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1.
In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology
of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels
using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.1 channels was more sensitive to solution ionic strength than was TEA block, a result
consistent with a contribution from an electrostatic potential near the blocking site. In contrast, TEA block of another type
of K channel (Kv2.1) was insensitive to solution ionic strength and these channels were resistant to block by gallamine. Neutralizing
either of two lysine residues in the outer vestibule of these Kv2.1 channels conferred ionic strength sensitivity to TEA block.
Kv2.1 channels with both lysines neutralized were sensitive to block by gallamine, and the ionic strength dependence of this
block was greater than that for TEA. These results demonstrate that Kv3.1 (like Shaker) channels contain negatively charged residues in the outer vestibule of the pore that influence quaternary ammonium pharmacology.
The presence of specific lysine residues in wild-type Kv2.1 channels produces an outer vestibule with little or no net charge,
with important consequences for quaternary ammonium block. Neutralizing these key lysines results in a negatively charged
vestibule with pharmacological properties approaching those of other types of K channels. 相似文献
2.
F. Gómez-Lagunas T. Olamendi-Portugal F.Z. Zamudio L.D. Possani 《The Journal of membrane biology》1996,152(1):49-56
Two novel peptides were purified from the venom of the scorpion Pandinus imperator, and were named Pi2 and Pi3. Their complete primary structures were determined and their blocking effects on Shaker B K+ channels were studied. Both peptides contain 35 amino acids residues, compacted by three disulfide bridges, and reversibly
block the Shaker B K+ channels. They have only one amino acid changed in their sequence, at position 7 (a proline for a glutamic acid). Whereas
peptide Pi2, containing the Pro7, binds the Shaker B K+ channels with a K
d
of 8.2 nm, peptide Pi3 containing the Glu7 residue has a much lower affinity of 140 nm. Both peptides are capable of displacing the binding of 125I-noxiustoxin to brain synaptosome membranes. Since these two novel peptides are about 50% identical to noxiustoxin, the present
results support previous data published by our group showing that the amino-terminal region of noxiustoxin, and also the amino-terminal
sequence of the newly purified homologues: Pi2, and Pi3, are important for the recognition of potassium channels.
Received: 13 November 1995/Revised: 11 March 1996 相似文献
3.
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
4.
Two inward-rectifier K+ channels, ROMK2 (Kir1.1b) and IRK1 (Kir2.1), were expressed in Xenopus oocytes and their gating properties were studied in cell-attached membrane patches. The gating properties depended strongly
on the ion being conducted (K+, NH4
+, Rb+, or Tl+), suggesting tight coupling between permeation and gating. Mean open times were strongly dependent on the nature of the conducted
ion. For ROMK2 the order from the longest to the shortest times was K+ > Rb+ > Tl+ > NH4
+. For IRK1 the sequence was K+ > NH4
+ > Tl+. In both cases the open times decreased monotonically as the membrane voltage was hyperpolarized. Both the absolute values
and the voltage dependence of closed times were dependent on the conducted species. ROMK2 showed a single closed state whose
mean lifetimes were biphasic functions of voltage. The maxima were at various voltages for different ions. IRK1 had at least
two closed states whose lifetimes decreased monotonically with K+, increased monotonically with Tl+, and were relatively constant with NH4
+ as the conducted ion. We explain the ion-dependence of gating by assuming that the ions bind to a site within the permeation
pathway, resulting in a stable, ion-dependent, closed state of the channel. The patterns of voltage-dependence of closed-state
lifetimes, which are specific for different ions, can be explained by variations in the rate at which the bound ions leave
the pore toward the inside or the outside of the cell.
Received: 18 April 2001/Revised: 28 June 2001 相似文献
5.
John G. Starkus Lioba Kuschel Martin D. Rayner Stefan H. Heinemann 《The Journal of general physiology》1998,112(1):85-93
C-type inactivation in Shaker potassium channels inhibits K+ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na+ and Li+ currents in K+-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na+ and Li+ currents (in K+-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na+ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions. 相似文献
6.
Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these
channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential
and their contribution to cation toxicities. The selectivity of the rca channel, a Ca2+-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K+, Na+, Ca2+ and Mg2+ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two
ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity
of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the
pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure
of the rca channel appeared to differ from that of L-type calcium channels from animal cell membranes since its ion binding sites had
a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the
rca channel. It described the apparent submillimolar K
m
for the relationship between unitary conductance and Ca2+ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the
changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca2+ on K+ and Na+ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that
would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of
the rca channel for Ca2+ influx under physiological conditions.
Received: 23 August 1999/Revised: 12 November 1999 相似文献
7.
Melanoma cells are transformed melanocytes of neural crest origin. K+ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize
ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify
voltage-gated (KV), Ca2+-activated (KCa), and inwardly rectifying (KIR) K+ channels; swelling-sensitive Cl− channels (Clswell); voltage-gated Ca2+ channels (CaV) and Ca2+ channels activated by depletion of intracellular Ca2+ stores (CRAC); and voltage-gated Na+ channels (NaV). The presence of Ca2+ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca2+]
i
. The expression of K+ channels varied widely between different cell lines and was also influenced by culture conditions. KIR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for KIR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). KCa channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). KCa channels in C8146 and SK28 cells were sensitive to CTX (K
d
= 4 nm), but were unaffected by apamin. KV channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed
CRAC channels and a novel Clswell channel. Clswell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1
in symmetrical Cl−). We conclude that different melanoma cell lines express a diversity of ion channel types.
Received: 2 April 1996/Revised: 22 August 1996 相似文献
8.
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
9.
U. Yermiyahu G. Rytwo D.K. Brauer T.B. Kinraide 《The Journal of membrane biology》1997,159(3):239-252
A general model for the sorption of trivalent cations to wheat-root (Triticum aestivum L cv. Scout 66) plasma membranes (PM) has been developed and includes the first published coefficients for La3+ and Al3+ binding to a biological membrane. Both ions are rhizotoxic, and the latter ion is the principal contributor to the toxicity
of acidic soils around the world. The model takes into account both the electrostatic attraction and the binding of cations
to the negatively charged PM surface. Ion binding is modeled as the reaction P
−+I
Z⇌PI
Z
−1 in which P
− represents a negatively charged PM ligand, located in an estimated area of 540 ?2, and I
Z represents an ion of charge Z. Binding constants for the reaction were assigned for K+ (1 m
−1) and Ca2+ (30 m
−1) and evaluated experimentally for La3+ (2200 m
−1) and H+ (21,500 m
−1). Al sorption is complicated by Al3+ hydrolysis that yields hydroxoaluminum species that are also sorbed. Binding constants of 30 and 1 m
−1 were assigned for AlOH2+ and Al(OH)+
2, respectively, then a constant for Al3+ (20,000 m
−1) was evaluated experimentally using the previously obtained values for K+, Ca2+ and H+ binding. Electrostatic attraction was modeled according to Gouy-Chapman theory. Evaluation of parameters was based upon the
sorption of ions to PM vesicles suspended in solutions containing variable concentrations of H+, Ca2+ and La3+ or Al3+. Use of small volumes, and improved assay techniques, allowed the measurement of concentration depletions caused by sorption
to vesicles. Some independent confirmation of our model is provided by substantial agreement between our computations and
two published reports of La3+ effects upon zeta potentials of plant protoplasts. The single published report concerning the electrostatic effects of Al
on cell membranes is in essential agreement with the model.
Received: 6 January 1997/Revised: 6 June 1997 相似文献
10.
T.E. DeCoursey S.Y. Kim M.R. Silver F.N. Quandt 《The Journal of membrane biology》1996,152(2):141-157
Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells
by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K+ currents, I
DR, present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K+ channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K+ channel (I
IR) and a Ca2+-activated maxi-K channel (I
BK). I
IR was a classical inward rectifier, conducting large inward currents negative to E
K and very small outward currents. I
IR was blocked in a voltage-dependent manner by Cs+, Na+, and Ba2+, block increasing with hyperpolarization. Block by Na+ and Ba2+ was time-dependent, whereas Cs+ block was too fast to resolve. Rb+ was sparingly permeant. In cell-attached patches with high [K+] in the pipette, the single I
IR channel conductance was ∼30 pS and no outward current could be detected. I
BK channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the
conductance was ∼200–250 pS and at potentials positive to ∼100 mV a negative slope conductance of the unitary current was
observed, suggesting block by intracellular Na+. I
BK was activated at large positive potentials in cell-attached patches; in inside-out patches the voltage-activation relationship
was shifted to more negative potentials by increased [Ca2+]. Macroscopic I
BK was blocked by external TEA+ with half block at 0.35 mm. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K+ channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the
same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold.
Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed.
Received: 19 September 1995/Revised: 14 March 1996 相似文献
11.
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
12.
In our previous studies on sheep parotid secretory cells, we showed that the K+ current evoked by acetylcholine (ACh) was not carried by the high-conductance voltage- and Ca2+-activated K+ (BK) channel which is so conspicuous in unstimulated cells, notwithstanding that the BK channel is activated by ACh. Since
several studies from other laboratories had suggested that the BK channel did carry the ACh-evoked K+ current in the secretory cells of the mouse mandibular gland, and that the current could be blocked with tetraethylammonium
(TEA), a known blocker of BK channels, we decided to investigate the ACh-evoked K+ current in mouse cells more closely. We studied whether the ACh-evoked K+ current in the mouse is inhibited by TEA and quinine. Using the whole-cell patch-clamp technique and microspectrofluorimetric
measurement of intracellular Ca2+, we found that TEA and quinine do inhibit the ACh-evoked K+ current but that the effect is due to inhibition of the increase in intracellular Ca2+ evoked by ACh, not to blockade of a K+ conductance. Furthermore, we found that the K+ conductance activated when ionomycin is used to increase intracellular free Ca2+ was inhibited only by quinine and not by TEA. We conclude that the ACh-evoked K+ current in mouse mandibular cells does not have the blocker sensitivity pattern that would be expected if it were being carried
by the high-conductance, voltage- and Ca2+-activated K+ (BK) channel. The properties of this current are, however, consistent with those of a 40 pS K+ channel that we have reported to be activated by ACh in these cells [16].
Received: 9 January 1996/Revised: 17 April 1996 相似文献
13.
The permeation properties of KAT1, an inward rectifying potassium channel from plant cells, were investigated with different
ions in the external medium. With either K+, NH+
4 or methylammonium (MA) in the external solution, the channel, expressed in Xenopus oocytes, appeared permeable to K+ and, to a lesser extent, to NH+
4 but not to the slightly bigger, methylated analogue of NH+
4, MA. Substituting NH+
4 for K+ shifted the voltage dependency of channel activation further negative and hastened activation kinetics. This suggests that
channel operation depends on the transported substrate. In mixed solution (50 mm K+, 50 mm MA) MA inhibited K+ current in a voltage-independent manner. The maximum block did not exceed 50% of the K+ current. In contrast, when NH+
4 was the permeant ion (50 mm NH+
4, 50 mm MA) MA caused a voltage-dependent, slowly developing open channel block, achieving complete inhibition at very negative voltages.
The latter block could be partially overcome by the addition of K+ in the external solution. The data support a model in which ions, after entering the channel pore, compete with different
affinities for binding sites on their permeation pathway.
Received: 6 October 1997/Revised: 28 January 1998 相似文献
14.
