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1.
Monovalent and divalent cation permeability and block of neuronal nicotinic receptor channels in rat parasympathetic ganglia 总被引:4,自引:0,他引:4
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《The Journal of general physiology》1995,105(6):701-723
Acetylcholine-evoked currents mediated by activation of nicotinic receptors in rat parasympathetic neurons were examined using whole-cell voltage clamp. The relative permeability of the neuronal nicotinic acetylcholine (nACh) receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements. The channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Cs+ > K+ > Rb+ > Na+ > Li+, and permeability ratios relative to Na+ (Px/PNa) ranging from 1.27 to 0.75. The selectivity of the alkaline earths was also weak, with the sequence of Mg2+ > Sr2+ > Ba2+ > Ca2+, and relative permeabilities of 1.10 to 0.65. The relative Ca2+ permeability (PCa/PNa) of the neuronal nACh receptor channel is approximately fivefold higher than that of the motor endplate channel (Adams, D. J., T. M. Dwyer, and B. Hille. 1980. Journal of General Physiology. 75:493-510). The transition metal cation, Mn2+ was permeant (Px/PNa = 0.67), whereas Ni2+, Zn2+, and Cd2+ blocked ACh-evoked currents with half-maximal inhibition (IC50) occurring at approximately 500 microM, 5 microM and 1 mM, respectively. In contrast to the muscle endplate AChR channel, that at least 56 organic cations which are permeable to (Dwyer et al., 1980), the majority of organic cations tested were found to completely inhibit ACh- evoked currents in rat parasympathetic neurons. Concentration-response curves for guanidinium, ethylammonium, diethanolammonium and arginine inhibition of ACh-evoked currents yielded IC50's of approximately 2.5- 6.0 mM. The organic cations, hydrazinium, methylammonium, ethanolammonium and Tris, were measureably permeant, and permeability ratios varied inversely with the molecular size of the cation. Modeling suggests that the pore has a minimum diameter of 7.6 A. Thus, there are substantial differences in ion permeation and block between the nACh receptor channels of mammalian parasympathetic neurons and amphibian skeletal muscle which represent functional consequences of differences in the primary structure of the subunits of the ACh receptor channel. 相似文献
2.
《The Journal of general physiology》1994,103(6):1055-1070
These studies examine the properties of an apical potassium (K+) channel in macula densa cells, a specialized group of cells involved in tubuloglomerular feedback signal transmission. To this end, individual glomeruli with thick ascending limbs (TAL) and macula densa cells were dissected from rabbit kidney and the TAL covering macula densa cells was removed. Using patch clamp techniques, we found a high density (up to 54 channels per patch) of K+ channels in the apical membrane of macula densa cells. An inward conductance of 41.1 +/- 4.8 pS was obtained in cell-attached patches (patch pipette, 140 mM K+). In inside- out patches (patch pipette, 140 mM; bath, 5 mM K+), inward currents of 1.1 +/- 0.1 pA (n = 11) were observed at 0 mV and single channel current reversed at a pipette potential of -84 mV giving a permeability ratio (PK/PNa) of over 100. In cell-attached patches, mean channel open probability (N,Po, where N is number of channels in the patch and Po is single channel open probability) was unaffected by bumetanide, but was reduced from 11.3 +/- 2.7 to 1.6 +/- 1.3 (n = 5, p < 0.02) by removal of bath sodium (Na+). Simultaneous removal of bath Na+ and calcium (Ca2+) prevented the Na(+)-induced decrease in N.Po indicating that the effect of Na+ removal on N.Po was probably mediated by stimulation of Ca2+ entry. This interpretation was supported by studies where ionomycin, which directly increases intracellular Ca2+, produced a fall in N.Po from 17.8 +/- 4.0 to 5.9 +/- 4.1 (n = 7, p < 0.02). In inside- out patches, the apical K+ channel was not sensitive to ATP but was directly blocked by 2 mM Ca2+ and by lowering bath pH from 7.4 to 6.8. These studies constitute the first single channel observations on macula densa cells and establish some of the characteristics and regulators of this apical K+ channel. This channel is likely to be involved in macula densa transepithelial Cl- transport and perhaps in the tubuloglomerular feedback signaling process. 相似文献
3.
