK channels similar to that observed
with verapamil only when applied to the internal side of the membrane, indicating that D890 binds to a site accessible from
the cytoplasmic side. Finally, the voltage dependence of D890 block was assessed. The experimental data fitted with a Langmuir
equation incorporating the Woodhull model for charged blockers confirms that the D890-binding site is accessed from the internal
mouth of the BK channel, and locates it approximately 40% of the membrane voltage drop along the permeation pathway.
Received: 11 April 2000/Revised: 17 October 2000 相似文献
18.
N.B. Brovtsyna D.B. Tikhonov O.B. Gorbunova V.E. Gmiro S.E. Serduk N.Y. Lukomskaya L.G. Magazanik B.S. Zhorov 《The Journal of membrane biology》1996,152(1):77-87
Structure-activity relationships of 56 pentamethylenbis-ammonium compounds, the blockers of the neuronal nicotinic acetylcholine
receptor (nAChR) ion channel, have been studied to estimate the cross-sectional dimensions of the channel pore. The cat superior
cervical sympathetic ganglion in situ and isolated guinea pig ileum were used to evaluate the potency of the compounds to block ganglionic transmission. Minimum-energy
conformations of each compound were calculated by the molecular mechanics method. A topographic model of the binding site
of the blockers was proposed. It incorporates two narrowings, a large and a small one. The small narrowing is located between
the large one and the cytoplasmic end of the pore. The cross-sectional dimensions of the large and small narrowings estimated
from the dimensions of the blockers are 6.1 × 8.3 ? and 5.5 × 6.4 ?, respectively, the distance between the narrowings along
the pore being approximately 7 ?. Most potent blockers would occlude the pore via binding to the channel at the levels of
both narrowings. Less potent blockers are either too large or too small to bind to both narrowings simultaneously: large blockers
would occlude the pore at the level of large narrowing, while small blockers would pass the large narrowing and occlude the
pore at the level of small narrowing only. A comparison of the topographic model with a molecular five-helix bundle model
of nAChR pore predicts Serine and Threonine rings to be the most probable candidates for the large and small narrowings, respectively.
Received: 6 September 1995/Revised: 12 March 1996 相似文献
19.
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
20.
An ion''s view of the potassium channel. The structure of the permeation pathway as sensed by a variety of blocking ions 总被引:11,自引:5,他引:6
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We have studied the block of potassium channels in voltage-clamped squid giant axons by nine organic and alkali cations, in order to learn how the channel selects among entering ions. When added to the internal solution, all of the ions blocked the channels, with inside-positive voltages enhancing the block. Cesium blocked the channels from the outside as well, with inside-negative voltages favoring block. We compared the depths to which different ions entered the channel by estimating the "apparent electrical distance" to the blocking site. Simulations with a three-barrier, double-occupancy model showed that the "apparent electrical distance," expressed as a fraction of the total transmembrane voltage, appears to be less than the actual value if the blocking ion can pass completely through the channel. These calculations strengthen our conclusion that sodium and cesium block at sites further into the channel than those occupied by lithium and the organic blockers. Our results, considered together with earlier work, demonstrate that the depth to which an ion can readily penetrate into the potassium channel depends both on its size and on the specific chemical groups on its molecular surface. The addition of hydroxyl groups to alkyl chains on a quaternary ammonium ion can both decrease the strength of binding and allow deeper penetration into the channel. For alkali cations, the degree of hydration is probably crucial in determining how far an ion penetrates. Lithium, the most strongly hydrated, appeared not to penetrate as far as sodium and cesium. Our data suggest that there are, minimally, four ion binding sites in the permeation pathway of the potassium channel, with simultaneous occupancy of at least two. 相似文献