Depolarization-Activated K+ Channel in Chara Droplets

A novel potassium channel was characterized in the droplet membrane of Chara gymnophylla. This channel has a conductance of about 90 pS (in symmetrical 0.15 M KCl), which is lower compared to the 170-pS K+ channel predominant in this preparation. In contrast to the large conductance K+ channel, the novel channel opened with a delay at depolarization and closed at hyperpolarization and did not require cytosolic Ca2+ for its opening. It also showed comparatively weak selectivity for K+ over other monovalent cations, although its cation to anion selectivity was high. Externally or internally applied Cs+ blocked the channel in a voltage-dependent manner, similarly to the 170-pS channel. The sensitivity of the 90-pS channel to external tetraethylammonium chloride (half-blocking concentration approximately 1.5 mM) was 20-fold higher compared to the large conductance channel. With respect to its voltage-gating kinetics, the 90-pS channel was identified as a "slow delayed rectifier."     

Slow membrane potential changes in skeletal muscle induced in Cl(-)-free medium.

1. Conventional microelectrode techniques were used to measure simultaneous changes in membrane potential (Vm) and conductance (Gm) induced by single electrical stimuli in muscles bathed in Cl(-)-free solution containing 40 mM of tetraethylammonium (TEA+). 2. Stimulation induced slow transient depolarizations (slow response) accompanied by increased calcium conductance, while the potassium conductance was first elevated and later reduced. 3. Stepwise elevation of [K+]0 from 2.5 to 5 or 10 mM during the slow response evoked an abrupt repolarization of 42.3 +/- 8.9 mV (n = 4; p less than 0.001), and 24.8 +/- 3.5 +/- mV (n = 5; p less than 0.001), respectively, while Gm was increased to 1.45 +/- 0.25-fold (n = 5; p less than 0.05). Neither the slow response nor K(+)-induced changes in Vm or Gm were sensitive to tetrodotoxin (3 microM), however, nifedipine (10 microM) abolised the slow response. 4. It was concluded that beyond the increase of calcium conductance, the ionic conductance of the inward rectifier K+ channel was reduced during the slow response, which could be restored by the elevation of [K+]0. The results suggest the possible contribution of these mechanisms to the electrical instability of myotonic muscles. Potential therapeutic consequences are discussed.     

Calcium- and voltage-dependent ion channels in Saccharomyces cerevisiae.

Ion channels in both the tonoplast and the plasma membrane of Saccharomyces cerevisiae have been characterized at the single channel level by patch-clamp techniques. The predominant tonoplast channel is cation selective, has an open-channel conductance of 120 pS in 100 mM KCl, and conducts Na+ or K+ equally well, and Ca2+ to a lesser extent. Its open probability (Po) is voltage-dependent, peaking at about -80 mV (cytoplasm negative), and falling to near zero at +80 mV. Elevated cytoplasmic Ca2+, alkaline cytoplasmic pH, and reducing agents activate the channel. The predominant plasma membrane channel is highly selective for K+ over anions and other cations, and shows strong outward rectification of the time-averaged current-voltage curves in cell-attached experiments. In isolated inside-out patches with micromolar cytoplasmic Ca2+, this channel is activated by positive going membrane voltages: mean Po is zero at negative membrane voltages and near unity at 100 mV. At moderate positive membrane voltages (20-40 mV), elevating cytoplasmic Ca2+ activates the channel to open in bursts of several hundred milliseconds duration. At higher positive membrane voltages, however, elevating cytoplasmic Ca2+ blocks the channel in a voltage-dependent fashion for periods of 2-3 ms. The frequency of these blocking events depends on cytoplasmic Ca2+ and membrane voltage according to second-order kinetics. Alternative cations, such as Mg2+ or Na+, block the yeast plasma-membrane K+ channel in a similar but less pronounced manner.     