Permeation of internal and external monovalent cations through the catfish cone photoreceptor cGMP-gated channel 总被引:2,自引:0,他引:2
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《The Journal of general physiology》1995,106(3):485-505
The permeation of monovalent cations through the cGMP-gated channel of catfish cone outer segments was examined by measuring permeability and conductance ratios under biionic conditions. For monovalent cations presented on the cytoplasmic side of the channel, the permeability ratios with respect to extracellular Na followed the sequence NH4 > K > Li > Rb = Na > Cs while the conductance ratios at +50 mV followed the sequence Na approximately NH4 > K > Rb > Li = Cs. These patterns are broadly similar to the amphibian rod channel. The symmetry of the channel was tested by presenting the test ion on the extracellular side and using Na as the common reference ion on the cytoplasmic side. Under these biionic conditions, the permeability ratios with respect to Na at the intracellular side followed the sequence NH4 > Li > K > Na > Rb > Cs while the conductance ratios at +50 mV followed the sequence NH4 > K approximately Na > Rb > Li > Cs. Thus, the channel is asymmetric with respect to external and internal cations. Under symmetrical 120 mM ionic conditions, the single-channel conductance at +50 mV ranged from 58 pS in NH4 to 15 pS for Cs and was in the order NH4 > Na > K > Rb > Cs. Unexpectedly, the single-channel current-voltage relation showed sufficient outward rectification to account for the rectification observed in multichannel patches without invoking voltage dependence in gating. The concentration dependence of the reversal potential for K showed that chloride was impermeant. Anomalous mole fraction behavior was not observed, nor, over a limited concentration range, were multiple dissociation constants. An Eyring rate theory model with a single binding site was sufficient to explain these observations. 相似文献
4.
Block by calcium of ATP-activated channels in pheochromocytoma cells 总被引:12,自引:0,他引:12
《The Journal of general physiology》1993,101(3):377-392
We have investigated the effects of Ca2+ on Na+ influx through ATP- activated channels in pheochromocytoma PC12 cells using single channel current recordings. Under cell-attached patch-clamp conditions with 150 mM Na+ and 2 mM Ca2+ in the pipette, the unitary current activity showed an open level of about -4.3 pA at -150 mV. The channel opening was interrupted by flickery noise as well as occasional transition to a subconducting state of about -1.7 pA at -150 mV. The open level was decreased with increased external Ca2+, suggesting that external Ca2+ blocks Na+ permeation. We assessed the block by Ca2+ as the mean amplitude obtained with heavy filtration according to Pietrobon et al. (Pietrobon, D., B. Prod'hom, and P. Hess, 1989. J. Gen. Physiol. 94:1- 21). The block was concentration dependent with a Hill coefficient of 1 and a half-maximal concentration of approximately 6 mM. A similar block was observed with other divalent cations, and the order of potency was Cd2+ > Mn2+ > Mg2+ not equal to Ca2+ > Ba2+. High Ca2+, Mg2+ and Ba2+ did not block completely, probably because they can carry current in the channel. The block by external Ca2+ did not exhibit voltage dependence between -100 and -210 mV. In the inside-out patch-clamp configuration, the amplitude of inward channel current obtained with 150 mM external Na+ was reduced by increased internal Ca2+. The reduction was observed at lower concentrations than that by external Ca2+. Internal Ba2+ and Cd2+ induced similar reduction in current amplitude. This inhibitory effect of internal Ca2+ was voltage dependent; the inhibition was relieved with hyperpolarization. The results suggest that both external and internal Ca2+ can block Na+ influx through the ATP-activated channel. A simple one-binding site model with symmetric energy barriers is not sufficient to explain the Ca2+ block from both sides. 相似文献
5.
