Cell-attached Patch Mode Research Articles

Intracellular acidification evoked by moderate extracellular acidosis attenuates transient receptor potential V1 (TRPV1) channel activity in rat dorsal root ganglion neurons

Transient receptor potential V1 (TRPV1) has been suggested to play an important role in detecting decreases in extracellular pH (pH(o)). Results from recent in vivo studies, however, have suggested that TRPV1 channels play less of a role in sensing a moderately acidic pH(o) (6.0 < pH < 7.0) than predicted from the in vitro experiments. A clear explanation for this discrepancy between the in vitro and in vivo data has not yet been provided. We report here that intracellular acidification induced by a moderately low pH(o) (6.4) almost completely inhibited the effect of extracellular acidosis on TRPV1 activity. In our experiments, sodium acetate (20 mm), which was used to induce intracellular acidosis, attenuated the capsaicin-evoked TRPV1 current (I(CAP)) in a reversible manner in whole-cell patch-clamp mode and shifted the concentration-response curve to the right. Likewise, the concentration-response curve was significantly shifted to the right by lowering the pH of the pipette solution from 7.2 to 6.5. In addition, application of an acidic bath solution (pH 6.4) to the intracellular side also significantly suppressed I(CAP) in inside-out patch mode. In cell-attached patch mode, the single-channel activity of i(CAP) was significantly attenuated by intracellular acidosis that was induced by a decrease in pH(o) (6.4). These results suggested that intracellular acidification induced by a low pH(o) inhibited TRPV1 activity. When studied in perforated patch mode or by acidifying the intracellular pipette solution, potentiation or activation of TRPV1 by extracellular acidosis (pH 6.4) at 37 °C was almost completely inhibited. Likewise, enhancement of neuronal excitability by a moderately acidic pH(o) (6.4) at a physiological temperature (37 °C) was attenuated by lowering the pH of the pipette solution to 6.5 or using perforated patch mode. Taken together, these results suggest that extracellular acidosis of moderate intensity may not significantly modulate TRPV1 activity in physiological conditions at which intracellular pH can be readily affected by pH(o), and this phenomenon is due to attenuation of TRPV1 channel activity by low-pH(o)-induced intracellular acidification.

Inhibition by α‐Tetrahydrodeoxycorticosterone (THDOC) of Pre‐Sympathetic Parvocellular Neurones in the Paraventricular Nucleus of Rat Hypothalamus

alpha-tetrahydrodeoxycorticosterone (THDOC) is an endogenous neuroactive steroid which increases in plasma and brain concentration during stress. It has both positive and negative modulatory effects on GABA activated GABAA currents, dependent upon the dose. We investigated the effects of THDOC on spinally-projecting "pre-sympathetic" neurones in the parvocellular subnucleus of the hypothalamic paraventricular nucleus (PVN), to determine whether it activates or inhibits these neurones, and by what mechanism. Rat spinally-projecting (parvocellular) PVN neurones were identified by retrograde labelling and the action of THDOC investigated with three modes of patch-clamp: cell-attached action current, whole-cell voltage-clamp and cell-attached single-channel recording. In cell-attached patch mode, parvocellular neurones fired action potentials spontaneously with an average frequency of 3.6 +/- 1.1 Hz. Bath application of THDOC reduced this with an EC50 of 67 nM (95% confidence limits: 54 to 84 nM), Hill coefficient 0.8 +/- 0.04, n = 5. In whole-cell patch-clamp mode, pressure ejection of GABA evoked inward currents. These were clearly GABAA currents, since they were inhibited by the GABAA receptor antagonist bicuculline, and reversed near the chloride equilibrium potential. THDOC significantly potentiated GABAA currents (1 microM THDOC: 148 +/- 15% of control, n = 5, p < or = 0.05, ANOVA). Single-channel analysis showed no differences in conductance or corrected mean open times in the presence of 1 microM THDOC. THDOC inhibited parvocellular neuronal activity without showing any evidence of the bidirectional activity demonstrated previously with cultured hypothalamic neurones. Our data are consistent with the hypothesis that THDOC acts by potentiating the post-synaptic activity of endogenously released GABA.

Expression of alpha 1D subunit mRNA is correlated with L-type Ca2+ channel activity in single neurons of hippocampal "zipper" slices.