Characterization of the calcium-sensitive voltage-gated delayed rectifier potassium channel in isolated guinea pig hepatocytes

The voltage-dependent K+ channel was examined in enzymatically isolated guinea pig hepatocytes using whole-cell, excised outside-out and inside- out configurations of the patch-clamp technique. The resting membrane potential in isolated hepatocytes was -25.3 +/- 4.9 mV (n = 40). Under the whole-cell voltage-clamp, the time-dependent delayed rectifier outward current was observed at membrane potentials positive to -20 mV at physiological temperature (37 degrees C). The reversal potential of the current, as determined from tail current measurements, shifted by approximately 57 mV per 10-fold change in the external K+ concentration. In addition, the current did not appear when K+ was replaced with Cs+ in the internal and external solutions, indicating that the current was carried by K+ ions. The envelope test of the tails demonstrated that the growth of the tail current followed that of the current activation. The ratio between the activated current and the tail amplitude was constant during the depolarizing step. The time course of growth and deactivation of the tail current were best described by a double exponential function. The current was suppressed in Ca(2+)-free, 5 mM EGTA internal or external solution (pCa > 9). The activation curve (P infinity curve) was not shifted by changing the internal Ca2+ concentration ([Ca2+]i). The current was inhibited by bath application of 4-aminopyridine or apamin. alpha 1-Adrenergic stimulation with noradrenaline enhanced the current but beta-adrenergic stimulation with isoproterenol had no effect on the current. In single- channel recordings from outside-out patches, unitary current activity was observed by depolarizing voltage-clamp steps whose slope conductance was 9.5 +/- 2.2 pS (n = 10). The open time distribution was best described by a single exponential function with the mean open lifetime of 18.5 +/- 2.6 ms (n = 14), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 2.0 +/- 0.3 ms (n = 14) and that for the slow component of 47.7 +/- 5.9 ms (n = 14). Ensemble averaged current exhibited delayed rectifier nature which was consistent with whole-cell measurements. In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The concentration of Ca2+ at the half-maximal activation was 0.031 microM. These results suggest that guinea pig hepatocytes possess voltage-gated delayed rectifier K+ channels which are modified by intracellular Ca2+.     

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.     

Regulation of the hyperpolarization-activated K+ channel in the lateral membrane of the cortical collecting duct [published erratum appears in J Gen Physiol 1995 Sep;106(3):579]

An intermediate-conductance K+ channel (I.K.), the activity of which is increased by hyperpolarization, was previously identified in the lateral membrane of the cortical collecting duct (CCD) of the rat kidney (Wang, W. H., C. M. McNicholas, A. S. Segal, and G. Giebisch. 1994. American Journal of Physiology. 266:F813-F822). The biophysical properties and regulatory mechanisms of this K+ channel have been further investigated with patch clamp techniques in the present study. The slope conductance of the channel in inside-out patches was 50 pS with 140 mM KCl in the pipette and 5 mM KCl, 140 mM NaCl (NaCl Ringer''s solution) in the bath. Replacement of the bath solution with symmetrical 140 mM KCl solution changed the slope conductance of the channel to 85 pS and shifted the reversal potential by 55 mV, indicating that the selectivity ratio of K+/Na+ was at least 10:1. Channel open probability (Po) in inside-out patches was 0.12 at 0 mV and was increased by hyperpolarization. The voltage-dependent Po was fitted with the Boltzmann''s equation: Po = 1/[1 + exp(V-V1/2)zF/RT], with z = 1.2 and V1/2 = -40 mV. Addition of 2 mM tetraethylammonium or 500 mM quinidine to the bath blocked the activity of the K+ channel in inside-out patches. In addition, decrease in the bath pH from 7.40 to 6.70 reduced Po by 30%. Addition of the catalytic subunit of protein kinase A (PKAc; 20 U/ml) and 100 microM [corrected] MgATP to the bath increased Po from 0.12 to 0.49 at 0 mV and shifted the voltage dependence curve of channel activity toward more positive potentials by 40 mV. Two exponentials were required to fit both the open-time and the closed-time histograms. Addition of PKAc increased the long open-time constant and shortened the long closed-time constant. In conclusion, PKA-mediated phosphorylation plays an important role in the regulation of the voltage dependence of the hyperpolarization-activated K+ channel in the basolateral membrane of CCD.     