A mechanosensitive K+ channel in heart cells. Activation by arachidonic acid 总被引:6,自引:0,他引:6
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D Kim 《The Journal of general physiology》1992,100(6):1021-1040
Mechanosensitive ion channels have been described in many types of cells. These channels are believed to transduce pressure signals into intracellular biochemical and physiological events. In this study, the patch-clamp technique was used to identify and characterize a mechanosensitive ion channel in rat atrial cells. In cell-attached patches, negative pressure in the pipette activated an ion channel in a pressure-dependent manner. The pressure to induce half-maximal activation was 12 +/- 3 mmHg at +40 mV, and nearly full activation was observed at approximately 20 mmHg. The probability of opening was voltage dependent, with greater channel activity at depolarized potentials. The mechanosensitive channel was identical to the K+ channel previously shown to be activated by arachidonic acid and other lipophilic compounds, as judged by the outwardly rectifying current-voltage relation, single channel amplitude, mean open time (1.4 +/- 0.3 ms), bursty openings, K+ selectivity, insensitivity to any known organic inhibitors of ion channels, and pH sensitivity. In symmetrical 140 mM KCl, the slope conductance was 94 +/- 11 pS at +60 mV and 64 +/- 8 pS at -60 mV. Anions and cations such as Cl-, glutamate, Na+, Cs+, Li+, Ca2+, and Ba2+ were not permeant. Extracellular Ba2+ (1 mM) blocked the inward K+ current completely. GdCl3 (100 microM) or CaCl2 (100 microM) did not alter the K+ channel activity or amplitude. Lowering of intracellular pH increased the pressure sensitivity of the channel. The K+ channel could be activated in the presence of 5 mM intracellular [ATP] or 10 microM glybenclamide in inside-out patches. In the absence of ATP, when the ATP-sensitive K+ channel was active, the mechanosensitive channel could further be activated by pressure, suggesting that they were two separate channels. The ATP-sensitive K+ channel was not mechanosensitive. Pressure activated the K+ channel in the presence of albumin, a fatty acid binding protein, suggesting that pressure and arachidonic acid activate the K+ channel via separate pathways. 相似文献
6.
Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells. 总被引:2,自引:0,他引:2
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We studied monovalent permeability of Ca2+ release-activated Ca2+ channels (ICRAC) in Jurkat T lymphocytes following depletion of calcium stores. When external free Ca2+ ([Ca2+]o) was reduced to micromolar levels in the absence of Mg2+, the inward current transiently decreased and then increased approximately sixfold, accompanied by visibly enhanced current noise. The monovalent currents showed a characteristically slow deactivation (tau = 3.8 and 21.6 s). The extent of Na+ current deactivation correlated with the instantaneous Ca2+ current upon readdition of [Ca2+]o. No conductance increase was seen when [Ca2+]o was reduced before activation of ICRAC. With Na+ outside and Cs+ inside, the current rectified inwardly without apparent reversal below 40 mV. The sequence of conductance determined from the inward current at -80 mV was Na+ > Li+ = K+ > Rb+ >> Cs+. Unitary inward conductance of the Na+ current was 2.6 pS, estimated from the ratios delta sigma2/delta Imean at different voltages. External Ca2+ blocked the Na+ current reversibly with an IC50 value of 4 microM. Na+ currents were also blocked by 3 mM Mg2+ or 10 microM La3+. We conclude that ICRAC channels become permeable to monovalent cations at low levels of external divalent ions. In contrast to voltage-activated Ca2+ channels, the monovalent conductance is highly selective for Na+ over Cs+. Na+ currents through ICRAC channels provide a means to study channel characteristics in an amplified current model. 相似文献
7.