L-type voltage-sensitive Ca(2+) channels (L-VSCCs) play an important role in developmental and aging processes, as well as during normal function of brain neurons. Here, we tested a prediction of the hypothesis that membrane density of functional L-VSCCs is regulated by the level of gene expression for its alpha(1D) pore-forming subunit. If so, alpha(1D) mRNA and L-VSCC activity should be positively correlated within individual neurons. Conventional methods of aspiration and/or acute cell dissociation used in prior single-cell studies have generally yielded variable and incomplete recovery of intracellular mRNA. Thus, quantitative relationships between channel function and expression have been difficult to define. In this study, we used the partially dissociated ("zipper") hippocampal slice preparation as a method for collecting a single neuron's mRNA complement. This preparation, developed to expose neuronal somata for recording, also enables the extraction of a neuron with major processes largely intact. Thus, single-cell measures of gene/mRNA expression can be based on approximately the cell's full set of mRNA transcripts. In adult and aged rat hippocampal zipper slices, L-VSCC activity was first recorded in CA1 neurons in cell-attached patch mode. The same neurons were then extracted and collected for semiquantitative reverse transcriptase-PCR analysis of alpha(1D) and calmodulin A (CaM) mRNA content. Across multiple single neurons, a significant, positive correlation was found between the rank orders of L-VSCC activity and of alpha(1D), but not CaM, mRNA expression. Thus, these studies support the possibility that the level of alpha(1D) gene expression regulates the density of functional L-VSCCs.

Membrane stretch increases the activity of Ca(2+)-activated K+ channels in rabbit coronary vascular smooth muscles.

It has been proposed that Ca(2+)-activated K+ channels play an essential role in maintaining vascular tone during stretch of blood vessel. However, the underlying mechanism of stretch-induced change of Ca(2+)-activated K+ channel activities are still unknown. The present experiment was designed to investigate the effect of membrane stretch on these channels whose activity was measured from rabbit coronary smooth muscle cells using a patch clamp technique. Ca(2+)-activated K+ channel were identified by their Ca2+ and voltage dependencies and its large conductances as in other preparations. Perfusion of cells with a hypotonic solution, which mimics stretching the cell membrane by making a cell swelling, produced an increase in channel activity in cell-attached patch mode. The similar increase was observed when negative pressure was applied into the patch pipette for stretching the cell membrane within a patch area. In inside-out patch, stretch still increased channel activity even under the conditions which exclude the possible involvement of secondary messengers, or of transmembrane Ca2+ influx via stretch-activated cation channels. Pretreatment of arachidonic acid or albumin showed no effect on stretch-induced channel activation, excluding the possibility of fatty acids mediated channel activation during membrane stretch. These results indicate that the stretch may directly increase the activity of Ca(2+)-activated K+ channels in our experimental condition.

Characterization of ATP-sensitive potassium channels in human corporal smooth muscle cells.

Potassium (K) channels play a significant role in modulating human corporal smooth muscle tone, and thus, erectile capacity. Recent pharmacological studies indicate that the metabolically-regulated K channel (KATP) may be an important modulator of human penile erection with significant therapeutic potential. The goal of these initial studies, therefore, was to utilize patch clamp techniques to characterize the putative KATP subtype(s) present in cultured and freshly isolated human corporal smooth muscle cells. In the cell-attached patch mode, two distinct unitary K+ currents were identified whose respective conductance values were similar in cultured and freshly isolated smooth muscle cells. In cultured myocytes, the measured conductance values in symmetric KCl (140 mM) solutions were 59.1 +/- 2.7 pS and 18.4 +/- 2.1 pS (n = 5 cells). Under identical experimental conditions in freshly isolated myocytes, corresponding conductance values were 59.2 +/- 3.7 pS and 18.5 +/- 2.4 pS, respectively (n = 4 cells). I-V curves constructed during step depolarization (-60 to +80 mV), revealed a linear I-V relationship for both unitary conductances. Single channel records documented that both conductances were reversibly inhibited by the application of ATP (1-3 mM) to the bath solution in the inside-out attached patch configuration. The unitary activity of both K channel subtypes was significantly increased by the application of pinacidil (10 microM) and levcromakalim (10 microM). Whole cell patch recordings documented a glibenclamide-sensitive, pinacidil- and levcromakalim-induced increase in the whole cell outward K+ current during step depolarization (-70 mV to +130 mV) of 105 +/- 37%, 139 +/- 42%, respectively. These data confirm and extend our previous observations, and provide the first evidence for the presence of KATP channel subtypes in human corporal smooth muscle cells.