Changes in densities and kinetics of delayed rectifier potassium channels during neuronal differentiation

Single-channel K+ currents were recorded from young and mature spinal neurons cultured from Xenopus embryos to examine the bases of the developmental increases in density and in rate of activation of the macroscopic voltage-dependent delayed rectifier K+ current (IKv). K+ channels of three conductance classes (integral of 80, 30, and 15 pS) are present at both ages, but only the intermediate and small conductance classes are voltage-dependent and thus underlie IKv. The increase in the density of IKv is due to increases in the numbers of intermediate and small channels per cell, but not to changes in their open probabilities. The increase in rate of activation of IKv results from a change in the activation kinetics of the intermediate channel class alone.     

Properties and modulation of alpha human atrial natriuretic peptide (alpha-hANP)-formed ion channels

Using the lipid bilayer technique we have optimized recording conditions and confirmed that alpha human atrial natriuretic peptide [alpha-hANP(1-28)] forms single ion channels. The single channel currents recorded in 250/50 mM KCl cis/trans chambers show that the ANP-formed channels were heterogeneous, and differed in their conductance, kinetic, and pharmacological properties. The ANP-formed single channels were grouped as: (i) H202- and Ba2+-sensitive channel with fast kinetics; the nonlinear current-voltage (I-V) relationship of this channel had a reversal potential (Erev) of -28.2 mV, which is close to the equilibrium potential for K+ (EK = -35 mV) and a maximal slope conductance (gmax) of 68 pS at positive potentials. Sequential ionic substitution (KCl, K gluconate and choline Cl) of the cis solution suggests that the current was carried by cations. The fast channel had three modes (spike mode, burst mode, and open mode) that differed in their kinetics but not in their conductance properties. (ii) A large conductance channel possessing several subconductance levels that showed time-dependent inactivation at positive and negative membrane potentials (Vm). The inactivation ratio of the current at the end of the voltage step (Iss) to the initial current (Ii) activated immediately after the voltage step, (Iss/Ii), was voltage dependent and described by a bell-shaped curve. The maximal current-voltage (I-V) relationship of this channel, which had an Erev of +17.2 mV, was nonlinear and the value of gmax was 273 pS at negative voltages. (iii) A transiently-activated channel: the nonlinear I-V relationship of this channel had an Erev of -29.8 mV and the value of gmax was 160 pS at positive voltages. We propose that the voltage-dependence of the ionic currents and the kinetic parameters of these channel types indicate that if they were formed in vivo and activated by cytosolic factors they could change the membrane potential and the electrolyte homeostasis of the cell.     

Multiple types of voltage-dependent Ca2+-activated K+ channels of large conductance in rat brain synaptosomal membranes

K+-selective ion channels from a mammalian brain synaptosomal membrane preparation were inserted into planar phospholipid bilayers on the tips of patch-clamp pipettes, and single-channel currents were measured. Multiple distinct classes of K+ channels were observed. We have characterized and described the properties of several types of voltage-dependent, Ca2+-activated K+ channels of large single-channel conductance (greater than 50 pS in symmetrical KCl solutions). One class of channels (Type I) has a 200-250-pS single-channel conductance. It is activated by internal calcium concentrations greater than 10(-7) M, and its probability of opening is increased by membrane depolarization. This channel is blocked by 1-3 mM internal concentrations of tetraethylammonium (TEA). These channels are similar to the BK channel described in a variety of tissues. A second novel group of voltage-dependent, Ca2+-activated K+ channels was also studied. These channels were more sensitive to internal calcium, but less sensitive to voltage than the large (Type I) channel. These channels were minimally affected by internal TEA concentrations of 10 mM, but were blocked by a 50 mM concentration. In this class of channels we found a wide range of relatively large unitary channel conductances (65-140 pS). Within this group we have characterized two types (75-80 pS and 120-125 pS) that also differ in gating kinetics. The various types of voltage-dependent, Ca2+-activated K+ channels described here were blocked by charybdotoxin added to the external side of the channel. The activity of these channels was increased by exposure to nanomolar concentrations of the catalytic subunit of cAMP-dependent protein kinase. These results indicate that voltage-dependent, charybdotoxin-sensitive Ca2+-activated K+ channels comprise a class of related, but distinguishable channel types. Although the Ca2+-activated (Type I and II) K+ channels can be distinguished by their single-channel properties, both could contribute to the voltage-dependent Ca2+-activated macroscopic K+ current (IC) that has been observed in several neuronal somata preparations, as well as in other cells. Some of the properties reported here may serve to distinguish which type contributes in each case. A third class of smaller (40-50 pS) channels was also studied. These channels were independent of calcium over the concentration range examined (10(-7)-10(-3) M), and were also independent of voltage over the range of pipette potentials of -60 to +60 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