Ion permeation and block of M-type and delayed rectifier potassium channels. Whole-cell recordings from bullfrog sympathetic neurons 总被引:1,自引:0,他引:1
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《The Journal of general physiology》1996,107(4):473-488
Ion permeation and conduction were studied using whole-cell recordings of the M-current (I(M)) and delayed rectifier (IDR), two K+ currents that differ greatly in kinetics and modulation. Currents were recorded from isolated bullfrog sympathetic neurons with 88 mM [K+]i and various external cations. Selectivity for extracellular monovalent cations was assessed from permeability ratios calculated from reversal potentials and from chord conductances for inward current. PRb/PK was near 1.0 for both channels, and GRb/GK was 0.87 +/- 0.01 for IDR but only 0.35 +/- 0.01 for I(M) (15 mM [Rb+]o or [K+]o). The permeability sequences were generally similar for I(M) and IDR: K+ approximately Rb+ > NH4+ > Cs+, with no measurable permeability to Li+ or CH3NH3+. However, Na+ carried detectable inward current for IDR but not I(M). Nao+ also blocked inward K+ current for IDR (but not IM), at an apparent electrical distance (delta) approximately 0.4, with extrapolated dissociation constant (KD) approximately 1 M at 0 mV. Much of the instantaneous rectification of IDR in physiologic ionic conditions resulted from block by Nao+. Extracellular Cs+ carried detectable inward current for both channel types, and blocked I(M) with higher affinity (KD = 97 mM at 0 mV for I(M), KD) approximately 0.2 M at 0 mV for IDR), with delta approximately 0.9 for both. IDR showed several characteristics reflecting a multi-ion pore, including a small anomalous mole fraction effect for PRb/PK, concentration-dependent GRb/GK, and concentration- dependent apparent KD's and delta's for block by Nao+ and Cso+. I(M) showed no clear evidence of multi-ion pore behavior. For I(M), a two- barrier one-site model could describe permeation of K+ and Rb+ and block by Cso+, whereas for IDR even a three-barrier, two-site model was not fully adequate. 相似文献
8.
K+ channels of stomatal guard cells. Characteristics of the inward rectifier and its control by pH 总被引:2,自引:0,他引:2
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M R Blatt 《The Journal of general physiology》1992,99(4):615-644
Intracellular microelectrode recordings and a two-electrode voltage clamp have been used to characterize the current carried by inward rectifying K+ channels of stomatal guard cells from the broadbean, Vicia faba L. Superficially, the current displayed many features common to inward rectifiers of neuromuscular and egg cell membranes. In millimolar external K+ concentrations (Ko+), it activated on hyperpolarization with half-times of 100-200 ms, showed no evidence of time- or voltage-dependent inactivation, and deactivated rapidly (tau approximately 10 ms) on clamping to 0 mV. Steady-state conductance-voltage characteristics indicated an apparent gating charge of 1.3-1.6. Current reversal showed a Nernstian dependence on Ko+ over the range 3-30 mM, and the inward rectifier was found to be highly selective for K+ over other monovalent cations (K+ greater than Rb+ greater than Cs+ much greater than Na+). Unlike the inward rectifiers of animal membranes, the current was blocked by charybdotoxin and alpha-dendrotoxin (Kd much less than 50 nM), as well as by tetraethylammonium chloride (K1/2 = 9.1 mM); gating of the guard cell K+ current was fixed to voltages near -120 mV, independent of Ko+, and the current activated only with supramillimolar K+ outside (EK+ greater than -120 mV). Most striking, however, was inward rectifier sensitivity to [H+] with the K+ current activated reversibly by mild acid external pH. Current through the K+ inward rectifier was found to be largely independent of intracellular pH and the current reversal (equilibrium) potential was unaffected by pHo from 7.4 to 5.5. By contrast, current through the K+ outward rectifier previously characterized in these cells (1988. J. Membr. Biol. 102:235) was largely insensitive to pHo, but was blocked reversibly by acid-going intracellular pH. The action of pHo on the K+ inward rectifier could not be mimicked by extracellular Ca2+ for which changes in activation, deactivation, and conductance were consonant with an effect on surface charge ([Ca2+] less than or equal to 1 mM). Rather, extracellular pH affected activation and deactivation kinetics disproportionately, with acid-going pHo raising the K+ conductance and shifting the conductance-voltage profile positive-going along the voltage axis and into the physiological voltage range. Voltage and pH dependencies for gating were consistent with a single, titratable group (pKa approximately 7 at -200 mV) residing deep within the membrane electric field and accessible from the outside.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
9.
