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1.
S Masetto E Mazzucchelli V Taglietti M Toselli 《Bollettino della Società italiana di biologia sperimentale》1990,66(1):51-58
The single K-channel current reported in a previous note was also studied in "outside-out" conditions. The electrode filling solutions used for the "cell-attached" experiments faced in this case the intracellular side of the membrane patches, the extracellular side facing the bath saline, i.e. Ringer standard. The most significant observations were obtained with filling solutions with varying proportions in K/Na concentrations solutions. In the absence of Na+ ([K+] = 110 mM), the elementary conductance was still around 90 pS and the I/V diagram was again somewhat bell shaped, though the distinctive reduction of the elementary conductance began at more positive potentials (+110 mV). No inward current could be detected upon membrane repolarization also in this case. The rectification became less evident and conductance increased with increasing Na+ concentration in the filling solution, until the I/V curve became a linear one and conductance was 270 pS with standard Ringer. Distinct inward elementary currents were evident upon repolarization in these conditions. Thus a complex interaction between Na+ and K+ takes place for conduction through the outward K channel in the frog oocyte, both cations probably competing for at least one active site inside. Another interesting observation concerns the process of gating of the OPC: the open times of the elementary currents were in fact much greater in outside out experiments as compared to cell-attached experiments, probably due to the presence of Ca++ in contact with the inner membrane side. Even increasing Na+ concentration prolonged the open time duration. The gating of the OPC in the membrane was not only voltage dependent, but also Ca++ and Na+ dependent. 相似文献
2.
H Lorkovi? 《Archives internationales de physiologie et de biochimie》1976,84(5):925-938
(1) Thin sheets of fibres from gastrocnemius and lumbricalis muscles of rats were washed in Tris-proponiate solutions containing 0.67 to 60 mM K. The voltage-current relationship was measured by the two microelectrode technique. (2) The V-C curve was S-shaped. The steep region, sometimes including a "forbidden" voltage zone, occurred between about -40 and -70 mV when the solution contained 2 mM K. In some fibres the steep region was found to occur at more positive currents and voltages in "upward" runs (steps of increasing depolarizing currents) then in "downward" runs. The V-C curves thus revealed hysteresis loops presumably covering a negative conductance region. (3) The voltage at which the steep region occurred was a function of [K]0. The mid-point of the steep region was 50 to 60 mV more positive than EK for a particular [K]0 was about 6 mM the steep region of the V-C relationship was not conspicuous. The steep V-C region is considered to reflect depolarizing K inactivation. The near disappearance of the phenomenon at 6 mM K is thought to result from an interference of delayed rectification and depolarizing K inactivation. 相似文献
3.
J E Hall 《Biophysical journal》1981,33(3):373-381
Alamethicin appears to allow voltage-dependent lipid exchange ("flip-flop") between leaflets of a planar bilayer. In membranes with one leaflet of phosphatidyl serine and one of phosphatidyl ethanolamine, the shape of the nonactin current-voltage curve accurately reports the difference in surface potential between the two sides of the membrane. The surface potential is itself a good measure of membrane asymmetry. Alamethicin added to the bathing solutions of an asymmetric membrane does not per se reduce the membrane asymmetry, but turning on the alamethicin conductance by application of a voltage pulse does. Immediately after application of a voltage pulse, large enough to turn on the alamethicin conductance, the asymmetry of the nonactin-K+ current voltage curve decreases, in some cases, nearly to zero. During the pulse, the alamethicin conductance activates if a decrease in surface potential favors turn-on of the alamethicin conductance or inactivates if a decrease in surface potential favors turn-off of the alamethicin conductance. After the pulse, the nonactin-K+ asymmetry returns to its original value if the alamethicin conductance is not turned on. The time-course of this return allows an estimate of the diffusion constant of lipid in the planar bilayer. The value obtained is 5.1 x 10(-8) cm2/s. 相似文献
4.
