III. Ion Channel Expression in PMA-differentiated Human THP-1 Macrophages

Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K⁺ currents, I _DR, present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K⁺ channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K⁺ channel (I _IR) and a Ca²⁺-activated maxi-K channel (I _BK). I _IR was a classical inward rectifier, conducting large inward currents negative to E _K and very small outward currents. I _IR was blocked in a voltage-dependent manner by Cs⁺, Na⁺, and Ba²⁺, block increasing with hyperpolarization. Block by Na⁺ and Ba²⁺ was time-dependent, whereas Cs⁺ block was too fast to resolve. Rb⁺ was sparingly permeant. In cell-attached patches with high [K⁺] in the pipette, the single I _IR channel conductance was ∼30 pS and no outward current could be detected. I _BK channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the conductance was ∼200–250 pS and at potentials positive to ∼100 mV a negative slope conductance of the unitary current was observed, suggesting block by intracellular Na⁺. I _BK was activated at large positive potentials in cell-attached patches; in inside-out patches the voltage-activation relationship was shifted to more negative potentials by increased [Ca²⁺]. Macroscopic I _BK was blocked by external TEA⁺ with half block at 0.35 mm. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K⁺ channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold. Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed. Received: 19 September 1995/Revised: 14 March 1996     

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     

Flow-Dependent Activation of Maxi K+ Channels in Apical Membrane of Rabbit Connecting Tubule

The Ca²⁺-activated maxi K⁺ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean number of open channels (NP _o ) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the stretch-activation of Ca²⁺-permeable cation channels, because the maxi K⁺ channel was not stretch-activated in both the cell-attached configuration using Ca²⁺-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K⁺ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K⁺ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K⁺ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV _t ) when luminal K⁺ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV _t was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced ΔV _t to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV _t to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV _t was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV _t =−7.2 ± 0.5 mV in control vs.ΔV _t =−6.7 ± 0.6 mV in AA). These results suggest that the Ca²⁺-activated maxi K⁺ channel is responsible for flow-dependent K⁺ secretion by coupling with the stretch-activated Ca²⁺-permeable cation channel in the rabbit CNT. Received: 21 August 1997/Revised: 20 March 1998     

Cloning and Function of the Rat Colonic Epithelial K+ Channel KVLQT1

K_VLQT1 (KCNQ1) is a voltage-gated K⁺ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K⁺ channel in the colonic epithelium. We now report cloning and expression of K_VLQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to K_VLQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of K_VLQT1 in crypt cells and surface epithelium. Expression of rK_VLQT1 in Xenopus oocytes induced a typical delayed activated K⁺ current, that was further activated by increase of intracellular cAMP but not Ca²⁺ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K⁺ conductance in the colonic mucosal epithelium and inhibited whole cell K⁺ currents in patch-clamp experiments on isolated colonic crypts. We conclude that K_VLQT1 is forming an important component of the basolateral cAMP-activated K⁺ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis. Received: 17 July 2000/Revised: 25 October 2000     

Membrane Potassium Channels and Human Bladder Tumor Cells. I. Electrical Properties

These experiments were conducted to determine the membrane K⁺ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K⁺ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca²⁺-dependent outward K⁺ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca²⁺-activated, voltage-dependent K⁺ channel (K_Ca) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K_Ca activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K⁺ channel, 21 ± 1 pS at positive transmembrane potential (V _m ) and 145 ± 13 pS at negative V _m . Glibenclamide (50 μm), Ba²⁺ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K⁺ channels in inside/out patches in a dose-dependent manner, and the IC₅₀= 46 μm. The identity of this K⁺ channel with an ATP-sensitive K⁺ channel (K_ATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the K_Ca and a lower conductance K⁺ channel with properties consistent with a sulfonylurea receptor-linked K_ATP. Received: 12 June 1997/Revised: 21 October 1997     

