The ACh-evoked,Ca2+-activated Whole-cell K+ Current in Mouse Mandibular Secretory Cells. Whole-cell and Fluorescence Studies

In our previous studies on sheep parotid secretory cells, we showed that the K⁺ current evoked by acetylcholine (ACh) was not carried by the high-conductance voltage- and Ca²⁺-activated K⁺ (BK) channel which is so conspicuous in unstimulated cells, notwithstanding that the BK channel is activated by ACh. Since several studies from other laboratories had suggested that the BK channel did carry the ACh-evoked K⁺ current in the secretory cells of the mouse mandibular gland, and that the current could be blocked with tetraethylammonium (TEA), a known blocker of BK channels, we decided to investigate the ACh-evoked K⁺ current in mouse cells more closely. We studied whether the ACh-evoked K⁺ current in the mouse is inhibited by TEA and quinine. Using the whole-cell patch-clamp technique and microspectrofluorimetric measurement of intracellular Ca²⁺, we found that TEA and quinine do inhibit the ACh-evoked K⁺ current but that the effect is due to inhibition of the increase in intracellular Ca²⁺ evoked by ACh, not to blockade of a K⁺ conductance. Furthermore, we found that the K⁺ conductance activated when ionomycin is used to increase intracellular free Ca²⁺ was inhibited only by quinine and not by TEA. We conclude that the ACh-evoked K⁺ current in mouse mandibular cells does not have the blocker sensitivity pattern that would be expected if it were being carried by the high-conductance, voltage- and Ca²⁺-activated K⁺ (BK) channel. The properties of this current are, however, consistent with those of a 40 pS K⁺ channel that we have reported to be activated by ACh in these cells [16]. Received: 9 January 1996/Revised: 17 April 1996     

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     

The ACh-induced whole-cell currents in sheep parotid secretory cells. Do BK channels really carry the ACh-evoked whole-cell K+ current?

As in other salivary glands, the secretory cells of the sheep parotid have a resting K⁺ conductance that is dominated by BK channels, which are activated by acetylcholine (ACh) and are blocked by tetraethylammonium (TEA). Nevertheless, perfusion studies indicate that TEA does not inhibit ACh-evoked fluid secretion or K⁺ efflux from intact sheep parotid glands. In the present study, we have used whole-cell patch clamp techniques to show that ACh activates K⁺ and Cl^– conductances in sheep parotid secretory cells by increasing intracellular free Ca²⁺, and we have compared the blocker sensitivity of the ACh-evoked whole-cell K⁺ current to the previously reported blocker sensitivity of the BK channels seen in these cells.The ACh-induced whole-cell K⁺ current was not blocked by TEA (10 mmol/l) or verapamil (100 mol/l), both of which block the resting K⁺ conductance and inhibit BK channels in these cells. Quinine (1 mmol/l) and quinidine (1 mmol/l), although only weak blockers of the resting K⁺ conductance, inhibited the ACh-evoked current at 0 mV (K⁺ current), by 68% and 78%, respectively. 4-Aminopyridine (10 mmol/l) partially inhibited the ACh-induced K⁺ current and caused it to fluctuate. It also caused the resting membrane currents to fluctuate, possibly by altering cytosolic free Ca²⁺. Ba²⁺ (100 mol/l), a blocker of the inwardly rectifying K⁺ conductance in sheep parotid cells, had no effect on the ACh-induced K⁺ current.We conclude that the ACh-induced K⁺ conductance in sheep parotid cells is pharmacologically distinct from both the outwardly rectifying (BK) K⁺ conductance and the inwardly rectifying K⁺ conductance seen in unstimulated cells. Given that in vitro perfusion and K⁺ efflux studies on other salivary glands in which BK channels dominate the resting conductance (e.g., the rat mandibular, rat parotid and mouse mandibular glands) have revealed an insensitivity to TEA, suggesting that BK channels do not carry the ACh-evoked K⁺ current, we propose that BK channels do not contribute substantially to the K⁺ current evoked by ACh in the secretory cells of most salivary glands.This project was supported by the Australian Research Council. We thank Dr. N. Sangster, Dr. J. Rothwell and Mr. R. Murphy for giving us access to their sheep.     