S Masetto E Mazzucchelli V Taglietti M Toselli 《Bollettino della Società italiana di biologia sperimentale》1990,66(1):43-50
A single channel current was studied in the membrane of the immature oocyte of the european frog (Rana esculenta) by using the "patch clamp" technique in the "cell attached" configuration. Single channel activity appeared as short outward currents when membrane potential was made positive inside; full activation required seconds to be complete, no inactivation being appreciable. Deactivation (or current block) upon membrane repolarization was so fast that no inward current could be detected in any case. The reversal potential, estimated by interpolating the I/V diagrams, was -30 mV using standard Ringer as electrode filling solution, and the elementary conductance was 95 pS. Neither reversal potential nor elementary conductance were affected by removal of external Ca2+ (Mg2+ or Ba2+ substitution) or external Cl- (methanesulphonate substitution). The reversal potential moved towards positive potentials by substituting external Na+ with K+, the magnitude of the shifts being consistent with a ratio PK/PNa = 6.4. A distinctive property of the current/voltage relation for this K-current is its anomalous bell-shape, the outward current displaying a maximum at membrane potentials around 75 mV with standard Ringer as electrode filling solution and tending to zero with more positive potentials. 相似文献
5.
Charge-pulse relaxation studies with lipid bilayer membranes modified by alamethicin 总被引:1,自引:0,他引:1
Charge-pulse relaxation studies with the alamethicin-lipid membrane system reveal a triphasic decay of membrane voltage. At short times (resolution time 2 microseconds), where a voltage decay due to the orientation of alamethicin dipoles from the interface into the membranes interior ("gating current") could possibly be expected, only a slow decrease with a time constant determined by the bare membrane conductance occurs. After approximately 1 ms (depending on the experimental conditions) the formation of alamethicin pores starts, leading to an increase in the voltage decay rate. When the characteristic voltage Vcpc is approached, pores close and after passing Vcpc the voltage decreases slowly again according to the bare membrane conductance. Vcpc is determined as a function of the initially applied voltage Vo, alamethicin and KCl concentration. Since the membrane voltage decreases continuously, the system does not reach the equilibrium states obtained at constant voltages. Taking the presented experimental results into account the estimate of the electrical potential at the functional membrane of photosynthesis induced by a saturating single turnover flash of deltaphio approximately 105-135 mV (Zickler, Witt and Boheim (1976) FEBS Lett. 66, 142-148) is changed to deltaphio approximately 200 mV. 相似文献
6.
Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium 总被引:1,自引:0,他引:1
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《The Journal of general physiology》1994,103(4):583-604
Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump. 相似文献
7.
The properties of two types of K+ channels in murine T lymphocytes are described on the basis of whole-cell and isolated-patch recordings using the gigohm-seal technique. Type l (standing for "lpr gene locus" or "large") channels were characterized mainly in T cells from mutant MRL/MpJ-lpr/lpr mice, in which they are present in large numbers. Type n ("normal") K+ channels are abundant and therefore most readily studied in concanavalin A-activated T cells from four strains of mice, MRL-+/+, CBA/J, C57BL/6J, and BALB/c. Type l channels, compared with type n, are activated at potentials approximately 30 mV more positive, and close much more rapidly upon repolarization. Type l channels inactivate more slowly and less completely than type n during maintained depolarization, but recover from inactivation more rapidly, so that little inactivation accumulates during repetitive pulses. Type l channels have a higher unitary conductance (21 pS) than type n (12 pS) and are less sensitive to block by external Co++, but are 100-fold more sensitive to block by external tetraethylammonium (TEA), with half-block of type l channels at 50-100 microM TEA compared with 8-16 mM for type n. TEA blocks both types of channels by reducing the apparent single channel current amplitude, with a dose-response relation similar to that for blocking macroscopic currents. Murine type n K+ channels resemble K+ channels in human T cells. 相似文献
8.
