烟草根皮层原生质体质膜钾通道的特性研究

采用膜片钳技术对烟草根皮层原生质体质膜上的钾通道进行全细胞记录,从而深入研究烟草K^＋的吸收机制和调控机理。结果表明,内向钾通道在膜电压低于-40mV时,可以被K^＋激活。内向电流可以被钾通道的专一抑制剂TEA^＋抑制。动力学分析表明内向钾电流产生的K^＋表观解离常数（Km）≈15．2mmol／L,类似于低亲和性钾通道。该通道具有依赖于胞外K^＋浓度的特性,对胞外NH4^＋、Ca^2＋、Mg^2＋浓度变化反应敏感,内向K^＋电流可被不同程度地抑制。     

Identification and characterization of inward K+ -channels in plasma membranes ofArabidopsis root cortex cells

Patch clamping whole-cell recording techniques were applied to study the inward K⁺ -channels inArabidopsis root cortex cells. The inward K⁺ -channels in the plasma membranes of the root cortex cell protoplasts were activated by hyperpolarized membrane potentials. The channels were highly selective for K⁺ ions over Na⁺ ions. The channel activity was significantly inhibited by the external TEA⁺ or Ba²⁺. The changes in cytoplasmic Ca²⁺ concentrations did not affect the whole-cell inward K⁺ -currents. The possible association between the channel selectivity to K⁺ and Na⁺ ions and plant salt-tolerance was also discussed.     

Measurement of Na-K pump current in acinar cells of rat lacrimal glands.

Isolated cells from rat lacrimal glands were voltage clamped using the tight-seal whole-cell recording technique. The intracellular solution contained ATP and an elevated Na concentration (70 mM). Removing external K ions elicited an inward current shift. Ouabain (0.5 mM) induced an inward current shift of identical amplitude, but with slower kinetics. In the presence of ouabain, removal of K ions did not alter the cell current. The potassium- and ouabain-sensitive current was outward between -120 and +20 mV, and its amplitude decreased below -60 mV. This current was highly sensitive to temperature, and was not affected by blockers of the K channels which are present in these cells. It was attributed to an inhibition of the Na-K pump. The Na-K pump current was estimated to be 15 pA for an average acinar cell at physiological temperature, with 70 mM internal Na ions and 20 mM external K ions. Implications of this value in terms of electrolyte secretion are discussed.     

Evidences for Regulation of the Inward K+ channels by CDPK in Vicia faba Guard Cells

Regulation of the inward K+ -channels in the guard cell plasma membranes plays impotant roles in regulation of stomatal movement in responses to exogenous and endogenous signals. It is well-known that elevation of cytosolic Ca2+ in guard cells inactivates these inward K + channels, and consequently inhibits stomatal opening or induces stomatal closing, yet the downstream molecular mechanism for the Ca2 + -mediated inhibition of the inward K+ channels remains unknown. The calmodulin-like domain protein kinases (CDPKs) have been identified as an unique group of protein kinases in higher plant cells. As a downstream regulator, CDPK may play roles in mediating Ca2+ regulation on the inward K+ -channels in stomatal guard cells. The authors have applied the patchclamp technique to investigate if CDPK be involved in the regulation of the inward K+ -channels in Vicia faba guard cells by cytosolic Ca2+ . The presence of the 1.5 μmol/L intracellular Ca2 + result-ed in inhibition of the inward K+ channel activity by 60%, while the addition of purified CDPK from the cytoplasmic side resulted in greater inhibition than Ca2+ alone. Histone Ⅲ-S and protamine, which is the substrate and substrate competitive inhibitor of CDPKs respectively, completely reversed the Ca2+ -induced inhibition of the inward K+ channel activities. These results are the first reported evidences for that CDPKs are involved in the Ca2+ -mediated inward K+ -channel regulation in guard cells.     

Identification and characterization of the inward K+ channel in the plasma membrane of Brassica pollen protoplasts.