Properties and the Cytoskeletal Control of Ca++-independent Large Conductance K+ Channels in Neonatal Rat Hippocampal Neurons

A member of the family of Ca⁺⁺-independent large conductance K⁺ channels (termed BK channels) was identified in patch clamp experiments with cultured neonatal rat hippocampal neurons. Permeation was characterized (at 5 mmol/l external, 140 mmol/l internal K⁺; 135 mmol/l external Na⁺) by a conductance of 107 pS, a ratio P_Na/P_K∼ 0.01, and outward rectification near the reversal potential. Channel activity was not voltage-dependent, could not be reduced by internal TEA or by a shift of internal pH from 7.4 to 6.8, i.e., discriminating features within the Ca⁺⁺-independent BK channel family. Cytosolic proteolysis abolished the functional state of hippocampal Ca⁺⁺-independent BK channels, in contrast to the pronase resistance of hippocampal Ca⁺⁺-activated BK channels which suggests structural dissimilarities between these related channels. Cytoskeletal alterations had an activating influence on Ca⁺⁺-independent BK channels and caused a 3–4-fold rise in P _o , but patch excision and channel isolation from the natural environment provoked the strongest increase in P _o , from 0.07 ± 0.03 to 0.73 ± 0.04. This activation process operated slowly, on a minute time scale and can be most easily explained with the loss of a membrane-associated inhibitory particle. Once activated, Ca⁺⁺-independent BK channels reacted sensitively to a Mg-ATP supplemented brain tissue extract with a P _o decline, from 0.60 ± 0.06 to 0.10 ± 0.05. Heated extracts failed to induce significant channel inhibition, providing evidence for a heat-unstable molecule with reassociates with the internal channel surface to reestablish channel inhibition. A dualistic channel control, by this membrane-associated molecule and by the cytoskeleton seems possible. Received: 16 July 1997/Revised: 3 November 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 the Driving Force as a Modulator of Gating in Cardiac ATP-sensitive K+ Channels — Evidence for Specific Elementary Properties

Single cardiac ATP-sensitive K⁺ channels and, comparatively, two other members of the inwardly rectifying K⁺ channel family, cardiac K⁺ _(ir) and K⁺ _(ACh) channels, were studied in the inside-out recording mode in order to analyze influence and significance of the electrochemical K⁺ gradient for open-state kinetics of these K⁺ channels. The conductive state of K⁺ _(ATP) channels was defined as a function of the electrochemical K⁺ gradient in that increased driving force correlates with shortened open-channel lifetime. Flux coupling of gating can be largely excluded as the underlying mechanism for two reasons: (i) τ_open proved identical in 23 pS, 56 pS and 80 pS channels; (ii) K⁺ _(ATP) channel protonation by an external pH shift from 9.5 to 5.5 reduced conductance without a concomitant detectable change of τ_open. Since gating continued to operate at E _K , i.e., in the absence of K⁺ permeation through the pore, K⁺ driving force cannot be causally involved in gating. Rather the driving force acts to modulate the gating process similar to Rb⁺ whose interference with an externally located binding site stabilizes the open state. In K⁺ _(ir) and K⁺ _(ACh) channels, the open state is essentially independent on driving force meaning that their gating apparatus does not sense the electrochemical K⁺ gradient. Thus, K⁺ _(ATP) channels differ in an important functional aspect which may be tentatively explained by a structural peculiarity of their gating apparatus. Received: 24 March 1997/Revised: 24 April 1998     

Small Inward Rectifying K+ Channels in Coleoptiles: Inhibition by External Ca2+ and Function in Cell Elongation

Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles. Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex. These currents are essentially mediated by K⁺ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage and inhibited by extracellular Ca²⁺ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca²⁺-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive states via independent first order reactions: a fast inactivation/activation by Ca²⁺-binding and -release, respectively (rate constants >>10³ sec⁻¹) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the range of 10³ sec⁻¹). With 10 mm K⁺ and 1 mm Ca²⁺ in the external solution, intact coleoptile cells have a membrane voltage (V) of −105 ± 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K⁺ uptake required for cell elongation. Extracellular TEA or Ca²⁺, which inhibit the K⁺ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects of Ca²⁺ and TEA on both processes and the similar Ca²⁺ concentration required for half maximal inhibition of growth (4.3 mm Ca²⁺) and for conductance (1.2 mm Ca²⁺) suggest that K⁺ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles. Received: 6 June 1995/Revised: 12 September 1995     