重组人白细胞介素—1β对小鼬有骨髓基质细胞K^＋通道的影响

In the present study, both cell-attached and inside-out mode of patch clamp technique were applied to detect the effect of recombinant human interleukin-1 beta (IL-1 beta) on K+ channels of mice bone marrow stromal cells. A 16.7 pS voltage-dependent K+ channel resembling the delayed rectifier K+ channel in excitable cells was identified and characterized. Under stimulation of IL-1 beta (1000 U/ml), the single channel conductance increased to 26.1 +/- 3.6 pS (P < 0.01). IL-1 beta also markedly increased the open time constant and the open probability, but decreased the closed time constant as compared to the same patches without IL-1 beta. In addition, IL-1 beta induced multi-channel activities in about 80 percent patches by inducing more K+ channels of the same type open. These results suggest that activation of K+ channels plays a role in signal transduction of interleukin-1.     

Trek-like Potassium Channels in Rat Cardiac Ventricular Myocytes Are Activated by Intracellular ATP

Large (111 +/- 3.0 pS) K+ channels were recorded in membrane patches from adult rat ventricular myocytes using patch-clamp techniques. The channels were not blocked by 4-AP (5 mM), intracellular TEA (5 mM) or glybenclamide (100 mM). Applying stretch to the membrane (as pipette suction) increased channel open probability (Po) in both cell-attached and isolated patches (typically, Po approximately equals 0.005 with no pressure; approximately equals 0.328 with 90 cm H2O: Vm = 40 mV, pHi = 7.2). The channels were activated by a decrease in intracellular pH; decreasing pHi to 5.5 from 7.2 increased Po to 0.16 from approx. 0.005 (no suction, Vm held at 40 mV). These properties are consistent with those demonstrated for TREK-1, a member of the recently cloned tandem pore family. We confirmed, using RT-PCR, that TREK-1 is expressed in rat ventricle, suggesting that the channel being recorded is indeed TREK-1. However, we show also that the channels are activated by millimolar concentrations of intracellular ATP. At a pH of 6 with no ATP at the intracellular membrane face, Po was 0.048 +/-0.023, whereas Po increased to 0.22 +/- 0.1 with 1 mM ATP, and to 0.348 +/- 0.13 with 3 mM (n = 5; no membrane stretch applied). The rapid time course of the response and the fact that we see the effect in isolated patches appear to preclude phosphorylation. We conclude that intracellular ATP directly activates TREK-like channels, a property not previously described.     

Basic properties and potential regulators of the apical K+ channel in macula densa cells

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.     

Regulation of action potential duration under acute heat stress by I(K,ATP) and I(K1) in fish cardiac myocytes