Patch clamp techniques have been used to identify and characterize the whole-cell currents carried by inward K+ channels in isolated matured pollen protoplasts of Brassica chinensis var. chinensis. The whole-cell inward currents in the isolated pollen protoplasts were activated at hyperpolarized membrane potentials more negative than -100 mV. The magnitudes of the whole-cell inward currents were strongly dependent on the external K+ concentration, and were highly selective for K+ over other monovalent cations. The inward currents were not observed when external K+ was replaced with the same concentration of Cs+ or Na+. The addition of 1 mM or 10 mM Ba2+ in external solutions resulted in 30% or 80% inhibition of the inward currents at -180 mV, respectively. These results demonstrated that the inward K+ currents mainly account for the recorded whole-cell inward currents in Brassica pollen protoplasts. Increase of cytoplasmic Ca2+ concentrations from 10 nM to 30 microM or even 5 mM did not affect the inward K+ currents. Decrease of external Ca2+ concentrations from 10 mM to 1 mM inhibited the inward K+ currents by 25%, while the increase of external Ca2+ from 10 mM to 50 mM almost completely blocked the inward K+ currents. Physiological importance of K+ transport into pollen and its possible regulatory mechanisms are also discussed.     

In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts

The focus of this study is to investigate the regulatory role of K(+) influx in Arabidopsis pollen germination and pollen tube growth. Using agar-containing media, in vitro methods for Arabidopsis pollen germination have been successfully established for the first time. The pollen germination percentage was nearly 75% and the average pollen tube length reached 135 microm after a 6 h incubation. A decrease in external K(+) concentration from 1 mM to 35 microM resulted in 30% inhibition of pollen germination and 40% inhibition of pollen tube growth. An increase in external K(+) concentration from 1 mM to 30 mM stimulated pollen tube growth but inhibited pollen germination. To study how K(+) influx is associated with pollen germination and tube growth, regulation of the inward K(+) channels in the pollen plasma membrane was investigated by conducting patch-clamp whole-cell recording with pollen protoplasts. K(+) currents were first identified in Arabidopsis pollen protoplasts. The inward K(+) currents were insensitive to changes in cytoplasmic Ca(2+) but were inhibited by a high concentration of external Ca(2+). A decrease of external Ca(2+) concentration from 10 mM (control) to 1 mM had no significant effect on the inward K(+) currents, while an increase of external Ca(2+) concentration from 10 mM to 50 mM inhibited the inward K(+) currents by 46%. Changes in external pH significantly affected the magnitude, conductance, voltage-independent maximal conductance, and activation kinetics of the inward K(+) currents. The physiological importance of potassium influx mediated by the inward K(+)-channels during Arabidopsis pollen germination and tube growth is discussed.     

Octopaminergic modulation of the membrane currents in the central feeding system of the pond snail Lymnaea stagnalis

Octopamine is released by the intrinsic OC interneurons in the paired buccal ganglia and serves both as a neurotransmitter and a neuromodulator in the central feeding network of the pond snail Lymnaea stagnalis. The identified B1 buccal motoneuron receives excitatory inputs from the OC interneurons and is more excitable in the presence of 10 microM octopamine in the bath. This modulatory effect of octopamine on the B1 motoneuron was studied using the two electrode voltage clamp method. In normal physiological saline depolarising voltage steps from the holding potential of -80 mV evoke a transient inward current, presumably carried by Na(+) ions. The peak values of this inward current are increased in the presence of 10 microM octopamine in the bath. In contrast, both the transient (IA) and delayed (IK) outward currents are unaffected by octopamine application. Replacing the normal saline with a Na(+)-free bathing solution containing K(+) channel blockers (50 mM TEACl, 4 mM 4AP) revealed the presence of an additional inward current of the B1 neurons, carried by Ca(2+). Octopamine (10 microM) in the bath decreased the amplitudes of this current. These results suggest that the membrane mechanisms which underlie the modulatory effect of octopamine on the B1 motoneuron include selective changes of the Na(+)- and Ca(2+)-channels.     