Single K+ Channels in Embryonic Leech Ganglion Cells

We investigated the properties of single K⁺ channels in the soma membrane of embryonic leech ganglion cells using the patch-clamp technique. We compared these K⁺ channels with the K⁺ channels found previously in Retzius neurons of the adult leech. In ganglion cells of 9- to 15-day-old embryos we characterized eight different types of K⁺ channels with mean conductances of 21, 55, 84, 111, 122, 132, 149 and 223 pS. The 55 pS and 84 pS channels showed flickering and were active for less than 2 min after excising the patch. The 111 pS channel was an outward rectifier, and the open state probability (p _o ) decreased in the inside-out configuration when the Ca²⁺ concentration was raised from pCa 7 to pCa 3. The 122 pS channel also showed outward rectification. This type of channel was activated after changing from the cell-attached to the inside-out configuration and it did not inactivate during more than 30 min. The p _o was Ca²⁺- and voltage-insensitive. One hundred μm glibenclamide reversibly reduced p _o . The 132 pS channel was an outward rectifier and was Ca²⁺-insensitive. The 149 pS channel inactivated in the inside-out configuration. The 149- and the 223 pS channel showed inward rectification. The 111 pS channel had similar properties to the Ca²⁺-dependent K⁺ channel and the 122 pS channel resembled the ATP-inhibited K⁺ channel found previously in Retzius neurons of the adult leech. Received: 20 April 1995/Revised: 18 January 1996     

Characterization of Angiotensin II-regulated K+ Conductance in Rat Adrenal Glomerulosa Cells

Nystatin perforated-patch clamp and single-channel recording methods were used to characterize macroscopic and single-channel K⁺ currents and the effects of angiotensin II (AngII) in cultured rat adrenal glomerulosa cells. Two basic patterns of macroscopic current-voltage relationships were observed: type 1 exhibited a rapidly activating, noninactivating, voltage-dependent outward current and type 2 exhibited an inactivating voltage-dependent outward current attributed to charybdotoxin sensitive Ca⁺⁺-dependent K⁺ channels. Most cells exhibited the type 1 pattern and experiments focused on this cell type. Cell-attached and inside-out patches were dominated by a single K⁺ channel class which exhibited an outward conductance of 12 pS (20 mm K⁺ pipette in cell-attached and inside-out configurations, 145 mm K⁺ _in), a mean open time of 2 msec, and a weakly voltage-dependent low open probability that increased with depolarization. Channel open probability was reversibly inhibited by bath stimulation with AngII. At the macroscopic level, type 1 cell macroscopic K⁺ currents appeared comprised of two components: a weakly voltage-dependent current controlling the resting membrane potential (−85 mV) which appeared mediated by the 12 pS K⁺ channel and a rapidly activating, noninactivating voltage-dependent current activated above −50 mV. The presence of the second voltage-dependent K⁺ channel class was suggested by the effects of AngII, the blocking effects of quinidine and Cs⁺, and the properties of the weakly voltage-dependent K⁺ channel described. The K⁺ selectivity of the macroscopic current was demonstrated by the dependence of current reversal potentials on the K⁺ equilibrium potential and by the effects of K⁺ channel blockers, Cs⁺ and quinidine. AngII (10 pm to 1 nm) reversibly inhibited macroscopic K⁺ currents and this effect was blocked by the AT₁ receptor antagonist losartin. Received: 6 August 1996/Revised: 15 November 1996     

Large Conductance Ca2+-Activated K+ Channels in Human Meningioma Cells

Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K⁺ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC₅₀= 5.3 mm). Raising the internal Ca²⁺ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V _1/2) of the K⁺ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca²⁺-activated (BK _Ca ) K⁺ channel with a conductance of 296 pS (130 mm K⁺ at both sides of the patch). V _1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca²⁺, respectively, at the internal face of the patch. In cell-attached patches the open probability (P _o ) of BK _Ca channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K⁺ currents with 10 nm Ca²⁺ in the pipette. Application of 20 μm cytochalasin D increased P _o of BK _Ca channels in cell-attached patches within minutes. These data suggest that the activation of BK _Ca channels in meningioma cells does not only depend on voltage and internal Ca²⁺ but is also controlled by the cytoskeleton. Received 18 June 1999/Revised: 18 January 2000     