Barium modulates the gating of batrachotoxin-treated Na+ channels in high ionic strength solutions.
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S Cukierman 《Biophysical journal》1993,65(3):1168-1173
Batrachotoxin-activated rat brain Na+ channels were reconstituted in neutral planar phospholipid bilayers in high ionic strength solutions (3 M NaCl). Under these conditions, diffuse surface charges present on the channel protein are screened. Nevertheless, the addition of extracellular and/or intracellular Ba2+ caused the following alterations in the gating of Na+ channels: (a) external (or internal) Ba2+ caused a depolarizing (or hyperpolarizing) voltage shift in the gating curve (open probability versus membrane potential curve) of the channels; (b) In the concentration range of 10-120 mM, extracellular Ba2+ caused a larger voltage shift in the gating curve of Na+ channels than intracellular Ba2+; (c) voltage shifts of the gating curve of Na+ channels as a function of external or internal Ba2+ were fitted with a simple binding isotherm with the following parameters: for internal Ba2+, delta V0.5,max (maximum voltage shift) = -11.5 mV, KD = 64.7 mM; for external Ba2+, delta V0.5,max = 13.5 mV, KD = 25.8 mM; (d) the change in the open probability of the channel caused by extracellular or intracellular Ba2+ is a consequence of alterations in both the opening and closing rate constants. Extracellular and intracellular divalent cations can modify the gating kinetics of Na+ channels by a specific modulatory effect that is independent of diffuse surface potentials. External or internal divalent cations probably bind to specific charges on the Na+ channel glycoprotein that modulate channel gating. 相似文献
10.
The conductance properties of organic cations in single gramicidin A channels were studied using planar lipid bilayers. From measurements at 10 mM and at 27 mV the overall selectivity sequence was found to be NH4+ > K+ > hydrazinium > formamidinium > Na+ > methylammonium, which corresponds to Eisenman polyatomic cation sequence X'. Methylammonium and formamidinium exhibit self block, suggesting multiple occupancy and single filing. Formamidinium has an apparent dissociation constant (which is similar to those of alkali metal cations) for the first ion being 22 mM from the Eadie-Hofstee plot (G0 vs. G0/C), 12 mM from the rate constants of a three-step kinetic model. The rate-limiting step for formamidinium is translocation judging from supralinear I-V relations at low concentrations. 1 M formamidinium solutions yields exceptionally long single channel lifetimes, 20-fold longer than methylammonium, which yields lifetimes similar to those found with alkali metal cations. The average lifetime in formamidinium solution significantly decreases with increasing voltage up to 100 mV but is relatively voltage independent between 100 and 200 mV. At lower voltages (< or = 100 mV), the temperature and concentration dependences of the average lifetime of formamidinium were steep. At very low salt concentrations (0.01 M, 100 mV), there was no significant difference in average lifetime from that formed with 0.01 M methylammonium or hydrazinium. We conclude that formamidinium very effectively stabilizes the dimeric channel while inside the channel and speculate that it does so by affecting tryptophan-reorientation or tryptophan-lipid interactions at binding sites. 相似文献
11.