Activation of inwardly rectifying potassium channels by muscarinic receptor-linked G protein in isolated human ventricular myocytes

Muscarinic receptor-linked G protein, G _i , can directely activate the specific K⁺ channel (I _K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G _i to the K⁺ channel in the ventricle has not been well defined. G protein regulation of K⁺ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization, mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I _K(ACh) and/or the background inwardly-rectifying K⁺ channel (I _K1) in ventricular cell membranes. I _K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I _K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I _K(ACh), G protein-activated I _K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I _K(ACh) and 1.2 μm in I _K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic receptor-linked PTX-sensitive G protein, G _i , is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action potential by activating I _K(ACh) and I _K1 via muscarinic receptor-linked G _i proteins in human ventricular myocytes. Received: 23 September 1996/Revised: 18 December 1996     

Clustered Distribution of Calcium Sensitivities: An Indication of Hetero-tetrameric Gating Components in Ca2+-activated K+ Channels Reconstituted from Avian Nasal Gland Cells

High-conductance, Ca²⁺-activated K⁺ channels from the basolateral membrane of rabbit distal colon epithelial cells were reconstituted into planar phospholipid bilayers to examine the effect of Mg²⁺ on the single-channel properties. Mg²⁺ decreases channel current and conductance in a concentration-dependent manner from both the cytoplasmic and the extracellular side of the channel. In contrast to other K⁺ channels, Mg²⁺ does not cause rectification of current through colonic Ca²⁺-activated K⁺ channels. In addition, cytoplasmic Mg²⁺ decreases the reversal potential of the channel. The Mg²⁺-induced decrease in channel conductance is relieved by high K⁺ concentrations, indicating competitive interaction between K⁺ and Mg²⁺. The monovalent organic cation choline also decreases channel conductance and reversal potential, suggesting that the effect is unspecific. The inhibition of channel current by Mg²⁺ and choline most likely is a result of electrostatic screening of negative charges located superficially in the channel entrance. But in addition to charge, other properties appear to be necessary for channel inhibition, as Na⁺ and Ba²⁺ are no (or only weak) inhibitors. Mg²⁺ and possibly other cations may play a role in the regulation of current through these channels. Received: 25 August 1995/Revised: 16 November 1995     

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     

Reconstitution in Lipid Bilayers of an ATP-Sensitive K+ Channel from Pig Coronary Smooth Muscle

A K⁺ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid bilayers. This channel was identified as an ATP-sensitive K⁺ channel (K_ATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na₂. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0 mV and with a 5/250 KCl gradient, the main conductance of the K_ATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels. In general, K_ATP channels were characterized by brief openings followed by long closings (open probability, P _o ≈ 0.02); only occasionally (3 out of 12 experiments) did the K_ATP channels have a high open probability (P _o ≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl₂ to the internal side of the channel. Received: 7 November 1995/Revised: 10 June 1996     

Large Conductance Ca2+-activated K+ Channels in the Soma of Rat Motoneurones

Properties of large conductance Ca²⁺-activated K⁺ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K⁺ _o //155 mm K⁺ _i ) and 231 ± 4 pS in external high-K _o solution (155 mm K⁺ _o //155 mm K⁺ _i ). The channels were activated by depolarization and by an increase in internal Ca²⁺ concentration. Potentials of half-maximum channel activation (E₅₀) were −13, −34, −64 and −85 mV in the presence of 10⁻⁶, 10⁻⁵, 10⁻⁴ and 10⁻³ m internal Ca²⁺, respectively. Using an internal solution containing 10⁻⁴ m Ca²⁺, averaged K_Ca currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged K_Ca currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with a 50% blocking concentration (IC₅₀) of 0.17 ± 0.02 mm. K_Ca channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that K_Ca channels activated by Ca²⁺ entry during the action potential play an important role in the excitability of motoneurones. Received: 7 November 1996/Revised: 29 October 1997     

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     

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     

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     

Control of the Amiloride-Sensitive Na+ Current in Salivary Duct Cells by Extracellular Sodium