Single inward rectifier K+ channels were studied in Xenopus laevis embryonic myocytes. We have characterized in detail the channel which is most frequently observed (Kir) although we routinely
observe three other smaller current levels with the properties of inward rectifier K+ channels (Kir(0.3), Kir(0.5) and Kir(0.7)). For Kir, slope conductances of inward currents were 10.3, 20.3, and 27.9 pS, in 60, 120 and 200 mM [K+]
o
respectively. Extracellular Ba2+ blocked the normally high channel activity in a concentration-dependent manner (K
A
= 7.8 μm, −90 mV). In whole-cell recordings of inward rectifier K+ current, marked voltage dependence of Ba2+ block over the physiological range of potentials was observed. We also examined current rectification. Following step depolarizations
to voltages positive to E
K
, outward currents through Kir channels were not observed even when the cytoplasmic face of excised patches were exposed to
Mg2+-free solution at pH 9.1. This was probably also true for Kir(0.3), Kir(0.5) and Kir(0.7) channels. We then examined the possibility of modulation of Kir channel activity and found neither ATP nor GTP-γS had any
effect on Kir channel activity when added to the solution perfusing the cytoplasmic face of a patch. Kinetic analysis revealed
Kir channels with a single open state (mean dwell time 72 msec) and two closed states (time constants 1.4, 79 msec). These
results suggest that the native Kir channels of Xenopus myocytes have similar properties to the cloned strong inward rectifier K+ channels, in terms of conductance, kinetics and barium block but does show some differences in the effects of modulators
of channel activity. Furthermore, skeletal muscle may contain either different inward rectifier channels or a single-channel
type which can exist in stable subconductance states.
Received: 16 September 1996/Revised: 14 March 1997 相似文献
15.
Brock MW Lebaric ZN Neumeister H DeTomaso A Gilly WF 《The Journal of membrane biology》2001,180(2):147-161
SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) α-subunit expressed only in the giant axon and the parental giant fiber lobe (GFL) neurons of the squid stellate ganglion.
We incorporated SqKv1A into a recombinant baculovirus for expression in the insect Sf9 cell line. Whole-cell patch-clamp recordings
reveal that very few cells display functional potassium current (I
K) if cultured at the standard postinfection temperature of 27°C. At 18°C, less SqKv1A protein is produced than at 27°C, but
cells with I
K currents are much more numerous and can survive for at least 20 days postinfection (vs. ∼5 days at 27°C). Activation and
deactivation kinetics of SqKv1A in Sf9 cells are slower (∼3- and 10-fold, respectively) than those of native channels in GFL
neurons, but have similar voltage dependencies. The two cell types show only subtle differences in steady-state voltage-dependence
of conductance and inactivation. Rates of I
K inactivation in 20 mm external K are identical in the two cell types, but the sensitivity of inactivation to external tetraethylammonium (TEA)
and K ions differ: inactivation of SqKv1A in Sf9 cells is slowed by external TEA and K ions, whereas inactivation of GFL I
K is largely insensitive. Functional differences are discussed in terms of factors that may be specific to cell-type, including
the presence of presently unidentified Kv1 subunits in GFL neurons that might form heteromultimers with SqKv1A. 相似文献
16.
P.M. Dunn 《The Journal of membrane biology》1998,165(2):133-143
The actions of clotrimazole and cetiedil, two drugs known to inhibit the Gardos channel, have been studied on single intermediate
conductance calcium-activated potassium (IKCa) channels in inside out patches from human red blood cells, and compared with those of TEA and Ba2+ applied to the cytoplasmic face of the membrane. TEA produced a fast block which was observed as a reduction in the amplitude
of the single channel current. This effect was weakly voltage dependent with the fraction of the membrane potential sensed
by TEA at its binding site (δ) of 0.18 and a K
d
at 0 mV of 20.5 mm. Ba2+ was a very potent blocker of the channel, breaking the single channel activity up into bursts, interspersed with silent periods
lasting several seconds. The effect of Ba2+ was very voltage sensitive, δ= 0.44, and a K
d
at 0 mV of 0.15 μm. Clotrimazole applied to the inner face of the membrane at a concentration ≤1 μm produced a slow block resulting in bursts of channel activity separated by quiescent periods lasting many seconds. The effect
of clotrimazole was mimicked by a quaternary derivative UCL 1559, in keeping with an action at the cytoplasmic face of the
channel. A high concentration of cetiedil (100 μm) produced only a weak block of the channel. The kinetics of this action were very slow, with burst and inter-burst intervals
lasting several minutes. While inhibition of the Gardos channel by cetiedil is unlikely to involve an intracellular site of
action, if clotrimazole is able to penetrate the membrane, part of its effect may result from binding to an intracellular
site on the channel.
Received; 18 February 1998/Received: 5 June 1998 相似文献
17.