Na+ and K+ transport at basolateral membranes of epithelial cells. III. Voltage independence of basolateral membrane Na+ efflux 总被引:2,自引:2,他引:0
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Na+ efflux across basolateral membranes of isolated epithelia of frog skin was tested for voltage sensitivity. The intracellular Na+ transport pool was loaded with 24Na from the apical solution and the rate of isotope appearance in the basolateral solution (JNa23) was measured at timed intervals of 30 s. Basolateral membrane voltage was depolarized by either 50 mM K+, 5 mM Ba++, or 80 mM NH+4. Whereas within 30 s ouabain caused inhibition of JNa23, depolarization of Vb by 30-60 mV caused no significant change of JNa23. Thus, both pump-mediated and leak Na+ effluxes were voltage independent. Although the pumps are electrogenic, pump-mediated Na+ efflux is voltage independent, perhaps because of a nonlinear relationship between pump current and transmembrane voltage. Voltage independence of the leak Na+ efflux confirms a previous suggestion (Cox and Helman, 1983. American Journal of Physiology. 245:F312-F321) that basolateral membrane Na+ leak fluxes are electroneutral. 相似文献
12.
Ion conduction in substates of the batrachotoxin-modified Na+ channel from toad skeletal muscle. 总被引:3,自引:1,他引:2
Batrachotoxin-modified Na+ channels from toad muscle were inserted into planar lipid bilayers composed of neutral phospholipids. Single-channel conductances were measured for [Na+] ranging between 0.4 mM and 3 M. When membrane preparations were made in the absence of protease inhibitors, two open conductance states were identified: a fully open state (16.6 pS in 200 mM symmetrical NaCl) and a substate that was 71% of the full conductance. The substate was predominant at [Na+] > 65 mM, whereas the presence of the fully open state was predominant at [Na+] < 15 mM. Addition of protease inhibitors during membrane preparation stabilized the fully open state over the full range of [Na+] studied. In symmetrical Na+ solutions and in biionic conditions, the ratio of amplitudes remained constant and the two open states exhibited the same permeability ratios of PLi/PNa and PCs/PNa. The current-voltage relations for both states showed inward rectification only at [Na+] < 10 mM, suggesting the presence of asymmetric negative charge densities at both channel entrances, with higher charge density in the external side. An energy barrier profile that includes double ion occupancy and asymmetric charge densities at the channel entrances was required to fit the conductance-[Na+] relations and to account for the rectification seen at low [Na+]. Energy barrier profiles differing only in the energy peaks can give account of the differences between both conductance states. Estimation of the surface charge density at the channel entrances is very dependent on the ion occupancy used and the range of [Na+] tested. Independent evidence for the existence of a charged external vestibule was obtained at low external [Na+] by identical reduction of the outward current induced by micromolar additions of Mg2+ and Ba2+. 相似文献
13.
Ion conduction properties of a de novo synthesized channel, formed from cyclic octa-peptides consisting of four alternate L-alanine (Ala) and N'-acylated 3-aminobenzoic acid (Aba) moieties, were studied in bilayer membranes. The single-channel conductance was 9 pS in symmetrical 500 mM KCl. The channel favored permeation of cations over anions with a permeability ratio (PCl-/PK+) of 0.15. The selectivity sequence among monovalent cations based on permeability ratio (PX+/PK+) fell into an order: NH4+(1.4) > Cs+(1. 1) >/= K+(1.0) > Na+(0.4) >> Li+(0). The conductance-activity relationship of the channel in K+ solutions followed simple Michaelis-Menten kinetics with a half-maximal saturating activity of 8 mM and a maximal conductance of 9 pS. The permeability ratio PNa+/PK+ remained constant ( approximately 0.40) under biionic concentrations from 10 to 500 mM. These results suggests that the channel is a one-ion channel. The pore diameter probed by a set of organic cations was approximately 6 A. The single-channel current was blocked by Ca2+ in a dose-dependent manner that followed a single-site titration curve with a voltage-dependent dissociation constant of 0.6 mM at 100 mV. The electric distance of the binding site for Ca2+ was 0.07 from both entrances of the channel, indicating the presence of two symmetrical binding sites in each vicinity of the channel entrance. Correlations between conduction properties and structural aspects of the channel are discussed in terms of a three-barrier and two-binding-site (3B2S) model of Eyring rate theory. All available structural information supported an idea that the channel was formed from a tail-to-tail associated dimer of the molecule, the pore of which was lined with hydrophobic acyl chains. This is the first report to have made a systematic analysis of ion permeation through a hydrophobic pore. 相似文献
14.