Regulation and Properties of KCNQ1 (KVLQT1) and Impact of the Cystic Fibrosis Transmembrane Conductance Regulator

The K⁺ channel KCNQ1 (K_VLQT1) is a voltage-gated K⁺ channel, coexpressed with regulatory subunits such as KCNE1 (IsK, mink) or KCNE3, depending on the tissue examined. Here, we investigate regulation and properties of human and rat KCNQ1 and the impact of regulators such as KCNE1 and KCNE3. Because the cystic fibrosis transmembrane conductance regulator (CFTR) has also been suggested to regulate KCNQ1 channels we studied the effects of CFTR on KCNQ1 in Xenopus oocytes. Expression of both human and rat KCNQ1 induced time dependent K⁺ currents that were sensitive to Ba²⁺ and 293B. Coexpression with KCNE1 delayed voltage activation, while coexpression with KCNE3 accelerated current activation. KCNQ1 currents were activated by an increase in intracellular cAMP, independent of coexpression with KCNE1 or KCNE3. cAMP dependent activation was abolished in N-terminal truncated hKCNQ1 but was still detectable after deletion of a single PKA phosphorylation motif. In the presence but not in the absence of KCNE1 or KCNE3, K⁺ currents were activated by the Ca²⁺ ionophore ionomycin. Coexpression of CFTR with either human or rat KCNQ1 had no impact on regulation of KCNQ1 K⁺ currents by cAMP but slightly shifted the concentration response curve for 293B. Thus, KCNQ1 expressed in Xenopus oocytes is regulated by cAMP and Ca²⁺ but is not affected by CFTR. Received: 13 December 2000/Revised: 30 March 2001     

Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells

The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K _Ca ) channel and (iii) a small K⁺ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na⁺ and K⁺, but not permeable to Cl⁻ or N-methyl-d-glucamine. Channel activity required the presence of Ca²⁺ at the cytosolic face, but was detected at Ca²⁺ concentrations as low as 10⁻⁷ m (open probability (P _o ) = 0.11 ± 0.03, n= 8). Gadolinium decreased P _o of the NSC channel from both the external and cytosolic side (IC₅₀∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K⁺ could be fitted to the I-V relationship in asymmetrical K⁺ and Na⁺ solutions. The channel was impermeable to Cl⁻ and N-methyl-d-glucamine. P _o of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca²⁺. TEA (20 mm), charybdotoxin (100 nm) and Ba²⁺ (1 mm) but not amiloride (1 mm) reduced P _o from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P _o and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca²⁺ activated, voltage-insensitive and involved in both constitutive K⁺ and Na⁺ reabsorption from endolymph while the BK channel might participate in the K⁺ pathway under stimulated conditions that produce an elevated intracellular Ca²⁺ or depolarized membrane potential. Received: 14 October 1999/Revised: 10 December 1999     

Effects of Cationic Substitutions on Delayed Rectifier Current in Type I Vestibular Hair Cells

The resting potassium current (I _KI ) in gerbil dissociated type I vestibular hair cells has been characterized under various ionic conditions in whole cell voltage-clamp. When all K⁺ in the patch electrode solution was replaced with Na⁺, (Na⁺) _in or Cs⁺, (Cs⁺) _in , large inward currents were evoked in response to voltage steps between −90 and −50 mV. Activation of these currents could be described by a Hodgkin-Huxley-type kinetic scheme, the order of best fit increasing with depolarization. Above ∼−40 mV currents became outward and inactivated with a monoexponential time course. Membrane resistance was inversely correlated with external K⁺ concentration. With (Na⁺) _in , currents were eliminated when K⁺ was removed from the external solution or following extracellular perfusion of 4-aminopyridine, indicating that currents flowed through I _KI channels. Also, reduction of K⁺ entry through manipulation of membrane potential reduced the magnitude of the outward current. Under symmetrical Cs⁺, 0 K⁺ conditions I _KI is highly permeable to Cs⁺. However, inward currents were reduced when small amounts of external K⁺ were added. Higher concentrations of K⁺ resulted in larger currents indicating an anomalous mole fraction effect in mixtures of external Cs⁺ and K⁺. Received: 23 June 1999/Revised: 27 September 1999     

