Constitutive inactivation of the hKv1.5 mutant channel, H463G, in K+-free solutions at physiological pH

Extracellular acidification and reduction of extracellular K⁺ are known to decrease the currents of some voltage-gated potassium channels. Although the macroscopic conductance of WT hKv1.5 channels is not very sensitive to [K⁺]_o at pH 7.4, it is very sensitive to [K⁺]_o at pH 6.4, and in the mutant, H463G, the removal of K⁺ _o virtually eliminates the current at pH 7.4. We investigated the mechanism of current regulation by K⁺ _o in the Kv1.5 H463G mutant channel at pH 7.4 and the wild-type channel at pH 6.4 by taking advantage of Na⁺ permeation through inactivated channels. Although the H463G currents were abolished in zero [K⁺]_o, robust Na⁺ tail currents through inactivated channels were observed. The appearnnce of H463G Na⁺ currents with a slow rising phase on repolarization after a very brief depolarization (2 ms) suggests that channels could activate directly from closed-inactivated states. In wild-type channels, when intracellular K⁺ was replaced by NMG⁺ and the inward Na⁺ current was recorded, addition of 1 mM K⁺ prevented inactivation, but changing pH from 7.4 to 6.4 reversed this action. The data support the idea that C-type inactivation mediated at R487 in Kv1.5 channels is influenced by H463 in the outer pore. We conclude that both acidification and reduction of [K⁺]_o inhibit Kv1.5 channels through a common mechananism (i.e., by increasing channel inactivation, which occurs in the resting state or develops very rapidly after activation).     

Lack of Negatively Charged Residues at the External Mouth of Kir2.2 Channels Enable the Voltage-Dependent Block by External Mg2+

Kir channels display voltage-dependent block by cytosolic cations such as Mg²⁺ and polyamines that causes inward rectification. In fact, cations can regulate K channel activity from both the extracellular and intracellular sides. Previous studies have provided insight into the up-regulation of Kir channel activity by extracellular K⁺ concentration. In contrast, extracellular Mg²⁺ has been found to reduce the amplitude of the single-channel current at milimolar concentrations. However, little is known about the molecular mechanism of Kir channel blockade by external Mg²⁺ and the relationship between the Mg²⁺ blockade and activity potentiation by permeant K⁺ ions. In this study, we applied an interactive approach between theory and experiment. Electrophysiological recordings on Kir2.2 and its mutants were performed by heterologous expression in Xenopus laevis oocytes. Our results confirmed that extracellular Mg²⁺ could reduce heterologously expressed WT Kir2.2 currents in a voltage dependent manner. The kinetics of inhibition and recovery of Mg²⁺ exhibit a 3∼4s time constant. Molecular dynamics simulation results revealed a Mg²⁺ binding site located at the extracellular mouth of Kir2.2 that showed voltage-dependent Mg²⁺ binding. The mutants, G119D, Q126E and H128D, increased the number of permeant K⁺ ions and reduced the voltage-dependent blockade of Kir2.2 by extracellular Mg²⁺.     

Properties of a potassium channel in the basolateral membrane of renal proximal convoluted tubule and the effect of cyclosporine on it

We studied the potassium channel in the basolateral membrane of the rat proximal convoluted tubule as affected by cyclosporine A. Proximal convoluted tubules were dissected from the rat kidney under a stereoscopic microscope, without a preliminary enzyme treatment. The standard configuration for single-channel tight seal patch-clamp technique was used to record channel currents. A small conductance, stretch-sensitive potassium channel could be observed spontaneously in most of the cell-attached patches as the gigaohm seal was formed. In the inside-out configuration, channel activity was diminished. The K(+) channel appeared to be an inward rectifier. The limiting inward slope conductance was 28.3+/-1.7 pS (Vp was between 40 mV and 80 mV, n=6) and the outward chord conductance was 5.6+/-0.3 pS (Vp was between -40 and -60 mV, n=5). The open dwell time constants of the potassium channel were 0.524 ms and 5.087 ms, while the closed dwell time constants were 1.029 ms and 16.500 ms. The opening probability of the channel decreased when the extracellular fluid was acidified. Cyclosporine A had no significant effect on the potassium channel of the proximal tubular cell in the basolateral membrane at concentrations of 10 and 50 microg/ml, while at 100 microg/ml, it decreased the opening probability.     