Internal Na+ and Mg2+ blockade of DRK1 (Kv2.1) potassium channels expressed in Xenopus oocytes. Inward rectification of a delayed rectifier 总被引:1,自引:0,他引:1
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《The Journal of general physiology》1994,103(2):203-216
Delayed rectifier potassium channels were expressed in the membrane of Xenopus oocytes by injection of rat brain DRK1 (Kv2.1) cRNA, and currents were measured in cell-attached and inside-out patch configurations. In intact cells the current-voltage relationship displayed inward going rectification at potentials > +100 mV. Rectification was abolished by excision of membrane patches into solutions containing no Mg2+ or Na+ ions, but was restored by introducing Mg2+ or Na+ ions into the bath solution. At +50 mV, half- maximum blocking concentrations for Mg2+ and Na+ were 4.8 +/- 2.5 mM (n = 6) and 26 +/- 4 mM (n = 3) respectively. Increasing extracellular potassium concentration reduced the degree of rectification of intact cells. It is concluded that inward going rectification resulting from voltage-dependent block by internal cations can be observed with normally outwardly rectifying DRK1 channels. 相似文献
15.
The conduction properties of the cloned Shaker K+ channel were studied using electrophysiological techniques. Single channel conductance increases in a sublinear manner with symmetric increases in K+ activity, reaching saturation by 0.6 M K+. The Shaker K+ channel is highly selective among monovalent cations; under bi-ionic conditions, its selectivity sequence is K+ > Rb+ > NH+4 > Cs+ > Na+, whereas, by relative conductance in symmetric solutions, it is K+ > NH+4 > Rb+ > Cs+. In Cs+ solutions, single channel currents were too small to be measured directly, so nonstationary fluctuation analysis was used to determine the unitary Cs+ conductance. The single channel conductance displays an anomalous molefraction effect in symmetric mixtures of K+ and NH+4, suggesting that the conducting pore is occupied by multiple ions simultaneously. 相似文献
16.
Cyclic nucleotide-gated channels of rat olfactory receptor cells: divalent cations control the sensitivity to cAMP
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《The Journal of general physiology》1994,103(1):87-106
cAMP-gated channels were studied in inside-out membrane patches excised from the apical cellular pole of isolated olfactory receptor cells of the rat. In the absence of divalent cations the dose-response curve of activation of patch current by cAMP had a KM of 4.0 microM at -50 mV and of 2.5 microM at +50 mV. However, addition of 0.2 or 0.5 mM Ca2+ shifted the KM of cAMP reversibly to the higher cAMP concentrations of 33 or 90 microM, respectively, at -50 mV. Among divalent cations, the relative potency for inducing cAMP affinity shifts was: Ca2+ > Sr2+ > Mn2+ > Ba2+ > Mg2+, of which Mg2+ (up to 3 mM) did not shift the KM at all. This potency sequence corresponds closely to that required for the activation of calmodulin. However, the Ca(2+)-sensitivity is lower than expected for a calmodulin-mediated action. Brief (60 s) transient exposure to 3 mM Mg2+, in the absence of other divalent cations, had a protective effect in that following washout of Mg2+, subsequent exposure to 0.2 mM Ca2+ no longer caused affinity shifts. This protection effect did not occur in intact cells and was probably a consequence of patch excision, possibly representing ablation of a regulatory protein from the channel cyclic nucleotide binding site. Thus, the binding of divalent cations, probably via a regulatory protein, controls the sensitivity of the cAMP-gated channels to cAMP. The influx of Ca2+ through these channels during the odorant response may rise to a sufficiently high concentration at the intracellular membrane surface to contribute to the desensitization of the odorant- induced response. The results also indicate that divalent cation effects on cyclic nucleotide-gated channels may depend on the sequence of pre-exposure to other divalent cations. 相似文献
17.