Rat diaphragm fibers were equilibrated for several hours in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 17 min. This repolarization was blocked by 5 mM BaCl2, a blocker of the inward rectifier K channel. On the other hand, 0.1 mM apamin and 0.02 mM glibenclamide which block the Ca-dependent and ATP sensitive K channels, respectively, and 0.1 mM 9-AC a blocker of the Cl- channel did not affect the repolarization. 5 mM barium decreased the K conductance measured under current-clamp conditions in diaphragm muscle fibers. The possible role of the inward rectifier system in the repolarization following return to normal [K]o is discussed. 相似文献
9.
The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (alpha-GPD) were studied in intrafusal muscle fibres of rat fast and slow muscles. The ATPase reaction was carried out after the three standard acid preincubations. The cold K2-EDTA preincubated ATPase reaction product was similar to that seen following the regular or alkali-preincubated ATPase reaction, except that the intermediate bag fibres exhibited much higher activity after cold K2-EDTA preincubation. Following either acetic acid solution or cold and room temperature K2-EDTA-preincubation, followed by the ATPase reaction, chain fibres of the fast muscles vastus lateralis and extensor digitorum longus exhibited a very low amount of reaction product as compared with those of the slow soleus. Veronal acetate and K2-EDTA preincubations (and equally preincubation in acetic acid solution) resulted in acid stable ATPase activity along the entire length of the typical bag fibres but only in the polar regions of the intermediate bag fibres. On the basis of differing alpha-GPD reaction, two sub populations of nuclear chain fibres were discovered in one spindle. It is a matter of conjecture, to what extent the histochemical differences of intrafusal fibres from fast and slow muscles reflects functional distinctions in the response to stretch of muscle spindles from fast and slow muscles. 相似文献
10.
Potassium-stimulated 45Ca entry into rat brain synaptosomes was measured at times ranging from 1 to 60 s. The K-rich solutions were used to depolarize the synaptosomes. Backflux of 45Ca from the synaptosomes was negligible during the first 10-20 s of incubation. An initial ("fast") phase of K-stimulated Ca entry, lasting from 1 to 2 s was observed. This phase was inhibited by low concentrations of La (KI approximately equal to 0.3 microM). It was also abolished ("inactivated") by incubating the synaptosomes in depolarizing solutions (containing veratridine, gramicidin, or elevated [K]o) before the addition of 45Ca. An additional long lasting ("slow") phase of K-stimulated Ca entry was also detected. This "slow" Ca entry was much less sensitive to La (KI > 100 microM) and was not affected by depolarizing the synaptosomes before the addition of 45Ca. The rate of influx during the fast phase was about four times the rate of Ca influx during the slow phase. Neither the fast nor slow phase of Ca entry was sensitive to tetrodotoxin (10 microM), a potent blocker of Na channels, but both phases were inhibited by Ni, Mn, Mg, and other agents that block Ca channels. The data are consistent with the presence of two distinct populations of voltage-regulated, divalent cation-selective pathways for Ca entry in presynaptic brain nerve endings. 相似文献
11.
Wegner LH Flickinger B Eing C Berghöfer T Hohenberger P Frey W Nick P 《Biochimica et biophysica acta》2011,1808(6):1728-1736
Permeabilization of biological membranes by pulsed electric fields ("electroporation") is frequently used as a tool in biotechnology. However, the electrical properties of cellular membranes at supra-physiological voltages are still a topic of intensive research efforts. Here, the patch clamp technique in the whole cell and the outside out configuration was employed to monitor current-voltage relations of protoplasts derived from the tobacco culture cell line "Bright yellow-2". Cells were exposed to a sequence of voltage pulses including supra-physiological voltages. A transition from a low-conductance (~0.1 nS/pF) to a high-conductance state (~5 nS/pF) was observed when the membrane was either hyperpolarized or depolarized beyond threshold values of around -250 to -300 mV and +200 to +250 mV, respectively. Current-voltage curves obtained with ramp protocols revealed that the electro-permeabilized membrane was 5-10 times more permeable to K+ than to gluconate. The K+ channel blocker tetraethylammonium (25 mM) did not affect currents elicited by 10 ms-pulses, suggesting that the electro-permeabilization was not caused by a non-physiological activation of K+ channels. Supra-physiological voltage pulses even reduced "regular" K+ channel activity, probably due to an increase of cytosolic Ca2+ that is known to inhibit outward-rectifying K+ channels in Bright yellow-2 cells. Our data are consistent with a reversible formation of aqueous membrane pores at supra-physiological voltages. 相似文献
12.