Novel type of ion channel activated by Pb2+, Cd2+, and Al3+ in cultured mouse neuroblastoma cells

Summary Superfusion with Pb²⁺ induces a slow, noninactivating and reversible inward current in voltage-clamped N1E-115 neuroblastoma cells. The amplitude of this inward current increases in the range of 1–200 m Pb²⁺. Single-channel patch-clamp experiments have revealed that this inward current is mediated by discrete ion channels. Reversal potentials from linearI–V relationships are close to 0 mV for whole-cell and single-channel currents and the single-channel conductance amounts to 24 pS. The Pb²⁺-induced membrane current is not mediated by various known types of ion channels, since it is not blocked by external tetrodotoxin, tetraethylammonium,d-tubocurarine, atropine, ICS 205-930 and by internal EGTA. In Na⁺-free solutions superfusion with Pb²⁺ neither evokes a whole-cell inward current, nor single-channel openings. At –80 mV the open-time distribution of the single channels activated by 1m Pb²⁺ is dual exponential with time constants of 17 and 194 msec. When the Pb²⁺ concentration is increased from 1 to 20 m these time constants decrease to 2 and 13 msec, but the amplitude of single-channel currents remains –1.9 nA. Cd²⁺ and Al³⁺ induce inward currents and single-channel openings similar to Pb²⁺. Time constants fitted to the open-time distribution of single channels are 14 and 135 msec in the presence of 1 m Cd²⁺ and 15 and 99 msec in the presence of 50 m Al³⁺. Conversely, Cu²⁺ induces an irreversible inward current in neuroblastoma cells. Single-channel openings are undetected in the presence of Cu²⁺ and in Na⁺-free solutions Cu²⁺ is still able to induce an inward current. It is concluded that Pb²⁺, Cd²⁺ and possibly Al³⁺ activate a novel type of metal ionactivated (MIA) channel in N1E-115 cells.     

Inward and outward currents of native and cloned K(ATP) channels (Kir6.2/SUR1) share single-channel kinetic properties

BackgroundThe ATP-sensitive K⁺ (K(ATP)) channel is found in a variety of tissues extending from the heart and vascular smooth muscles to the endocrine pancreas and brain. Common to all K(ATP) channels is the pore-forming subunit Kir6.x, a member of the family of small inwardly rectifying K⁺ channels, and the regulatory subunit sulfonylurea receptor (SURx). In insulin secreting β-cells in the endocrine part of the pancreas, where the channel is best studied, the K(ATP) channel consists of Kir6.2 and SUR1. Under physiological conditions, the K(ATP) channel current flow is outward at membrane potentials more positive than the K⁺ equilibrium potential around ?80 mV. However, K(ATP) channel kinetics have been extensively investigated for inward currents and the single-channel kinetic model is based on this type of recording, whereas only a limited amount of work has focused on outward current kinetics.MethodsWe have estimated the kinetic properties of both native and cloned K(ATP) channels under varying ionic gradients and membrane potentials using the patch-clamp technique.ResultsAnalyses of outward currents in K(ATP) and cloned Kir6.2ΔC26 channels, alone or co-expressed with SUR1, show openings that are not grouped in bursts as seen for inward currents. Burst duration for inward current corresponds well to open time for outward current.ConclusionsOutward K(ATP) channel currents are not grouped in bursts regardless of membrane potential, and channel open time for outward currents corresponds to burst duration for inward currents.     

Single-Channel Analysis of a Delayed Rectifier K+ Channel in Xenopus Myelinated Nerve

Single-channel properties of a delayed rectifier voltage-gated K⁺ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E₅₀, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105 mm K⁺, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C. In Na⁺-rich solution with 2.5 mm extracellular K⁺γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K⁺ over Na⁺. γ depended on extracellular K⁺ concentration (K _D = 19.6 mm) and temperature (Q ₁₀= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested with a half-maximal inhibiting concentration (IC₅₀) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC₅₀= 6.8 nm) and mast cell degranulating peptide (MCDP, IC₅₀= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential. Received: 2 June 1995/Revised: 13 October 1995     

Modulation of Voltage-dependent Properties of a Swelling-activated Cl? Current

We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl⁻ current (I _Cl,swell) in BC₃H1 myoblasts. This Cl⁻ current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl⁻ currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (∼60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10–80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg²⁺ concentration or reducing the Cl⁻ concentration of the extracellular solution. Replacing extracellular Cl⁻ by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN⁻ > I⁻ > NO₃ ⁻ > Cl⁻ >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg²⁺, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of I _Cl,swell are strongly influenced by external pH , external divalent cations, and by the nature of the permeant anion.     

Slow components of potassium tail currents in rat skeletal muscle

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.     