Omega-conotoxin blockade of calcium currents in cultured neonatal rat cardiomyocytes: different action on EGTA-modified calcium channels

Calcium currents from neonatal rat ventricular heart muscle cells grown in primary culture were examined using the "whole-cell" voltage clamp technique. An inward current characterized by large amplitude and slow inactivation decay was induced when the extracellular Ca2+ concentration was reduced by EGTA. This current was suppressed by extracellular Na+ removal, or by calcium antagonists, and increased by epinephrine and BAY K 8644. These findings suggest that this current is carried by sodium ions through Ca channels. Both Ca and Na currents through calcium channels were irreversibly blocked by omega-conotoxin. Complete blockade developed 10-15 minutes after the toxin introduction in the extracellular solution. Blockade of Na currents through calcium channels was characterized by a transient increase of current amplitude without any changes in its kinetics and voltage-dependent properties. Structural differences between calcium channels in rat and guinea-pig and frog cardiomyocytes were suggested.     

Ion distribution in roots of barley seedlings measured by electron probe x-ray microanalysis

The distribution of ions, particularly K and Na, was studied in roots of barley seedlings grown on various ionic solutions. Analyses were made by means of electron probe x-ray microanalysis using frozen, fractured bulk specimens. By this technique, it was demonstrated that there can be variability in the ratio K/Na measured in the vacuoles of cortical cells, with this ratio often being lower in epidermal cells of the root than in the inner cortex. A sharp difference in the K/Na ratio was also found between cells of the endodermis and those of the adjacent cortex, and generally higher ratios of K/Na occurred in the stele than in the cortex. Estimation of the concentrations in the cytoplasm was at the limit of resolution of this technique, but it can be shown that the K/Na ratio in the cytoplasm was higher than that in the vacuole. In low salt roots, the K concentration in the cytoplasm was higher than that in the vacuoles. The results with the x-ray microprobe confirm other measurements based on flux analysis or analysis of small samples of the root.     

Patch-clamp studies in human macrophages: Single-channel and whole-cell characterization of two K+ conductances

Summary Human peripheral blood monocytes cultured for varying periods of time were studied using whole-cell and single-channel patch-clamp recording techniques. Whole-cell recordings revealed both an outward K current activating at potentials >20 mV and an inwardly rectifying K current present at potentials negative to –60 mV. Tail currents elicited by voltage steps that activated outward current reversed nearE _K, indicating that the outward current was due to a K conductance. TheI–V curve for the macroscopic outward current was similar to the mean single-channelI–V curve for the large conductance (240 pS in symmetrical K) calcium-activated K channel present in these cells. TEA and charybdotoxin blocked the whole-cell outward current and the single-channel current. Excised and cell-attached single-channel data showed that calcium-activated K channels were absent in freshly isolated monocytes but were present in >85% of patches from macrophages cultured for >7 days. Only 35% of the human macrophages cultured for >7 days exhibited whole-cell inward currents. The inward current was blocked by external barium and increased when [K] _o increased. Inward-rectifying single-channel currents with a conductance of 28 pS were present in cells exhibiting inward whole-cell currents. These single-channel currents are similar to those described in detail in J774.1 cells (L.C. McKinney & E.K. Gallin,J. Membrane Biol. 103:41–53, 1988).     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantification of Rat Cerebral Cortex Na+,K+-ATPase: Effect of Age and Potassium Depletion