Mechanisms of modulation of voltage-dependent calcium channels by G proteins.

Voltage-dependent calcium channels (VDCCs) are key components in the complex functioning of excitable cells. Because of this, their regulation is of paramount importance to the control of cellular activity. VDCCs consist of a pore-forming α1 subunit, which has four domains each containing six putative transmembrane segments. From purification studies, these are associated with an intracellular β subunit. They also co-purify with an α2 subunit, which is entirely extracellular, linked into the membrane by S-S bonding to a transmembrane δ subunit (Witcher et al. 1993; Liu, De Waard, Scott, Gurnett, Lennon & Campbell, 1996) (Fig. 1). A number of different α1 subunits have been cloned; α1C, D and S all form 1,4-dihydropyridine (DHP)-sensitive L-type calcium channels, whereas α1A, B and E form P/Q-, N- and possibly R- or T-type channels, respectively. There are several means by which these channels may be modulated, but for neuronal channels, particularly N and P/Q, a major mechanism involves inhibitory modulation via the activation of heterotrimeric G proteins by seven transmembrane (7TM) receptors (for review see Dolphin, 1995). The key features of this inhibition are that it is always partial and is typified by a slowing of the current activation kinetics, which is thought to be due to a time-dependent recovery from voltage-dependent inhibition (Bean, 1989). The voltage dependence is manifested by a shift to more depolarized potentials of the current activation-voltage relationship, and the loss of inhibition at large depolarizations (Bean, 1989). There may also be additional mechanisms that are not voltage dependent, manifested by an incomplete ability of a depolarizing prepulse to reverse the inhibition (Diverse-Pierluissi & Dunlap, 1993), and the continuing presence of inhibition at large depolarizations measured from the tail current amplitude. Figure 1 The VDCC oligomeric complex Calcium channels are present in many tissues, where they fulfil a number of different specialized roles. In neurons the distribution of the channels is non-uniform, with α1B and α1A being particularly concentrated at synaptic terminals (Westenbroek, Hell, Warner, Dubel, Snutch & Catterall, 1992; Westenbroek et al. 1995). G protein-mediated modulation of these channels has been shown to occur at presynaptic terminals (Toth, Bindokas, Bleakman, Colmers & Miller, 1993). Evidence suggests that this mechanism may be responsible for at least some of the presynaptic inhibition of synaptic transmission mediated by a wide variety of 7TM receptors in many areas of the nervous system (Man-Son-Hing, Zoran, Lukowiak & Haydon, 1989; Hille, 1992; Toth et al. 1993; Dolphin, 1995). Activation of such receptors will reduce calcium entry into presynaptic terminals via VDCCs, but the effect should be frequency dependent. Inhibition will be reduced during a high frequency train as a result of the voltage dependence of the inhibitory modulation, providing a gain-setting mechanism. Relief of inhibition of calcium currents, evoked by action potential-like voltage waveforms, has been reported during high frequency trains (Williams, Serafin, Muhlethaler & Bernheim, 1997), and might contribute to the modulation of presynaptic inhibition depending on input frequency. The search for the molecular mechanism of this modulation is hotting up. Nevertheless, it should not be forgotten that indirect mechanisms such as presynaptic hyperpolarization, or direct mechanisms involving inhibition of exocytosis, are also likely to play a role in the modulation of presynaptic release of transmitter (Man-Son-Hing et al. 1989).

A large-conductance voltage-dependent potassium channel in cultured pig articular chondrocytes