Marcroscopic and single-channel studies of two Ca2+ channel types in oocytes of the ascidianCiona intestinalis

Summary Whole-cell and single-channel patch-clamp experiments were performed on unfertilized oocytes of the ascidianCiona intestinalis to investigate the properties of two voltage-dependent Ca²⁺ currents found in this cell. The peak of the low threshold current (channel I) occurred at –20 mV, the peak of the high-threshold current (channel II) at +20 mV. The two currents could be distinguished by voltage dependence, kinetics of inactivation and ion selectivity. During large depolarizing voltage pulses, a transient outward current was recorded which appeared to be due to potassium efflux through channel II. When the external concentrations of Ca²⁺ and Mg²⁺ were reduced sufficiently, large inward Na currents flowed through both channels I and II. Using divalent-free solutions in cell-attached patch recordings, single-channel currents representing Na influx through channels I and II were recorded. The two types of unitary events could be distinguished on the basis of open time (channel I longer) and conductance (channel I smaller). Blocking events during changel I openings were recorded when micromolar concentrations of Ca²⁺ or Mg²⁺ were added to the patch pipette solutions. Slopes of the blocking rate constantvs. concentration gave binding constants of 6.4×10⁶ m ^–1 sec^–1 for Mg²⁺ and 4.5×10⁸ m ^–1 sec^–1 for Ca²⁺. The Ca²⁺ block was somewhat relieved at negative potentials, whereas the Mg²⁺ block was not, suggesting that Ca²⁺, but not Mg²⁺, can exit from the binding site toward the cell interior.     

ATP-sensitive potassium channels are altered in ventricular myocytes from diabetic rats

Hypoxia-induced shortening of the action potential duration, attributed to activation of the ATP-sensitive potassium (K_ATP) channels, occurs to a much greater extent in ventricular cells from diabetic rats. This study examined whether the K_ATP channels are altered in streptozotocin-diabetic myocardium. In inside-out patches from ventricular myocytes (with symmetrical 140 mM [K+]), inward K_ATP currents (at potentials negative to the K+ reversal potential) were similar in amplitude in control and diabetic patches (slope conductances: 69 and 74 pS, respectively). However, outward single-channel currents were larger for channels from diabetic heart cells than from control cells (e.g., at +75 mV the diabetic channel currents were 3.7 ± 0.3 pA vs. 2.7 ± 0.1 pA for control currents, p < 0.05), due to reduced inward rectification of diabetic channel currents. There was no difference in open and closed times between control and diabetic channels. The IC₅₀ for ATP inhibition of the K_ATP channel single-channel currents was 11.4 M for control currents and 4.7 M for diabetic channel currents. Thus, the major difference found between K_ATP channels from control and diabetic hearts was the greater outward diabetic single-channel current, which may contribute to the enhanced sensitivity to hypoxia (or ischemia) in diabetic hearts.     

Characterization of Single Inward Rectifier Potassium Channels from Embryonic Xenopus laevis Myocytes

Single inward rectifier K⁺ channels were studied in Xenopus laevis embryonic myocytes. We have characterized in detail the channel which is most frequently observed (Kir) although we routinely observe three other smaller current levels with the properties of inward rectifier K⁺ channels (Kir_(0.3), Kir_(0.5) and Kir_(0.7)). For Kir, slope conductances of inward currents were 10.3, 20.3, and 27.9 pS, in 60, 120 and 200 mM [K⁺] _o respectively. Extracellular Ba²⁺ blocked the normally high channel activity in a concentration-dependent manner (K _A = 7.8 μm, −90 mV). In whole-cell recordings of inward rectifier K⁺ current, marked voltage dependence of Ba²⁺ block over the physiological range of potentials was observed. We also examined current rectification. Following step depolarizations to voltages positive to E _K , outward currents through Kir channels were not observed even when the cytoplasmic face of excised patches were exposed to Mg²⁺-free solution at pH 9.1. This was probably also true for Kir_(0.3), Kir_(0.5) and Kir_(0.7) channels. We then examined the possibility of modulation of Kir channel activity and found neither ATP nor GTP-γS had any effect on Kir channel activity when added to the solution perfusing the cytoplasmic face of a patch. Kinetic analysis revealed Kir channels with a single open state (mean dwell time 72 msec) and two closed states (time constants 1.4, 79 msec). These results suggest that the native Kir channels of Xenopus myocytes have similar properties to the cloned strong inward rectifier K⁺ channels, in terms of conductance, kinetics and barium block but does show some differences in the effects of modulators of channel activity. Furthermore, skeletal muscle may contain either different inward rectifier channels or a single-channel type which can exist in stable subconductance states. Received: 16 September 1996/Revised: 14 March 1997     

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).     