Na+,K(+)-ATPase concentration in rat cerebral cortex was studied by vanadate-facilitated [3H]ouabain binding to intact samples and by K(+)-dependent 3-O-methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [3H]Ouabain binding to cerebral cortex obtained from 12-week-old rats measured incubating samples in buffer containing [3H]ouabain, and ouabain at a final concentration of 1 x 10(-6) mol/L gave a value of 11,351 +/- 177 (n = 5) pmol/g wet weight (mean +/- SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. K(+)-dependent 3-O-methylfluorescein phosphatase activity in crude cerebral homogenates of age-matched rats was 7.24 +/- 0.14 (n = 5) mumol/min/g wet weight, corresponding to a Na+,K(+)-ATPase concentration of 12,209 +/- 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na+,K(+)-ATPase. The concentration of rat cerebral cortex Na+,K(+)-ATPase showed approximately 10-fold increase within the first 4 weeks of life to reach a plateau of approximately 11,000-12,000 pmol/g wet weight, indicating a larger synthesis of Na+,K+ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K+ depletion induced by K(+)-deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K+ content (6%, p > 0.5) and Na+,K(+)-ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K+ content and Na+,K(+)-ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K+ depletion, cerebral cortex can maintain almost normal K+ homeostasis, whereas K+ as well as Na+,K+ pumps are lost from skeletal muscles.     

Polyamines improve K+/Na+ homeostasis in barley seedlings by regulating root ion channel activities

Polyamines are known to increase in plant cells in response to a variety of stress conditions. However, the physiological roles of elevated polyamines are not understood well. Here we investigated the effects of polyamines on ion channel activities by applying patch-clamp techniques to protoplasts derived from barley (Hordeum vulgare) seedling root cells. Extracellular application of polyamines significantly blocked the inward Na(+) and K(+) currents (especially Na(+) currents) in root epidermal and cortical cells. These blocking effects of polyamines were increased with increasing polycation charge. In root xylem parenchyma, the inward K(+) currents were blocked by extracellular spermidine, while the outward K(+) currents were enhanced. At the whole-plant level, the root K(+) content, as well as the root and shoot Na(+) levels, was decreased significantly by exogenous spermidine. Together, by restricting Na(+) influx into roots and by preventing K(+) loss from shoots, polyamines were shown to improve K(+)/Na(+) homeostasis in barley seedlings. It is reasonable to propose that, therefore, elevated polyamines under salt stress should be a self-protecting response for plants to combat detrimental consequences resulted from imbalance of Na(+) and K(+).     

Permeation of Na+ through a delayed rectifier K+ channel in chick dorsal root ganglion neurons

In whole-cell patch clamp recordings from chick dorsal root ganglion neurons, removal of intracellular K+ resulted in the appearance of a large, voltage-dependent inward tail current (Icat). Icat was not Ca2+ dependent and was not blocked by Cd2+, but was blocked by Ba2+. The reversal potential for Icat shifted with the Nernst potential for [Na+]. The channel responsible for Icat had a cation permeability sequence of Na+ >> Li+ >> TMA+ > NMG+ (PX/PNa = 1:0.33:0.1:0) and was impermeable to Cl-. Addition of high intracellular concentrations of K+, Cs+, or Rb+ prevented the occurrence of Icat. Inhibition of Icat by intracellular K+ was voltage dependent, with an IC50 that ranged from 3.0-8.9 mM at membrane potentials between -50 and -110 mV. This voltage- dependent shift in IC50 (e-fold per 52 mV) is consistent with a single cation binding site approximately 50% of the distance into the membrane field. Icat displayed anomolous mole fraction behavior with respect to Na+ and K+; Icat was inhibited by 5 mM extracellular K+ in the presence of 160 mM Na+ and potentiated by equimolar substitution of 80 mM K+ for Na+. The percent inhibition produced by both extracellular and intracellular K+ at 5 mM was identical. Reversal potential measurements revealed that K+ was 65-105 times more permeant than Na+ through the Icat channel. Icat exhibited the same voltage and time dependence of inactivation, the same voltage dependence of activation, and the same macroscopic conductance as the delayed rectifier K+ current in these neurons. We conclude that Icat is a Na+ current that passes through a delayed rectifier K+ channel when intracellular K+ is reduced to below 30 mM. At intracellular K+ concentrations between 1 and 30 mM, PK/PNa remained constant while the conductance at -50 mV varied from 80 to 0% of maximum. These data suggest that the high selectivity of these channels for K+ over Na+ is due to the inability of Na+ to compete with K+ for an intracellular binding site, rather than a barrier that excludes Na+ from entry into the channel or a barrier such as a selectivity filter that prevents Na+ ions from passing through the channel.     