The patch-clamp techniques were used to study voltage-dependent potassium channels in cultured pig articular chondrocytes. A predominant single-channel conductance of 125 pS was found. These channels were reversibly blocked by tetraethylammonium. In cell-attached patches, transient increases in the channel activity were observed, and defined as a switching between low and high activity modes (LAM and HAM). Open-time distributions could be described with two kinetics components (in LAM and HAM) having similar time constants (fast tau1 and slow tau2). In HAM, the area of the slow component was larger. The mean burst length was significantly longer in HAM than in LAM. In both modes, the burst-length distributions were fitted with a sum of three exponentials. In LAM and HAM, the time constants tau1 and tau2 were indistinguishable from those of the open-time distributions. The slowest time constant, tau3, was strongly voltage dependent, and was significantly longer in HAM than in LAM. In both LAM and HAM, the ensemble currents were characterised by a rapid rising phase followed by fast and profound inactivation. The activation kinetics were similar, but the inactivation was faster in HAM. In the outside-out configuration no evidence for mode switching was found. The kinetics of the rising phase of the ensemble currents were also similar to those observed using the cell-attached configuration, but the channels did not inactivate. In the whole-cell configuration, the mode switching was not present. The inactivation time constant showed a large scattering, and was much slower than that measured in the cell-attached patch mode. These currents were blocked by tetraethylammonium and 4-aminopyridine. Our results indicate that intracellular factors are involved in controlling the mode switching and the kinetics of the inactivation of potassium channels in pig articular chondrocytes.

Caffeine- and histamine-induced oscillations of K(Ca) current in single smooth muscle cells of rabbit cerebral artery.

In the present experiment, we characterized the intracellular Ca2+ oscillations induced by caffeine (1mM) or histamine (1-3microM) in voltage-clamped single smooth muscle cells of rabbit cerebral (basilar) artery. Superfusion of caffeine or histamine induced periodic oscillations of large whole-cell K+ current with fairly uniform amplitudes and intervals. The oscillatory K+ current was abolished by inclusion of ethylenebis(oxonitrilo)tetraacetate (EGTA, 5mM) in the pipette solution. Caffeine- and histamine-induced periodic activation of the large-conductance Ca2+-activated K+ [K(Ca)] channel was recorded in the cell-attached patch mode. These results suggest that the oscillations of K+ current are carried by the K(Ca) channel and reflect the oscillations of intracellular Ca2+ concentration ([Ca2+]i). Ryanodine (1-10microM) abolished both caffeine- and histamine-induced oscillations. Caffeine-induced oscillations were abolished by the sarcoplasmic reticulum Ca2+-adenosine 5'-triphosphatase (Ca2+-ATPase) inhibitor, cyclopiazonic acid (10microM), and a high concentration of caffeine (10mM). Inclusion of heparin (3mg/ml) in the pipette solution blocked histamine-induced oscillations, but did not block caffeine-induced oscillations. By the removal of extracellular Ca2+, but not by the addition of verapamil and Cd2+, the caffeine-induced oscillations were abolished. Increasing Ca2+ influx rate increased the frequencies of caffeine-induced oscillations. Spontaneous oscillations were also observed in cells that were not superfused with agonists, and had similar characteristics to the caffeine-induced oscillations. From the above results, it is concluded, that in smooth muscle cells of the rabbit cerebral (basilar) artery, ryanodine-sensitive Ca2+-induced Ca2+ release pools play key roles in the generation of caffeine- and histamine-induced intracellular Ca2+ oscillations.

Properties and regulation of pH-dependent cation channels in the apical membrane of cultured proximal tubule cells.

The established opossum kidney (OK) cell line serves as a model system for ion and substrate transport in the renal proximal tubule. Previous experiments on OK cells revealed a channel-mediated Na+ conductance which is regulated by intracellular pH (pHi). In this study we report on patch clamp experiments determining the properties and pHi dependence of a cation channel located in the apical membrane. This channel is selective for sodium over chloride but discriminates poorly between the monovalent cations Na+,K+,Li+ and Cs+. Its open probability (P(o)) rises at depolarising membrane potentials. Under normal conditions the channel is inactive in the cell-attached patch mode and is activated upon excision. However, after excision the channel usually runs down within 30-90 s which cannot be overcome by either altering the Ca(2+)-concentration (10(-3) mol/l, 10(-6) mol/l, Ca(2+)-free) or adding 1 mmol/l Mg-ATP to the bath solution. In the cell-attached patch mode the channel could be activated by decreasing pHi from pH 7.4 to pH 6.5, by either the ammonium prepulse technique or the nigericin K+ method, in more than 50% of the experiments performed. In the renal proximal tubule such a non-selective cation channel would constitute a functional Na+ channel and might therefore support Na+ reabsorption especially during the intracellular acidification due to hormonal inhibition of the Na+/H+ exchanger.