We have previously reported that intralobular salivary duct cells contain an amiloride-sensitive Na⁺ conductance (probably located in the apical membranes). Since the amiloride-sensitive Na⁺ conductances in other tight epithelia have been reported to be controlled by extracellular (luminal) Na⁺, we decided to use whole-cell patch clamp techniques to investigate whether the Na⁺ conductance in salivary duct cells is also regulated by extracellular Na⁺. Using Na⁺-free pipette solutions, we observed that the whole-cell Na⁺ conductance increased when the extracellular Na⁺ was increased, whereas the whole-cell Na⁺ permeability, as defined in the Goldman equation, decreased. The dependency of the whole-cell Na⁺ conductance on extracellular Na⁺ could be described by the Michaelis-Menten equation with a K _m of 47.3 mmol/1 and a maximum conductance (G _max) of 2.18 nS. To investigate whether this saturation of the Na⁺ conductance with increasing extracellular Na⁺ was due to a reduction in channel activity or to saturation of the single-channel current, we used fluctuation analysis of the noise generated during the onset of blockade of the Na⁺ current with 200 μmol/l 6-chloro-3,5-diaminopyrazine-2-carboxamide. Using this technique, we estimated the single channel conductance to be 4 pS when the channel was bathed symmetrically in 150 mmol/l Na⁺ solutions. We found that Na⁺ channel activity, defined as the open probability multiplied by the number of available channels, did not alter with increasing extracellular Na⁺. On the other hand, the single-channel current saturated with increasing extracellular Na⁺ and, consequently, whole-cell Na⁺ permeability declined. In other words, the decline in Na⁺ permeability in salivary duct cells with increasing extracellular Na⁺ concentration is due simply to saturation of the single-channel Na⁺ conductance rather than to inactivation of channel activity. Received: 27 July 1995/Revised: 7 December 1995     

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     

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     

An inwardly rectifying potassium channel in the basolateral membrane of sheep parotid secretory cells

Summary Using whole-cell patch-clamp techniques, we demonstrate that sheep parotid secretory cells have both inwardly and outwardly rectifying currents. The outwardly rectifying current, which is blocked by 10 mmol/liter tetraethylammonium (TEA) applied extracellularly, is probably carried by the 250 pS Ca²⁺-and voltage-activated K⁺ (BK) channel which has been described in previous studies. In contrast, the inwardly rectifying current, which is also carried by K⁺ ions, is not sensitive to TEA. It is similar to the inwardly rectifying currents observed in many excitable tissues in that (i) its conductance is dependent on the square root of the extracellular K⁺, (ii) the voltage range over which it is activated is influenced by the extracellular K⁺ concentration and (iii) it is blocked by the addition of Cs⁺ ions (670 µmol/liter) to the bathing solution. Our previously published cell-attached patch studies have shown that the channel type most commonly observed in the basolateral membrane of unstimulated sheep parotid secretory cells is a K⁺ channel with a conductance of 30 pS and, in this study, we find that its conductance also depends on the square root of the extracellular K⁺ concentration. It thus seems likely that it carries the inwardly rectifying K⁺ current seen in the whole-cell studies.     

Anion Channels in Chara corallina Tonoplast Membrane: Calcium Dependence and Rectification

Tonoplast K⁺ channels of Chara corallina are well characterized but only a few reports mention anion channels, which are likely to play an important role in the tonoplast action potential and osmoregulation of this plant. For experiments internodal cells were isolated. Cytoplasmic droplets were formed in an iso-osmotic bath solution according to a modified procedure. Ion channels with conductances of 48 pS and 170 pS were detected by the patch-clamp technique. In the absence of K⁺ in the bath solution the 170 pS channel was not observed at negative pipette potential values. When Cl⁻ on either the vacuolar side or the cytoplasmic side was partly replaced with F⁻, the reversal potential of the 48 pS channel shifted conform to the Cl⁻ equilibrium potential with similar behavior in droplet-attached and excised patch mode. These results showed that the 48 pS channel was a Cl⁻ channel. In droplet-attached mode the channel rectified outward current flow, and the slope conductance was smaller. When Chara droplets were formed in a bath solution containing low (10⁻⁸ m) Ca²⁺, then no Cl⁻ channels could be detected either in droplet-attached or in inside-out patch mode. Channel activity was restored if Ca²⁺ was applied to the cytoplasmic side of inside-out patches. Rectification properties in the inside-out patch configuration could be controlled by the holding pipette potential. Holding potential values negative or positive to the calculated reversal potential for Cl⁻ ions induced opposite rectification properties. Our results show Ca²⁺-activated Cl⁻ channels in the tonoplast of Chara with holding potential dependent rectification. Received: 30 March 1999/Revised: 10 August 1999