The mechanism underlying temperature-dependent shortening of action potential (AP) duration was examined in the fish (Carassius carassius L.) heart ventricle. Acute temperature change from +5 to +18 degrees C (heat stress) shortened AP duration from 2.8 +/- 0.3 to 1.3 +/- 0.1 s in intact ventricles. In 56% (18 of 32) of enzymatically isolated myocytes, heat stress also induced reversible opening of ATP-sensitive K+ channels and increased their single-channel conductance from 37 +/- 12 pS at +8 degrees C to 51 +/- 13 pS at +18 degrees C (Q10 = 1.38) (P < 0.01; n = 12). The ATP-sensitive K+ channels of the crucian carp ventricle were characterized by very low affinity to ATP both at +8 degrees C [concentration of Tris-ATP that produces half-maximal inhibition of the channel (K1/2)= 1.35 mM] and +18 degrees C (K1/2 = 1.85 mM). Although acute heat stress induced ATP-sensitive K+ current (IK,ATP) in patch-clamped myocytes, similar heat stress did not cause any glibenclamide (10 microM)-sensitive changes in AP duration in multicellular ventricular preparations. Examination of APs and K+ currents from the same myocytes by alternate recording under current-clamp and voltage-clamp modes revealed that changes in AP duration were closely correlated with temperature-specific changes in the voltage-dependent rectification of the background inward rectifier K+ current IK1. In approximately 15% of myocytes (4 out of 27), IK,ATP-dependent shortening of AP followed the IK1-induced AP shortening. Thus heat stress-induced shortening of AP duration in crucian carp ventricle is primarily dependent on IK1. IK,ATP is induced only in response to prolonged temperature elevation or perhaps in the presence of additional stressors.     

Ion channels in human endothelial cells.

Ion channels were studied in human endothelial cells from umbilical cord by the patch clamp technique in the cell attached mode. Four different types of ion channels were recorded: i) potassium channel current that rectifies at positive potentials in symmetrical potassium solutions (inward rectifier); ii) low-conductance non-selective cation channel with a permeability ratio K:Na:Ca = 1:0.9:0.2; iii) high-conductance cation-selective channel that is about 100 times more permeable for calcium than for sodium or potassium; iv) high-conductance potassium channel with a permeability ratio K:Na = 1:0.05. The extrapolated reversal potential of the inwardly rectifying current was near to the potassium equilibrium potential. The slope conductance decreased from 27 pS in isotonic KCl solution to 7 pS with 5.4 mmol/l KCl and 140 mmol/l NaCl in the pipette but 140 mmol/l KCl in the bath. The low-conductance non-selective cation channel showed a single-channel conductance of 26 pS with 140 mmol/l Na outside, 28 pS with 140 mmol/l K outside, and rectified in inward direction in the presence of Ca (60 mmol/l Ca, 70 mmol/l Na, 2.7 mmol/l K in the pipette) at negative potentials. The current could be observed with either chloride or aspartate as anion. The high-conductance non-selective channel did not discriminate between Na and K. The single-channel conductance was about 50 pS. The extrapolated reversal potential was more positive than +40 mV (140 K or 140 Na with 5 Ca outside). Both the 26 and 50 pS channel showed a run-down, and they rapidly disappeared in excised patches. The high-conductance potassium channel with a single-channel conductance of 170 pS was observed only rarely. It reversed near the expected potassium equilibrium potential. The 26 pS channel could be stimulated with histamine and thrombin from outside in the cell-attached mode. Both the 26 pS as well as the 50 pS channel can mediate calcium flux into the endothelial cell.     

Gating mechanism of a cloned potassium channel expressed in frog oocytes and mammalian cells

We have cloned a cDNA coding for a delayed rectifier K+ channel from rat brain (RCK1) and rat muscle (RMK1) and expressed it in Xenopus oocytes and in a myoblast cell line (Sol-8). Stably transfected Sol-8 cells exhibited large outward K+ currents, which were indistinguishable from the K+ currents induced in Xenopus oocytes by injection of mRNA transcribed in vitro. RCK1 encodes a K+ channel with a unitary conductance of approximately 14 pS. The steep voltage dependence of channel opening resides in transitions between closed states, whereas the direct transitions into and out of the open state are very rapid and not markedly voltage-dependent. Channel inactivation is very slow, voltage-independent, and occurs from the open state only. We present a simple model that incorporates our findings and is consistent with the presumed structural symmetry of a functional K+ channel.     