TOK homologue in Neurospora crassa: first cloning and functional characterization of an ion channel in a filamentous fungus

In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K⁺ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K⁺ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K⁺ efflux. NcTOKA channel gating was sensitive to extracellular K⁺ such that channel activation was dependent on the reversal potential for K⁺. However, expression of NcTOKA was able to overcome the K⁺ auxotrophy of a yeast mutant missing the K⁺ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K⁺ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K⁺ currents at potentials negative of E_K. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca²⁺, verapamil, quinine, and TEA⁺ but was insensitive to Cs⁺, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.     

Voltage-sensitive chloride ion channels in Anopheles gambiae Sua-1B cells

In this study, we performed electrophysiological analysis of Anopheles gambiae Sua-1B cells having “neuron-like” morphologies using the patch clamp method. The recorded cells (n = 79) had processes resembling axons/dendrites, with 63 % unipolar, 22 % bipolar, and 15 % multipolar. While no inward currents were observed following step depolarizations (holding potential = ?80 mV), a slowly activating outward current was observed in 96 % of the cells, especially at depolarized potentials. The amplitude of the current was attenuated nearly 70 % by reducing extracellular Cl^? ion concentration, or by incubating with 100 μM DIDS, a known voltage-sensitive chloride channel blocker, suggesting that the current was mediated by chloride ions. No qualitative difference was found between recordings made with Cs⁺ ions in the intracellular pipette solution (inhibits K⁺ currents) and those made with normal physiological solution, indicating a deficiency of potassium channels. Additionally, recordings made with Ca²⁺-free extracellular bath solution eliminated the slowly activating outward current. A subset of cells (n = 3) lacked this current, but had outward currents with voltage-dependent properties similar to those of volume-regulated chloride channels. Taken together, our results suggest that the voltage-sensitive currents observed in the majority of Sua-1B cells are mediated primarily by chloride channels of the calcium-dependent subtype.     

Quantification of Transmembrane Currents during Action Potential Propagation in the Heart

The measurement, quantitative analysis, theory, and mathematical modeling of transmembrane potential and currents have been an integral part of the field of electrophysiology since its inception. Biophysical modeling of action potential propagation begins with detailed ionic current models for a patch of membrane within a distributed cable model. Voltage-clamp techniques have revolutionized clinical electrophysiology via the characterization of the transmembrane current gating variables; however, this kinetic information alone is insufficient to accurately represent propagation. Other factors, including channel density, membrane area, surface/volume ratio, axial conductivities, etc., are also crucial determinants of transmembrane currents in multicellular tissue but are extremely difficult to measure. Here, we provide, to our knowledge, a novel analytical approach to compute transmembrane currents directly from experimental data, which involves high-temporal (200 kHz) recordings of intra- and extracellular potential with glass microelectrodes from the epicardial surface of isolated rabbit hearts during propagation. We show for the first time, to our knowledge, that during stable planar propagation the biphasic total transmembrane current (I_m) dipole density during depolarization was ∼0.25 ms in duration and asymmetric in amplitude (peak outward current was ∼95 μA/cm² and peak inward current was ∼140 μA/cm²), and the peak inward ionic current (I_ion) during depolarization was ∼260 μA/cm² with duration of ∼1.0 ms. Simulations of stable propagation using the ionic current versus transmembrane potential relationship fit from the experimental data reproduced these values better than traditional ionic models. During ventricular fibrillation, peak I_m was decreased by 50% and peak I_ion was decreased by 70%. Our results provide, to our knowledge, novel quantitative information that complements voltage- and patch-clamp data.     

Anions permeate and gate GCAC1, a voltage-dependent guard cell anion channel

GCAC1 is a strongly voltage-dependent anion channel in the guard-cell plasma membrane of Vicia faba . In patch–clamp experiments, we have investigated the permeation and gating properties of GCAC1 with respect to its anion dependence in the whole-cell and excised-patch configuration. The relative permeability followed the order SCN^– > NO₃^– > Br^– > Cl^–, while the single-channel conductances in symmetrical anionic solutions exhibited a nearly inverse sequence. The Cl^– dependence of inward currents (Cl^– release) is characterized by a maximum single-channel conductance of 89 pS half-saturating at 87 mM cytoplasmic chloride. In addition to this substrate saturation, anion release was also dependent on the external Cl^– activity ( K _m = 16 mM). In the presence of SCN^– and Cl^–, the single-channel conductance exhibited an anomalous mole-fraction dependence, identifying GCAC1 as a multi-ion single-file pore. Using anions with increasing ionic size, a minimum pore diameter of 0.5 nm was assumed from their relative permeabilities. In line with an anion-selective channel, a tenfold increase in the extracellular anion activity shifted the reversal potential by –59.8 mV. Simultaneously, the half-activation potential shifted negatively by about 23 mV. A further analysis of the anion dependence revealed that extracellular rather than cytosolic anions affect the gating process of GCAC1. From anion substitution experiments, we conclude that anion concentration and species determines both permeation and gating of the plant anion channel GCAC1.     