A Component of Platypus (Ornithorhynchus anatinus) Venom Forms Slow-Kinetic Cation Channels

The lipid bilayer technique is used to examine the biophysical properties of anion and cation channels frequently formed by platypus (Ornithorhynchus anatinus) venom (OaV). The OaV-formed anion channel in 250/50 mm KCl cis/trans has a maximum conductance of 857 ± 23 pS (n= 5) in 250/50 mm KCl cis/trans. The current-voltage relationship of this channel shows strong inward rectification. The channel activity undergoes time-dependent inactivation that can be removed by depolarizing voltage steps more positive than the reversal potential for chloride, E _Cl , (+40 mV). The reversal potential of the OaV-formed slow current activity in 250/50 mm KCl cis/trans is close to the potassium equilibrium potential (E _K ) of −40 mV. The conductance values for the slow channel are 22.5 ± 2.6 pS and 41.38 ± 4.2 pS in 250/50 and 750/50 mm cis/trans, respectively. The gating kinetics of the slow ion channels are voltage-dependent. The channel open probability (P _o ) is between 0.1 and 0.8 at potentials between 0 and +140 mV. The channel frequency (F _o ) increases with depolarizing voltages between 0 and +140 mV, whereas mean open time (T _o ) and mean closed time (T _c ) decrease. Ion substitution experiments of the cis solution show that the channel has conductance values of 21.47 ± 2.3 and 0.53 ± 0.1 pS in 250 mm KCl and choline Cl, respectively. The amplitude of the single channel current is dependent on [K⁺] _cis and the current reversal potential (E _rev ) responds to increases in [K⁺] _cis by shifting to more negative voltages. The increase in current amplitude as a function of increasing [K⁺] _cis can be best described by a third order polynomial fit. At +140 mV, the values of the maximal single channel conductance (γ _max ) and the concentration for half maximal γ (K _s ) are 38.6 pS and 380 mm and decline to 15.76 pS and 250 mm at 0 mV, respectively. The ion selectivity of the channel to K⁺, Na⁺, Cs⁺ and choline⁺ was determined in ion substitution experiments. The permeability values for P _K+ :P _Na+ :P _Cs+ :P _choline+ were 1:1:0.63:0.089, respectively. On the other hand, the activity of the slow channel was eliminated (Fig. 7B). The slow channel was reversibly inhibited by [TEA⁺] _trans and the half-maximal inhibitory concentration (K _i ) was ∼48 mm. Received: 26 April 1999/Revised: 19 July 1999     

Ion Channel Phenotype of Melanoma Cell Lines

Melanoma cells are transformed melanocytes of neural crest origin. K⁺ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify voltage-gated (K_V), Ca²⁺-activated (K_Ca), and inwardly rectifying (K_IR) K⁺ channels; swelling-sensitive Cl⁻ channels (Cl_swell); voltage-gated Ca²⁺ channels (Ca_V) and Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores (CRAC); and voltage-gated Na⁺ channels (Na_V). The presence of Ca²⁺ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca²⁺] _i . The expression of K⁺ channels varied widely between different cell lines and was also influenced by culture conditions. K_IR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for K_IR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). K_Ca channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). K_Ca channels in C8146 and SK28 cells were sensitive to CTX (K _d = 4 nm), but were unaffected by apamin. K_V channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed CRAC channels and a novel Cl_swell channel. Cl_swell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1 in symmetrical Cl⁻). We conclude that different melanoma cell lines express a diversity of ion channel types. Received: 2 April 1996/Revised: 22 August 1996     