Malev VV Kaulin YA Bezrukov SM Gurnev PA Takemoto JY Shchagina LV 《Membrane & cell biology》2001,14(6):813-829
A cyclic lipodepsipeptide, syringomycin E (SME), incorporated into planar lipid membranes forms two types of channels ("small" and "large") different in their conductance by approximately a factor of six (Biophys. J. 74:2918-2925 (1998)). We analysed the dynamics of the SME-induced transmembrane current under voltage-clamp conditions to clarify the mechanisms of formation of these channels. The voltage-dependent opening/closure of SME channels in lipid bilayers are interpreted in terms of transitions between three types of clusters including 6-7 SME molecules and some lipid molecules. The initial cluster, the precursor of the other two, was in equilibrium with SME monomer molecules at the membrane surface. The other two types of clusters (State 1 and State 2) were formed from the precursor and also during their interconversions (the consecutive-parallel mechanism of transitions). State 1 was a non-conducting state in equilibrium with small channels, which partially determined the ionic conductance of lipid bilayers modified by SME. State 2 corresponded to large SME channels, major contributors to the conductance of a bilayer. The results of the theoretical analysis based on the chemical kinetics concepts were consistent with experimental observations. Such properties of the SME-induced channels as cluster organisation, voltage dependence and the existence of a non-conducting state are all features shared by many ion channels in biological membranes. This makes it possible to use SME channels as a model to study naturally occurring ion channels. 相似文献
13.
Summary The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (-GPD) were studied in intrafusal muscle fibres of rat fast and slow muscles. The ATPase reaction was carried out after the three standard acid preincubations. The cold K2-EDTA preincubated ATPase reaction product was similar to that seen following the regular or alkalipreincubated ATPase reaction, except that the intermediate bag fibres exhibited much higher activity after cold K2-EDTA preincubation. Following either acetic acid solution or cold and room temperature K2-EDTA-preincubation, followed by the ATPase reaction, chain fibres of the fast muscles vastus lateralis and extensor digitorum longus exhibited a very low amount of reaction product as compared with those of the slow soleus. Veronal acetate and K2-EDTA preincubations (and equally preincubation in acetic acid solution) resulted in acid stable ATPase activity along the entire length of the typical bag fibres but only in the polar regions of the intermediate bag fibres. On the basis of differing -GPD reaction, two sub populations of nuclear chain fibres were discovered in one spindle. It is a matter of conjecture, to what extent the histochemical differences of intrafusal fibres from fast and slow muscles reflects functional distinctions in the response to stretch of muscle spindles from fast and slow muscles. 相似文献
14.
Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and channel currents were measured in 250/50 mmol/l KCl cis/trans solutions. The channel currents measured from -40 to +40 mV had various linear voltage-current relationships and K(+)/Cl(-) permeability ratios at distinct voltage ranges. The channels possessed K(+)-Cl(-) promiscuous property. Depending on voltage, membrane permeability suddenly switched from K(+) over Cl(-) to Cl(-) over K(+) and back. The channels had Cl(-)/K(+) > 1 permeability at potentials around 0 mV and the permeability was switched to K(+)/Cl(-) > 1 at more negative and positive potentials. The chloride channel blocker, 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 5 x 10(-5) mol/l), influenced properties of the promiscuous channels - it activated potassium conductance of the channels. 相似文献
15.