V.M. Baizabal-Aguirre S. Clemens N. Uozumi J.I. Schroeder 《The Journal of membrane biology》1999,167(2):119-125
The Arabidopsis thaliana cDNA, KAT1 encodes a hyperpolarization-activated K+ (K+
in
) channel. In the present study, we identify and characterize dominant negative point mutations that suppress K+
in
channel function. Effects of two mutations located in the H5 region of KAT1, at positions 256 (T256R) and 262 (G262K), were
studied. The co-expression of either T256R or G262K mutants with KAT1 produced an inhibition of K+ currents upon membrane hyperpolarization. The magnitude of this inhibition was dependent upon the molar ratio of cRNA for
wild-type to mutant channel subunits injected. Inhibition of KAT1 currents by the co-expression of T256R or G262K did not
greatly affect the ion selectivity of residual currents for Rb+, Na+, Li+, or Cs+. When T256R or G262K were co-expressed with a different K+ channel, AKT2, an inhibition of the channel currents was also observed. Voltage-dependent Cs+ block experiments with co-expressed wild type, KAT1 and AKT2, channels further indicated that KAT1 and AKT2 formed heteromultimers.
These data show that AKT2 and KAT1 are able to co-assemble and suggest that suppression of channel function can be pursued
in vivo by the expression of the dominant negative K
+
in
channel mutants described here.
Received: 2 July 1998/Revised: 23 October 1998 相似文献
18.
P2X2 purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied
the channel's selectivity for the alkali metal ions and organic monovalent cations NMDG+, Tris+, TMA+, and TEA+. The selectivity sequence for currents carried by alkali metal ions is: K+ > Rb+ > Cs+ > Na+ > Li+, which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction
between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the
Stokes radius. The organic ions NMDG+, Tris+, TMA+ and TEA+ were virtually impermeant. The divalent ions (Mn2+, Mg2+, Ca2+ and Ba2+) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the
divalent ions exhibited an equilibrium affinity sequence of Mn2+ > Mg2+ > Ca2+ > Ba2+.
Received: 3 May 1999/Revised: 23 August 1999 相似文献
19.
Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through
the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to
investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles.
Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex.
These currents are essentially mediated by K+ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage
and inhibited by extracellular Ca2+ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca2+-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various
models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As
a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive
states via independent first order reactions: a fast inactivation/activation by Ca2+-binding and -release, respectively (rate constants >>103 sec−1) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the
range of 103 sec−1).
With 10 mm K+ and 1 mm Ca2+ in the external solution, intact coleoptile cells have a membrane voltage (V) of −105 ± 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K+ uptake required for cell elongation. Extracellular TEA or Ca2+, which inhibit the K+ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects
of Ca2+ and TEA on both processes and the similar Ca2+ concentration required for half maximal inhibition of growth (4.3 mm Ca2+) and for conductance (1.2 mm Ca2+) suggest that K+ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles.
Received: 6 June 1995/Revised: 12 September 1995 相似文献
20.
Kummerow D Hamann J Browning JA Wilkins R Ellory JC Bernhardt I 《The Journal of membrane biology》2000,176(3):207-216
The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive
fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K+ efflux and Cl− loss. When human erythrocytes were suspended in a physiological NaCl solution (pH
o
= 7.4), the measured pH
i
was 7.19 ± 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution,
an immediate alkalinization increased the pH
i
to 7.70 ± 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed
to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl− content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison
of the calculated H+ influx with the measured unidirectional K+ efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were
enhanced by decreasing the ionic strength of the solution resulting in a H+ influx and a K+ efflux in LIS solution of 108.2 ± 20.4 mmol (l
cells
hr)−1 and 98.7 ± 19.3 mmol (l
cells
hr)−1, respectively. For bovine and porcine erythrocytes, in LIS media, H+ influx and K+ efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions.
Quinacrine, a known inhibitor of the mitochondrial K+(Na+)/H+ exchanger, inhibited the K+ efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K+(Na+)/H+ exchanger in the human erythrocyte membrane.
Received: 22 December 1999/Revised: 10 April 2000 相似文献