Patch clamp analysis of the dominant plasma membrane K+ channel in root cell protoplasts of Plantago media L. Its significance for the P and K state

Ion channels in the plasma membrane of root cell protoplasts of Plantago media L. were studied with the patch clamp technique in the cell-attached patch and outside-out patch configuration. An outward rectifying potassium channel was dominantly present in the plasma membrane. It appears responsible for the diffusional part, dominated by the K+ diffusion potential, of the cell membrane potential, in vivo. This channel is activated at potentials near to and more positive than the K+ diffusion potential. The dependence of this ion channel on K+ activity and voltage has been characterized. The current-voltage relationships of the open channel at various K+ concentrations are described by a four-state model. The membrane potential of intact protoplasts appears either dominated by the K+ diffusion potential, the protoplast is then said to be in the K state, or by the pump potential generated by the plasma membrane-bound proton pump/H+ ATPase, the P state. An experimental procedure is described to determine in cell-attached patch mode the state of the protoplast, either K or P state.

Prostaglandin E 2 excites neurons of the nucleus tractus solitarius by activating cation channels

Nucleus tractus solitarius (NTS) has a high density of prostaglandin E 2 (PGE 2)-binding sites. Action of PGE 2 (10 −9–10 −6 M) was tested on neurons in a NTS slice with patch-clamp recording under synaptic blockade. PGE 2 raised the firing rate in approximately half of the neurons in cell-attached patch mode. In whole-cell current clamp, PGE 2 depolarized membrane potential accompanied by an increase in firing rate. In whole-cell voltage clamp (−58 mV), PGE 2 induced the inward current with an increase in conductance. The current was linearly related to voltage from −100 mV to −10 mV and suppressed between −10 mV and 20 mV. The current-voltage curve remained similar under low external Cl − or high internal Cl − conditions and after external Na + was replaced by Cs +. It is concluded that PGE 2 excites NTS neurons by activating cation conductance.

Vasopressin modulates K(+)-channel activities of cultured smooth muscle cells from porcine coronary artery.

The ATP-sensitive K+ channel (KATP channel) and the Ca(2+)-activated K+ channel (KCa channel) were active in cell-attached and excised inside-out patch configurations in cultured smooth muscle cells of the porcine coronary artery. Vasopressin activated the KCa channel (240 pS) when it was applied in the bath in the cell-attached patch mode presumably because of an increase in intracellular Ca2+, but it had no direct effect on the KCa channel. However, vasopressin directly blocked the KATP channel from outside the cell membranes in a concentration-dependent manner in both outside-out and cell-attached patch configurations; the K(+)-channel opener, nicorandil, reversed this effect. The KATP channel (30 pS) was highly active in the intact cell-attached patch configuration when the pipette contained a physiological concentration of Ca2+, suggesting that this channel may control the resting membrane potential. (The block might produce depolarization of the cells and might result in the contraction of smooth muscle cells.) These observations suggest that the KATP channel may play a role, at least in part, in controlling the contraction of smooth muscle cells of the coronary artery and that the control of vascular tone by vasopressin may be related to its ability to block the KATP channel.

Potassium rectifier currents differ in myocytes of endocardial and epicardial origin.