Evidence for a Multi-ion Pore Behavior in the Plant Potassium Channel KAT1

KAT1 is a cloned voltage-gated K⁺ channel from the plant Arabidopsis thaliana L., which displays an inward rectification reminiscent of `anomalous' rectification of the i <sub>f</sub> pacemaker current recorded in animal cells. Macroscopic conductance of KAT1 expressed in Xenopus oocytes was 5-fold less in pure Rb<sup>+</sup> solution than in pure K<sup>+</sup> solution, and negligible in pure Na<sup>+</sup> solution. Experiments in different K<sup>+</sup>/Na<sup>+</sup> or K<sup>+</sup>/Rb<sup>+</sup> mixtures revealed deviations from the principle of independence and notably two anomalous effects of the K<sup>+</sup>/Rb<sup>+</sup> mole fraction (i.e., the ratio [K<sup>+</sup>]/([K<sup>+</sup>]+[Rb<sup>+</sup>])). First, the KAT1 deactivation time constant was both voltage- and mole fraction-dependent (a so-called `foot in the door' effect was thus observed in KAT1 channel). Second, when plotted against the K⁺/Rb⁺ mole fraction, KAT1 conductance values passed through a minimum. This minimum is more important for two pore mutants of KAT1 (T259S and T260S) that displayed an increase in P_Rb/P_K. These results are consistent with the idea that KAT1 conduction requires several ions to be present simultaneously within the pore. Therefore, this atypical `green' member of the Shaker superfamily of K⁺ channels further shows itself to be an interesting model as well for permeation as for gating mechanism studies. Received: 9 February 1998/Revised: 28 July 1998     

Plasmalemmal voltage-activated K + currents in protoplasts from tobacco BY-2 cells: possible regulation by actin microfilaments?

Summary.  Plasmalemmal ionic currents from enzymatically isolated protoplasts of suspension-cultured tobacco ‘Bright Yellow-2’ cells were investigated by whole-cell patch-clamp techniques. In all protoplasts, delayed rectifier outward K⁺ currents having sigmoidal activation kinetics, no inactivation, and very slow deactivation kinetics were activated by step depolarization. Tail current reversal potentials were close to equilibrium potential E_K when external [K⁺] was either 6 or 60 mM. Several channel blockers, including external Ba²⁺, niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, inhibited this outward K⁺ current. Among the monovalent cations tested (NH₄ ⁺, Rb⁺, Li⁺, Na⁺), only Rb⁺ had appreciable permeation (P_Rb/P_K ⁼ 0.7). In addition, in 60 mM K⁺ solutions, a hyperpolarization-activated, time-dependent, inwardly rectifying K⁺ current was observed in most protoplasts. This inward current activated very slowly, did not inactivate, and deactivated quickly upon repolarization. The tail current reversal potential was very close to E_K, and other monovalent cations (NH₄ ⁺, Rb⁺, Li⁺, Na⁺) were not permeant. The inward current was blocked by external Ba²⁺ and niflumic acid. External Cs⁺ reversibly blocked the inward current without affecting the outward current. The amplitude of the inward rectifier K⁺ current was generally small compared to the amplitude of the outward K⁺ current in the same cell, although this was highly variable. Similar amplitudes for both currents occurred in only 4% of the protoplasts in control conditions. Microfilament-depolymerizing drugs shifted this proportion to about 12%, suggesting that microfilaments participate in the regulation of K⁺ currents in tobacco ‘Bright Yellow-2’ cells. Received December 7, 2001; accepted April 15, 2002; published online July 4, 2002 RID="*" ID="*" Correspondence and reprints: Pharmacologie et Physicochimie, UMR CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur, 74 route du Rhin, BP 24, 67401 Illkirch, France. Abbreviations: TBY-2 Tobacco ‘Bright Yellow-2’; DHCB dihydrocytochalasin B; I_Kin inward rectifier K⁺ current; I_Kout outward K⁺ current; MFs microfilaments; MTs microtubules; NPPB 5-nitro-2-(3-phenylpropylamino)-benzoic acid.     