Competitive Mg2+ block of a large-conductance, Ca(2+)-activated K+ channel in rat skeletal muscle. Ca2+, Sr2+, and Ni2+ also block
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W B Ferguson 《The Journal of general physiology》1991,98(1):163-181
The patch-clamp technique was used to investigate the effect of intracellular Mg2+ (Mgi2+) on the conductance of the large-conductance, Ca(2+)-activated K+ channel in cultured rat skeletal muscle. Measurements of single-channel current amplitudes indicated that Mgi2+ decreased the K+ currents in a concentration-dependent manner. Increasing Mgi2+ from 0 to 5, 10, 20, and 50 mM decreased channel currents by 34%, 44%, 56%, and 73%, respectively, at +50 mV. The magnitude of the Mgi2+ block increased with depolarization. For membrane potentials of -50, +50, and +90 mV, 20 mM Mgi2+ reduced the currents 22%, 56%, and 70%, respectively. Mgi2+ did not change the reversal potential, indicating that Mg2+ does not permeate the channel. The magnitude of the Mgi2+ block decreased as the concentration of K+ was increased. At a membrane potential of +50 mv, 20 mM Mgi2+ reduced the currents 71%, 56%, and 25% for Ki+ of 75, 150, and 500 mM. These effects of Mgi2+, voltage, and K+ were totally reversible. Although the Woodhull blocking model could approximate the voltage and concentration effects of the Mgi2+ block (Kd approximately 30 mM with 150 mM symmetrical K+; electrical distance approximately 0.22 from the inner surface), the Woodhull model could not account for the effects of K+. Double reciprocal plots of 1/single channel current vs. 1/[K+] in the presence and absence of Mgi2+, indicated that the Mgi2+ block is consistent with apparent competitive inhibition between Mgi2+ and Ki+. Cai2+, Nii2+, and Sri2+ were found to have concentration- and voltage-dependent blocking effects similar, but not identical, to those of Mgi2+. These observations suggest the blocking by Mgi2+ of the large-conductance, Ca(2+)-activated K+ channel is mainly nonspecific, competitive with K+, and at least partially electrostatic in nature. 相似文献
18.
The relation between ion permeation and recovery from inactivation of ShakerB K+ channels. 总被引:3,自引:1,他引:2
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We have studied the relation between permeation and recovery from N-type or ball-and-chain inactivation of ShakerB K channels. The channels were expressed in the insect cell line Sf9, by infection with a recombinant baculovirus, and studied under whole cell patch clamp. Recovery from inactivation occurs in two phases. The faster of the two lasts for approximately 200 ms and is followed by a slow phase that may require seconds for completion. The fast phase is enhanced by both permeant ions (K+, Rb+) and by the blocking ion Cs+, whereas the impermeant ions (Na+, Tris+, choline+) are ineffective. The relative potencies are K+ > Rb+ > Cs+ > NH4+ >> Na+ approximately choline+ approximately Tris+. Ion permeation through the channels is not essential for recovery. The results suggest that cations influence the fast phase of recovery by binding in a site with an electrical distance greater than 0.5. Recovery from fast inactivation is voltage-dependent. With Na+, choline+, or Tris+ outside, about 15% of the channels recover in the fast phase (-80 mV), and the other 85% apparently enter a second inactivated state from which recovery is very slow. Recovery in this phase is not influenced by external ions, but is speeded by hyperpolarization. 相似文献
19.
Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation. 总被引:1,自引:1,他引:0
The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel. 相似文献
20.
Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers 总被引:1,自引:0,他引:1
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《The Journal of general physiology》1995,106(3):445-466
Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride- sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12- 13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (< 30 mM) and 10-100 microM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans. Buffering of the cis compartment [Ca2+] to nearly zero (< 1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 +/- 0.02 vs 0.02 +/- 0.01 in control), but markedly reduced the influence of both cis- and trans-[Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCa2+] of 10-100 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels. 相似文献