钾通道活化剂对豚鼠气道平滑肌电压依赖性钾通道特性的影响

钾通道活化剂可激活钾离子通道并松驰支气管平滑肌,在急性分离的豚鼠支气管平滑肌细胞上,用膜片钳技术的细胞贴附式和内面向外式研究了其对电压依赖性钾通道的直接作用。结果证实：在全细胞记录条件下,卡吗克林和拉吗克林不影响静息膜电位。但在去极化时可使通道电导从７５．２±５．１ｐＳ分别增大到８５．９±１１．８ｐＳ和８２．１±５．５ｐＳ。通道动力学特性也发生了改变,通道平均开放时间的τｏ２值延长和开放概率显著增加,其中拉吗克林的作用更为强。两者均可诱发通道出现多级开放。表明这两类活化剂可使去极化时钾离子外流增加。     

A patch-clamp study of histamine-secreting cells

The ionic conductances in rat basophilic leukemia cells (RBL-2H3) and rat peritoneal mast cells were investigated using the patch-clamp technique. These two cell types were found to have different electrophysiological properties in the resting state. The only significant conductance of RBL-2H3 cells was a K+-selective inward rectifier. The single channel conductance at room temperature increased from 2-3 pS at 2.8 mM external K+ to 26 pS at 130 mM K+. This conductance, which appeared to determine the resting potential, could be blocked by Na+ and Ba2+ in a voltage-dependent manner. Rat peritoneal mast cells had a whole-cell conductance of only 10-30 pS, and the resting potential was close to zero. Sometimes discrete openings of channels were observed in the whole-cell configuration. When the Ca2+ concentration on the cytoplasmic side of the membrane was elevated, two types of channels with poor ion specificity appeared. A cation channel, observed at a Ca2+ concentration of approximately 1 microM, had a unit conductance of 30 pS. The other channel, activated at several hundred micromolar Ca2+, was anion selective and had a unit conductance of approximately 380 pS in normal Ringer solution and a bell-shaped voltage dependence. Antigenic stimulation did not cause significant changes in the ionic conductances in either cell type, which suggests that these cells use a mechanism different from ionic currents in stimulus-secretion coupling.     

Identification of a stretch-activated monovalent cation channel from teleost urinary bladder cells.

The urinary bladder of euryhaline teleost is an important osmoregulatory organ which absorbs Na+, Cl-, and water from urine. Using patch clamp technique, single stretch-activated channels, which were permeable to K+ and Na+ (PNa/PK approximately 0.75) and had conductances of 55 and 116 pS, were studied. In excised, inside-out patches which were voltage-clamped in the physiological range of membrane potential, the single-channel open probability (Po) was low (approximately 0.02), and increased to a maximum of 0.9 with applied pipette suction. Single-channel conductance also increased with suction. The channels showed adaptation to applied suction and relaxed to a steady-state activity about 20 seconds after application of suction. The Po increased up to 0.9 with strong membrane depolarization (Vm = 0 to +80 mV); however, there was little dependence of Po on membrane potential in the physiological range. The kinetic data suggest that there is one conducting state and at least two non-conducting states of the channel. The open-time constant increased with suction but remained unchanged with membrane potential (Vm = -70 to +60 mV). The mean closed-time of the channel decreased with suction and membrane depolarization. These results demonstrate the presence of a non-selective monovalent cation channel which may be involved in cell volume regulation in the goby urinary bladder. Additionally, this channel may function as an enhancer of Na+ influx and K+ efflux across the bladder cell as part of transepithelial ion transport if it is located in apical membrane.     

Electrophysiological characterization of a new member of the RCK family of rat brain K+ channels

A novel member of the RCK family of rat brain K+ channels, called RCK2, has been sequenced and expressed in Xenopus oocytes. The K+ currents were voltage-dependent, activated within 20 ms (at 0 mV), did not inactivate in 5 s, and had a single channel conductance in frog Ringers of 8.2 pS. Compared to other members of the RCK family the pharmacological profile of RCK2 was unique in that the channel was resistant to block (IC50 = 3.3 microM) by charybdotoxin [(1988) Proc. Natl. Acad. Sci. USA 85, 3329-3333] but relatively sensitive to 4-aminopyridine (0.3 mM), tetraethylammonium (1.7 mM), alpha-dendrotoxin (25 nM), noxiustoxin (200 nM), and mast cell degranulating peptide (200 nM). Thus, RCK2 is a non-inactivating delayed rectifier K+ channel with interesting pharmacological properties.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.