A heterogeneous electrophysiological profile of bone marrow-derived mast cells

Electrophysiological properties of mouse bone marrow-derived mast cells (BMMC) were studied under the whole-cell clamp configuration. About one third of the cells were quiescent, but others expressed either inward or outward currents. Inwardly rectifying (IR) currents were predominant in 14% of the cells, and outwardly rectifying (OR) currents in 24%. The rest (22%) of the cells exhibited both inward and outward currents. The IR currents were eliminated by 1 mm Ba²⁺, and were partially inhibited by 100 μm quinidine. The reversal potential was dependent on extracellular K⁺, thereby indicating that K⁺ mediated the IR currents. The negative conductance region was seen at potentials positive to E _K. The OR currents did not apparently depend on the extracellular K⁺ concentration, but were reduced by lowering the extracellular Cl^? concentration. The OR currents were partially blocked by 1 mm Ba²⁺, and were further blocked by a Cl^? channel blocker, 4,4′-diisothiocyano-2, 2′-stilbenedisulfonate (DIDS). In addition, the reversal potential of the OR currents was positively shifted by decreasing the ratio of external and internal Cl^? concentrations, suggesting that Cl^? was a major ion carrier. In cells exhibiting IR currents, the membrane potential varied among cells and tended to depolarize by elevating the external K⁺ concentration. In cells with OR currents, the resting potential was hyperpolarized in association with an increase in conductance. These results suggest that BMMC have a heterogeneous electrophysiological profile that may underlie a variety of ion channels expressed in different phenotypes of mast cells. Activities of both the inwardly rectifying K⁺ channel and the outwardly rectifying Cl^? channel seem to contribute to the regulation of the membrane potential.     

Single transient K channels in mammalian sensory neurons.

A single-channel recording of the transient outward current (A-current) was obtained from dorsal root ganglion cells in culture using patch-clamp techniques. Depolarization of the membrane patch elicited pulse like current of a uniform amplitude in an outward direction, of which the unitary conductance was 20 pS. Alteration of extracellular ionic compositions indicated that the charge carriers were K ions. A systematic study was made on the voltage-dependence of the ensemble average current; (a) the activation started at a potential around -60 mV; (b) the time course of the activation was relatively rapid; (c) the channel was completely inactivated at a potential positive to -40 mV. Two time constants (tau f = 100 ms and tau s = 4,000 ms) were detected in the decay of the current indicating that the channels had two different states of inactivation. A convulsant, 4-aminopyridine (4-AP), acted on the channel only from the intracellular side of the membrane. 4-AP (5 mM) reduced not only mean open time (by 50%) but also the single-channel conductance (by 20%). The properties of the channel were independent of Ca ions in the intracellular space.     

Potassium inward rectifier and acetylcholine receptor channels in embryonic Xenopus muscle cells in culture

Embryonic muscle cells of the frog Xenopus laevis were isolated and grown in culture and single-channel recordings of potassium inward rectifier and acetylcholine (ACh) receptor currents were obtained from cell-attached membrane patches. Two classes of inward rectifier channels, which differed in conductance, were apparent. With 140 mM potassium chloride in the electrode, one channel class had a conductance of 28.8 ± 3.4 pS (n = 21), and, much more infrequently, a smaller channel class with a conductance of 8.6 ± 3.6 pS (n = 7) was recorded. Both channel classes had relatively long mean channel open times, which decreased with membrane hyperpolarization. The probability of finding a patch of membrane with an inward rectifier channel was high (66%) and many membrane patches contained more than one inward rectifier channel. The open state probability (with no applied potential) was high for both inward rectifier channel classes so that 70% of the time there was a channel open. Seventy-three percent of the membrane patches with ACh receptor channels (n = 11) also had at least one inward rectifier channel present when the patch electrode contained 0.1 μM ACh. Inward rectifier channels were also found at 71% of the sites of high ACh receptor density (n = 14), which were identified with rhodamine-conjugated α-bungarotoxin. The results indicate that the density of inward rectifier channels in this embryonic skeletal muscle membrane was relatively high and includes sites of membrane that have synaptic specializations. © 1996 John Wiley & Sons, Inc.