Calcium-dependent sodium current in the marine ciliateEuplotes vannus

Summary Ca and Na inward currents were recorded upon depolarizations inEuplotes after the blockage of K outward currents with intracellular Cs ions. The Na current was analyzed under voltage clamp and had the following properties: it activated to a maximum within 150 msec and partly inactivated during sustained voltage steps. It had a positive equilibrium potential between 25 and 30 mV and could be carried by Na or Li ions but not by K, choline or Tris ions. The current revealed a prominent associated inward tail current which deactivated with a single-exponential time constant of 118 msec. Both the current and its tail were strongly reduced after reduction of the extracellular Na concentration. Externally applied K channel blocker tetraethylammonium chloride did not block the current. Either EGTA injection into the cell or nonlethal deciliation with ethanol eliminated the current and its tail. These results indicate the existence of a Na conductance within the membrane ofEuplotes which is activated by the intracellular level of free Ca²⁺.     

Modulation of voltage-gated sodium channels by recombinant interferon-alpha2b in the human neuroblastoma cell line IMR-32

Clonal human neuroblastoma cells imr-32 are a suitable model system for studies of neuronal excitability modulation. The ability interferon-alpha 2b "laferon" to modulate the mechanisms of electrical activity was studied in whole-cell patch-clamped undifferentiated human neuroblastoma cells IMR-32. It was shown that 1 h incubation of IMR-32 cells at 37 degrees C in medium with laferon (600 U/ml) exerted changes in voltage-dependent properties of Na(+)-channels. The results of the present study demonstrate that laferon decreased of Na(+)-channels sensitivity to changes of membrane potential leading of IMR-32 cells electrical excitability decrease.     

An amiloride-sensitive and voltage-dependent Na+ channel in an HLA-DR-restricted human T cell clone

We investigated changes in voltage-gated Na+ currents and effects of extracellular Na+ on proliferation in HLA-DR-restricted human CD4+ alphabeta T cells after stimulation with a non-self antigenic peptide, M12p54-68. In the absence of antigenic peptide, neither single (n = 80) nor APC-contacted (n = 71) T cells showed voltage-gated inward currents recording with whole-cell patch-clamp techniques, even with Ca2+ and Na+ ions present in the perfusion solution. However, with the same recording conditions, 31% (26 of 84) of APC-contacted T cells stimulated with the antigenic peptide showed voltage-dependent inward currents that were elicited from -60 mV. The inward currents were not inhibited in extracellular Ca2+-free conditions or in the presence of 1 mM NiCl2. However, they were completely inhibited in extracellular Na+-free conditions, which were made by replacing Na+ with iso-osmotic N-methyl-d -glucamine or choline. The Na+ currents were insensitive to tetrodotoxin, a classical blocker of Na+ channels, but were dose-dependently inhibited by amiloride, a potassium-sparing pyrazine diuretic. Furthermore, the Ag-specific proliferative response of T cells was completely inhibited in Na+-free Tyrode's solution and was suppressed by amiloride in a dose-dependent manner. Our findings suggest that activation of amiloride-sensitive and voltage-gated Na+ channels would be an important step to allow an adequate influx of Na+ and maintain a sustained high Ca2+ level during T cell activation.     