Whole-cell voltage-clamp experiments and single-channel current recordings in cell-attached patch mode were performed on enzymatically dissociated single ventricular myocytes harvested from feline endocardial and epicardial surfaces. The studies were designed to compare the characteristics of inward rectifier K+ current (IK1) and delayed rectifier K+ current (IK) between endocardial and epicardial cells and to test the hypothesis that the differential characteristics of IK1 and/or IK are responsible for the differences in action potential configuration between the two cell types. IK1 in endocardial cells displayed a distinct N-shaped current-voltage (I-V) relation, with a prominent outward current at potentials between -80 and -30 mV. In epicardial cells, an outward current region was much smaller, and the I-V relation demonstrated a blunted N-shaped I-V relation. In single-channel current recordings in cell-attached patch mode, neither unitary current amplitude of IK1 nor probability of channel opening was different between endocardial and epicardial cells, suggesting that the difference in the number of functional channels might be responsible for the differential IK1 I-V relations. The characteristics of IK also differed between endocardial and epicardial cells. The time course of growth of tail current of IK (IK,tail) (activation of IK) was significantly enhanced and that of IK,tail deactivation was delayed in epicardial cells compared with endocardial cells. The time constant of the slow component of IK activation at +20 mV was 3,950 +/- 787 msec in endocardial cells and 2,746 +/- 689 msec in epicardial cells (p less than 0.05); the corresponding values for IK deactivation at -50 mV were 1,041 +/- 387 msec and 1,959 +/- 551 msec, respectively (p less than 0.01). The voltage dependence of steady-state activation of IK,tail was similar between endocardial and epicardial cells, suggesting that the probability of channel opening at any potential was not different in the two cell types. The amplitude and density of fully activated IK (IK,full) were significantly greater in epicardial cells than in endocardial cells. At repolarization to -20 mV, IK,full amplitude was 452 +/- 113 pA in endocardial cells and 578 +/- 135 pA in epicardial cells (p less than 0.05), and the corresponding values for IK,full density were 2.86 +/- 0.73 and 4.21 +/- 0.83 microA/cm2, respectively (p less than 0.05). A nonstationary fluctuation analysis revealed that the amplitude of IK unitary current was similar between endocardial and epicardial cells (0.23 +/- 0.07 versus 0.22 +/- 0.03 pA, p = NS).(ABSTRACT TRUNCATED AT 400 WORDS)

Protein kinase c may regulate resting anion conductance in vascular smooth muscle cells

The blocker of protein kinase C(PKC) activated large-conductance channel(337.1 pS) in cell attached patch mode in cultured vascular smooth muscle cells. The channel showed time-dependent inactivation whose time course became faster as the amplitude of the command potential was increased. These characteristics of large-conductance channel activated by the application of the PKC blocker were very similar to those of voltage-dependent Cl channels in these cells, indicating the channel activated by the drug is Cl channel. Since voltage-dependent Cl channels were reported to be only activated by forming inside-out patch, these findings suggest Cl permeability of vascular smooth muscle cells is at least partially regulated by protein kinase C.

Metabolic regulation of the K(ATP) and a maxi-K(V) channel in the insulin-secreting RINm5F cell.

K channels in the cell membrane of the insulin-secreting RINm5F cell line were studied using the patch-clamp technique in cell-attached patch mode. With 140 mM K in the pipette, two channels displaying different conductive and kinetics properties were observed. A voltage-independent, inward-rectifying, 55-pS channel was active at rest (no glucose, -70 mV), but was almost completely inhibited by 5 mM glucose. A 140-pS channel was seen in the absence of glucose only after cell membrane depolarization with high (30 mM) K. This channel was voltage dependent, with a linear slope conductance between -60 and +60 mV, and was completely inhibited only by greater than 15 mM glucose. The former channel we identify as an ATP-sensitive channel previously described in excised patches and refer to it as the K(ATP) channel. The latter, because of its large conductance and voltage-dependent kinetics, will be referred to as the maxi-K(V) channel, adopting a nomenclature previously used to classify highly conductive K channels (Latorre, R., and C. Miller, 1983, Journal of Membrane Biology, 71:11-30). In addition to glucose, mannose and 2-ketoisocaproate, which also initiate insulin secretion and electrical activity in the islet beta cell, reduced the activity of both the K(ATP) and the maxi-K(V) channel. Lactate and arginine, which potentiate but do not initiate insulin secretion or beta cell electrical activity in normal islets, each caused a large reduction in maxi-K(V) channel activity, without consistently affecting the activity of K(ATP) channels. Another agonist that potentiates insulin secretion and electrical activity in normal cells, the tumor-promoting phorbol ester TPA, blocked maxi-K(V) channel activity while stimulating the activity of the K(ATP) channel, thereby implicating phosphorylation in the control of channel activity. These results indicate that metabolic substrates that initiate electrical activity and insulin secretion in normal beta cells reduce the activity of both the K(ATP) and the maxi-K(V) channel, while potentiating agents reduce only the maxi-K(V) channel. The possible role of these two channels in the processes of initiation and potentiation of the beta cell response is discussed.

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

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 = 4.5 mM, NaCl = 145 mM), and the electrode contained Na-free K Hanks' (KCl = 145 mM) single-channel currents were observed at potentials below -40 mV which showed inward rectification, were K-selective, and were blocked by 2.5 mM 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 (4.5 mM) and the other containing 145 mM 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.