Loss of shaker K channel conductance in 0 K+ solutions: role of the voltage sensor. 总被引:2,自引:0,他引:2
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In potassium-free solutions some types of K channels enter a long-lasting nonconducting or "defunct" state. It is known that Shaker K channels must open in K+-free solutions to become defunct. Gating current studies presented here indicate an abnormal conformation in the defunct state that restricts S4 movement and alters its kinetics. Thus an abnormality initiated in the P region spreads to the gating apparatus. We find that channels most readily become defunct on repolarization to an intermediate voltage, thus prolonging occupancy of one of the several intermediate closed states. The state dependence of becoming defunct was further dissected by using the gating mutant L382A. Simply closing this channel at 0 mV (reversing the last activation step) does not make the mutant channel defunct. Instead, it is necessary to move further left (more fully closed) in the activation sequence. This was confirmed with ShIR experiments showing that channels become defunct only if there is inward gating charge movement. Rapid transit through the intermediate states, achieved at very negative voltage, is relatively ineffective at making channels defunct. Several mutations that removed C-type inactivation also made the channels resistant to becoming defunct. Our results show that normal gating current cannot be stably recorded in the absence of K+. 相似文献
16.
The first part of this paper describes the current voltage curves of bimolecular membranes of oxidized cholesterol formed between two aqueous solutions of tetrabutylammonium chloride. These membranes are selectively permeable for cations and the membrane interfaces are electrically uncharged. The dependence of the membrane conductivity on the membrane potential can be described as the product of the conductivity at zero current ("zero conductivity") and a function called "overlinearity". The zero conductivity increases linearly with the concentration of tetrabutylammonium chloride. The overlinearity is independent of the concentration of tetrabutylammonium chloride. In the second part the Nernst-Planck and Poisson equations are integrated numerically for a three-phase system consisting of an aqueous electrolyte solution, a membrane and an aqueous electrolyte solution. Each phase is characterized by material constants. Appropriate boundary conditions cause the electric current to build up electrical double layers on both sides of the membrane. The opposing double layers with opposite electrical signs inject the soluble ions into the membrane. This ion injection accounts for the overlinearity of the current voltage curves, thus explaining the measured characteristics. 相似文献
17.
J M Diamond 《The Journal of experimental zoology》1975,194(1):227-239
This paper reviews the history of understanding how biological systems can discriminate so strikingly among physically similar ions, especially alkali cations. Appreciation of qualitative regularities ("permitted sequences") and quantitative regularities ("selectivity isotherms") in ion selectivity grew first from studies of ion exchangers and glass electrodes, then of biological systems such as enzymes and cell membranes, and most recently of lipid bilayers doped with model pores and carriers. Discrimination of ions depends on both electrostatic and steric forces. "Black-box" studies on intact biological membranes have in some cases yielded molecular clues to the structure of the actual biological pores and carriers. Major current problems involve the extraction of these molecules; how to do it, what to do when it is achieved, and how (and if) it is relevant to the central problems of membrane function. Further advances are expected soon from studies of rate barriers within membranes, of voltage-dependent ("excitable") conducting channels, and of increasingly complex model systems and biological membranes. 相似文献
18.
Single crab (Callinectes danae) fibers were equilibrated with isotonic, high KCl solutions and were subsequently returned to the control saline. This caused marked swelling of the T tubules. Fibers treated with 100 mM KCl had a 2.5-mV residual depolarization, a 50% decrease in effective membrane resistance (Reff) and a 75% reduction in membrane time constant (tau m). These fibers exhibited large increases in membrane conductance upon depolarization and were inexcitable; membrane depolarization with current pulses elicited no contraction. The effects of the KCl treatment on membrane properties were not reproduced by treatment with high potassium gluconate solutions, which did not cause tubular swelling. Tetrabutylammonium (10 mM) or Ba ions (10-20 mM), but not tetraethylammonium (40-100 mM), Sr ions (15-70 mM), or procaine (1-8 mM) reversed the effects of the KCl treatment on Reff, tau m, membrane excitability, and excitation-contraction coupling. The time course of the Ba effects was consistent with the suggestion that the KCl treatment increases the K conductance of the tubular membranes, which in turn prevents the activation of voltage-dependent Ca channels located in the membranes of the T system. This results in inhibition of the Ca-dependent electrogenesis and consequently, the absence of contraction upon depolarization of the plasma membrane. 相似文献
19.