The Impermeant Ion Methylammonium Blocks K+ and NH+ 4 Currents through KAT1 Channel Differently: Evidence for Ion Interaction in Channel Permeation

The permeation properties of KAT1, an inward rectifying potassium channel from plant cells, were investigated with different ions in the external medium. With either K⁺, NH⁺ ₄ or methylammonium (MA) in the external solution, the channel, expressed in Xenopus oocytes, appeared permeable to K⁺ and, to a lesser extent, to NH⁺ ₄ but not to the slightly bigger, methylated analogue of NH⁺ ₄, MA. Substituting NH⁺ ₄ for K⁺ shifted the voltage dependency of channel activation further negative and hastened activation kinetics. This suggests that channel operation depends on the transported substrate. In mixed solution (50 mm K⁺, 50 mm MA) MA inhibited K⁺ current in a voltage-independent manner. The maximum block did not exceed 50% of the K⁺ current. In contrast, when NH⁺ ₄ was the permeant ion (50 mm NH⁺ ₄, 50 mm MA) MA caused a voltage-dependent, slowly developing open channel block, achieving complete inhibition at very negative voltages. The latter block could be partially overcome by the addition of K⁺ in the external solution. The data support a model in which ions, after entering the channel pore, compete with different affinities for binding sites on their permeation pathway. Received: 6 October 1997/Revised: 28 January 1998     

Two Distinct K+ Channels in Lamprey (Lampetra fluviatilis) Erythrocyte Membrane Characterized by Single Channel Patch Clamp

Two channels, distinguished by using single-channel patch-clamp, carry out potassium transport across the red cell membrane of lamprey erythrocytes. A small-conductance, inwardly rectifying K⁺-selective channel was observed in both isotonic and hypotonic solutions (osmolarity decreased by 50%). The single-channel conductance was 26 ± 3 pS in isotonic (132 mm K⁺) solutions and 24 ± 2 pS in hypotonic (63 mm K⁺) solutions. No outward conductance was found for this channel, and the channel activity was completely inhibited by barium. Cell swelling activated another inwardly rectifying K⁺ channel with a larger inward conductance of 65 pS and outward conductance of 15 pS in the on-cell configuration. In this channel, rectification was due to the block of outward currents by Mg²⁺ and Ca²⁺ ions, since when both ions were removed from the cytosolic side in inside-out patches the conductance of the channel was nearly ohmic. In contrast to the small-conductance channel, the swelling-activated channel was observed also in the presence of barium in the pipette. Neither type of channel was dependent on the presence of Ca²⁺ ions on the cytosolic side for activity. Received: 18 July 1997/Revised: 30 January 1998     

Activation by Angiotensin II of Ca2+-dependent K+ and Cl− Currents in Zona Fasciculata Cells of Bovine Adrenal Gland

The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These effects were abolished by Losartan (10⁻⁵ m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method, a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau current. In Cl⁻-free solution, the outward current reversed at −78.5 mV, a value close to E _K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K⁺-free solution, the transient outward current, sensitive to Cl⁻ channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E _Cl. Its magnitude changed in the same direction as the driving force for Cl⁻. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca²⁺-free solution (47% of cells) and was reversibly blocked by Cd²⁺ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca²⁺ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin II is due to Ca²⁺-dependent K⁺ and Cl⁻ currents. These two membrane currents are co-activated in response to an internal increase of [Ca²⁺] _i originating from intra- and extracellular stores. Received: 29 May 1997/Revised: 4 November 1997