Demonstration of Voltage-Dependent and TTX-Sensitive Na+-Channels in Human Melanocytes

The electrophysiological properties of cultured human melanocytes were investigated using the whole-cell configuration of the patch-clamp technique. Depolarizations to membrane potentials more positive than -30 mV resulted in the rapid development (<1 ms to peak) of an inward current. The maximum peak current was observed at +10 mV and reached an average amplitude of about 270 pA. During the depolarizations, the current inactivated with a time constant of about 2 ms. The current was abolished by the addition of 0.3 μM tetrodotoxin, a blocker of voltage-gated Na⁺-channels, and disappeared when Na⁺ was omitted from the extracellular medium. In addition, the melanocytes contain at least two types of outward K⁺-current. The first type, observed in every cell, was highly sensitive (K_i 1 mM) to the K⁺-channel blocker TEA, required depolarizations beyond zero to be activated and did not inactivate. The second type was less regularly observed (10% of the cells). This current activated at more negative voltages (–20 mV), was resistant to TEA (20 mM) but was blocked by 2 mM 4-aminopyridine and inactivated rapidly during depolarizations. We conclude that human melanocytes are equipped with voltage-dependent Na⁺-channels, a delayed rectifying K⁺-current and a K⁺-current similar to the A-current in neurones.     

Novel regulation of Na, K-ATPase by Src tyrosine kinases in cortical neurons

The Na+, K+-ATPase or Na+, K+-pump plays a critical role in ion homeostasis and many cellular events. The Na+, K+-pump activity is regulated by serine/threonine phosphorylation, the role of tyrosine kinases in the regulation, however, is obscure. We now present novel evidence showing that tyrosine phosphorylation activates the Na+, K+-pump in cortical neurons. The electrogenic activity of the Na+, K+-pump was measured using whole-cell voltage clamp. A tonic activity was revealed by an inward current induced by the specific inhibitor ouabain or strophanthidin; an outward current due to activation of the pump was triggered by raising extracellular K+. The inward and outward currents were attenuated by the tyrosine kinase inhibitor genistein, herbimycin A, or lavendustin A, while blocking tyrosine phosphatases increased the pump current. Down-regulation of the pump current was also seen with the Src inhibitor PP1 and intracellularly applied anti-Lyn or anti-Yes antibody. Consistently, intracellular application of Lyn kinase up-regulated the pump current. Immunoprecipitation and western blotting showed tyrosine phosphorylation and a direct interaction between Lyn and the alpha3 subunit of the Na+, K+-pump. The tyrosine phosphorylation of the alpha3 subunit was reduced by serum deprivation. These data suggest that the Na+, K+-ATPase activity in central neurons is regulated by specific Src tyrosine kinases via a protein-protein mechanism and may play a role in apoptosis.     

Inwardly rectifying whole-cell and single-channel K currents in the murine macrophage cell line J774.1

Summary Inward currents in the murine macrophage-like cell line J774.1 were studied using the whole-cell and cell-attached variations of the patch-clamp technique. When cells were bathed in Na Hanks' (KCl=4.5mm, NaCl=145mm), and the electrode contained Na-free K Hanks' (KCl=145mm) single-channel currents were observed at potentials below –40 mV which showed inward rectification, were K-selective, and were blocked by 2.5mm Ba in the pipette. Single-channel conductance was 29 pS, and was proportional to the square root of [K] _o . Channels manifested complex kinetics, with multiple open and closed states. The steady-state open probability of the channel was voltage dependent, and declined from 0.9 to 0.45 between –40 and –140 mV. When hyperpolarizing voltage pulses were repetitively applied in the cell-attached patch mode, averaged single-channel currents showed inactivation. Inactivation of inwardly rectifying whole-cell current was measured in Na Hanks' and in two types of Na-free Hanks': one with a normal K concentration (4.5mm) and the other containing 145mm K. Inactivation was shown to have Na-dependent and Na-independent components. Properties of single-channel current were found to be sufficient to account for the behavior of the macroscopic current, except that single-channel current showed a greater degree of Na-independent inactivation than whole-cell current.