Summary The three types of porin (matrix-proteins) fromSalmonella typhimurium with molecular weights of 38,000, 39,000 and 40,000 were reconstituted with lipid bilayer membranes either as a trimer or as an oligomer (complex I). The specific conductance of the membranes increased several orders of magnitude after the addition of the porins into the aqueous phase bathing the membranes. A linear relationship between protein concentration in the aqueous phase and membrane conductance was found. In the case of lower protein concentrations (10–12
m), the conductance increased in a stepwise fashion with a single conductance increment of 2.3 nS in 1m KCl. For a given salt the conductance increment was found to be largely independent of the particular porin (38 K, 39K or 40 K) and on the state of aggregation, although porin oligomers showed an up to 10 times smaller conductance increase in macroscopic conductance measurements. The conductance pathway has an ohmic current voltage characteristic and a poor selectivity for different alkali ions. Further information on the structure of the pores formed by the different porins fromSalmonella was obtained from the selectivity for various ions. From the permeability of the pore for large ions (Tris+, glucosamine+, Hepes–_ a minimum pore diameter of 0.8 nm is estimated. This value is in agreement with the size of the pore as calculated from the conductance data for 1m KCl (1.4 nm for a pore length of 7.5 nm). The pore diameter may well account for the sugar permeability which has been found in reconstituted vesicles. The findings reported here are consistent with the assumption that the different porins form large aqueous channels in the lipid bilayer membranes and that the single condutance unit is a trimer. In addition, it is suggested that one trimer contains only one pore rather than a bundle of pores. 相似文献
20.
Properties and modulation of alpha human atrial natriuretic peptide (alpha-hANP)-formed ion channels
Using the lipid bilayer technique we have optimized recording conditions and confirmed that alpha human atrial natriuretic peptide [alpha-hANP(1-28)] forms single ion channels. The single channel currents recorded in 250/50 mM KCl cis/trans chambers show that the ANP-formed channels were heterogeneous, and differed in their conductance, kinetic, and pharmacological properties. The ANP-formed single channels were grouped as: (i) H202- and Ba2+-sensitive channel with fast kinetics; the nonlinear current-voltage (I-V) relationship of this channel had a reversal potential (Erev) of -28.2 mV, which is close to the equilibrium potential for K+ (EK = -35 mV) and a maximal slope conductance (gmax) of 68 pS at positive potentials. Sequential ionic substitution (KCl, K gluconate and choline Cl) of the cis solution suggests that the current was carried by cations. The fast channel had three modes (spike mode, burst mode, and open mode) that differed in their kinetics but not in their conductance properties. (ii) A large conductance channel possessing several subconductance levels that showed time-dependent inactivation at positive and negative membrane potentials (Vm). The inactivation ratio of the current at the end of the voltage step (Iss) to the initial current (Ii) activated immediately after the voltage step, (Iss/Ii), was voltage dependent and described by a bell-shaped curve. The maximal current-voltage (I-V) relationship of this channel, which had an Erev of +17.2 mV, was nonlinear and the value of gmax was 273 pS at negative voltages. (iii) A transiently-activated channel: the nonlinear I-V relationship of this channel had an Erev of -29.8 mV and the value of gmax was 160 pS at positive voltages. We propose that the voltage-dependence of the ionic currents and the kinetic parameters of these channel types indicate that if they were formed in vivo and activated by cytosolic factors they could change the membrane potential and the electrolyte homeostasis of the cell. 相似文献