Protein Kinase C and Regulatory Volume Decrease in Mudpuppy Red Blood Cells

This study examined whether protein kinase C (PKC) stimulates K⁺ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 μm), but not with the cyclic nucleotide-dependent kinase antagonists N-(2′-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 μm) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 μm). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 μm). The effect of these three antagonists and H-7 was reversed with gramicidin (5 μm in a choline Ringer), indicating PKC was linked to K⁺ efflux (gramicidin is a cationophore that was used to ensure a high K⁺ permeability). We also measured cell volume recovery from hypotonic shock (0.5× Ringer) with a Coulter counter and estimated cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 μm), sphingosine (100 nm), chelerythrine (10 μm), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 μm) nor H-8 (5 μm). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 μm). Furthermore, using the patch clamp technique, we found H-7 (10 μm) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K⁺ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K⁺ loss during RVD is regulated, at least in part, by protein kinase C. Received: 20 January 1998/Revised: 2 September 1998     

Ca2+-dependent K+ Channels in Bovine Adrenal Chromaffin Cells are Modulated by Lipoxygenase Metabolites of Arachidonic Acid

Fatty acids play an important role in a variety of physiological processes including ion channel modulation and catecholamine release. Using patch-clamp techniques we show that arachidonic acid (AA) is converted to lipoxygenase metabolites (LOMs) to potentiate activity of the Ca²⁺ and voltage-dependent, large-conductance K⁺ channel (BK) in bovine adrenal medullary chromaffin cells (BAMCCs). AA and LOM potentiation of BK current and recovery from potentiation were unaffected by the nonhydrolyzable ATP analogue AMP-PNP, or by exclusion of nucleotides in excised patch recordings. Also, AA and LOM potentiation of BK channel activity in outside-out patches exposed to strong Ca²⁺ buffering ruled out cytoplasmic messengers or changes in intracellular Ca²⁺ levels as causative factors. Lipoxygenase inhibitor attenuated AA, but not LOM potentiation of BK activity in outside-out patches, indicating that lipoxygenase processing of AA is possible in excised membrane patches, possibly via a membrane associated lipoxygenase. AA and LOM release have been implicated in the mechanics of catecholamine secretion from BAMCCs. By limiting action potential duration and thus voltage-gated Ca²⁺ influx, fatty acid potentiation of BK current may serve an inhibitory feedback function in regulating secretion from BAMCCs. Received: 15 July 1997/Revised: 27 January 1997     

Activation of Maxi-K Channels by Parathyroid Hormone and Prostaglandin E2 in Human Osteoblast Bone Cells

Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuration (CAP) no channel activity was observed in 2 mm Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon addition of 50 nm parathyroid hormone (PTH), 5 nm prostaglandin E₂ (PGE₂) or 0.1 mm dibutyryl cAMP + 1 μm forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that in symmetrical solutions (140 mm K) the channel has a conductance of 246 ± 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (P_K/P_Na) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability (P _o ) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the P _o vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mm ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE₂ or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (⁴⁵Ca⁺⁺) by MG-63 cells was stimulated in the presence of PTH and PGE₂, an effect inhibited by Nitrendipine (10 μm). Second, whereas PGE₂ stimulated the calcium-activated maxi-K channel in 2 mm Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE₂ did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mm Ca Ringer and 10 μm Nitrendipine in CAP configuration, PGE₂ (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of this channel, it is concluded that activation of this channel by PTH, PGE₂ or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx. Received: 17 September 1995/Revised: 7 December 1995     

ADH Action on Whole-Cell Currents by Cytosolic Ca2+-dependent Pathways in Aldosterone-treated A6 Cells

We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca²⁺ concentrations ([Ca²⁺] _c , 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca²⁺] _c , basal conductances mainly consisted of Cl⁻ conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca²⁺] _c to 2 nm abolished the basal Cl⁻ conductance. AVT evoked Cl⁻ conductances at 2 as well as 30 nm [Ca²⁺] _c . In addition to Cl⁻ conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca²⁺] _c but not at 2 nm [Ca²⁺] _c . Thus, cytosolic Ca²⁺ regulates NSC and Cl⁻ conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca²⁺] _c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells. Received: 6 May 1996/Revised: 28 June 1996     

Mechanically Activated Currents in Chick Heart Cells

As predicted from stretch-induced changes of rate and rhythm in the heart, acutely isolated embryonic chick heart cells exhibit whole-cell mechanosensitive currents. These currents were evoked by pressing on cells with a fire polished micropipette and measured through a perforated patch using a second pipette. The currents were carried by Na⁺ and K⁺ but not Cl⁻, and were independent of external Ca²⁺. The currents had linear I/V curves reversing at −16 mV and were completely blocked by Gd³⁺≥ 30 μm and Grammostola spatulata venom at a dilution of 1:1000. Approximately 20% of cells showed time dependent inactivation. In contrast to direct mechanical stimulation, hypotonic volume stress produced an increase in conductance for anions rather than cations—the two stimuli are not equivalent. The cells had two types of stretch-activated ion channels (SACs): a 21 pS nonspecific cation-selective reversing at −2 mV and a 90 pS K⁺ selective reversing at −70 mV in normal saline. The activity of SACs was strongly correlated with the presence of whole-cell currents. Both the whole-cell currents and SACs were blocked by Gd³⁺ and by Grammostola spatulata spider venom. Mechanical stimulation of spontaneously active cells increased the beating rate and this effect was blocked by Gd³⁺. We conclude that physiologically active mechanosensitive currents arise from stretch activated ion channels. Received: 8 April 1996/Revised: 8 August 1996     

Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells

Stimulation with leukotriene D₄ (LTD₄) (3–100 nm) induces a transient increase in the free intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in Ehrlich ascites tumor cells. The LTD₄-induced increase in [Ca²⁺] _i is, however, significantly reduced in Ca²⁺-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD₄ concentration (3 nm) does not result in any detectable increase in [Ca²⁺] _i . Addition of LTD₄ (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD₄-induced (100 nm) acceleration of the RVD response is also seen in Ca²⁺-free medium and also at 3 nm LTD₄, indicating that LTD₄ can open K⁺- and Cl⁻-channels without any detectable increase in [Ca²⁺] _i . Buffering cellular Ca²⁺ with BAPTA almost completely blocks the LTD₄-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca²⁺] _i level after BAPTA-loading or buffering of [Ca²⁺] _i seems to inhibit the LTD₄-induced stimulation of the RVD response even though the LTD₄-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca²⁺] _i . The LTD₄ receptor antagonist L649,923 (1 μm) completely blocks the LTD₄-induced increase in [Ca²⁺] _i and inhibits the RVD response as well as the LTD₄-induced acceleration of the RVD response. When the LTD₄ receptor is desensitized by preincubation with 100 nm LTD₄, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD₄ plays a role in the activation of the RVD response. LTD₄ seems to activate K⁺ and Cl⁻ channels via stimulation of a LTD₄ receptor with no need for a detectable increase in [Ca²⁺] _i . Received: 25 September 1995/Revised: 25 January 1996     

NaF Potentiates a K+-selective Ion Channel in G292 Osteoblastic Cells

Clinical studies have established that NaF increases mineral content in bone, although the cellular mechanisms underlying its osteoinductive effects remain unclear. Because metabolic effects of fluoride have been linked to ion flux and alterations in membrane potential, we used patch-clamp recording techniques to examine the electrophysiological response of osteoblastic cells to NaF. In these experiments, we show that NaF increased the amplitude and P _open of a 73 pS potassium-selective ion channel. The effect of NaF depended on extracellular Ca²⁺ and could be blocked by a combination of calcium-channel blocking agents, suggesting that potentiation of channel activity was dependent on external calcium. Because all patches were in the cell-attached configuration, the effect of NaF was presumably indirect. Although the underlying cellular mechanisms remain unclear, our findings suggest that activity of calcium and/or potassium-selective channels via second messenger cascades may mediate many of the early events involved in the response of bone cells to inorganic fluoride. Received: 30 March 1995/Revised: 13 October 1995     

Influence of Hypo-osmolality on the Activity of Short-Chain Neutral Amino Acid Carriers in Trout (Salmo trutta) Red Blood Cells

The present study shows that in trout red blood cells the activity of some amino acid carriers, not directly involved in cell volume regulation, is affected by external osmolality. Glycine uptake has been used as the experimental approach because it was shown previously that it is effected by different carriers, namely the Na⁺-dependent ASC and Gly systems, as well as the Na⁺-independent asc and L systems. An increase in the uptake through the Gly system and the two Na⁺-independent carriers was found, while the ASC system appeared to be downregulated. Those systems whose activities were increased by hypo-osmolality did not share the mechanism by which this increase was obtained. Thus, the Gly system was sensitive to intracellular ionic changes, while the Na⁺- independent systems were mechanically stimulated, as assessed by the iso-osmotic swelling caused by ammonium chloride. On the other hand, a volume-sensitive transporter may be present in trout red blood cells, which is involved in the swelling-induced glycine movement, as can be deduced from the effect of some inhibitors such as pyridoxal phosphate, DIDS (4,4′-diisothiocyanate-stilbene-2,2′-disulfonic acid) and quinine. Received: 12 February 1996/Revised: 9 September 1996     

Transitional Changes in Membrane Potential and Intracellular [Ca2+] in Rat Basophilic Leukemia Cells

Using whole-cell current-clamp measurements we have found that thapsigargin-mediated activation of store-regulated Ca²⁺ entry in rat basophilic leukemia cells is accompanied by complex changes in membrane potential. These changes consisted of: (i) an initial slow, small depolarization, (ii) a transitional change in potential to a depolarized value and (iii) transitional changes between a hyperpolarized and a depolarized potential. These complex changes in potential can be explained by the interaction between the endogenous inwardly rectifying K⁺ conductance and the generation of a small inward current. To investigate the possible influence of these changes of potential on [Ca²⁺] _i , single cell measurements of fura2 fluorescence were undertaken alone or in combination with current-clamp measurements. Thapsigargin-mediated activation of the store-regulated Ca²⁺ entry pathway was accompanied by a marked increase of [Ca²⁺] _i . During this increase, transient, abrupt declines in [Ca²⁺] _i were detected in approximately 60% of the cells investigated. These changes of [Ca²⁺] _i are consistent with the observed changes of membrane potential recorded under current-clamp. Received: 1 December 1998/Revised: 30 March 1999     

A Nonselective High Conductance Channel in Bovine Pigmented Ciliary Epithelial Cells

An ion channel activated by hyperpolarization was identified in excised patches of bovine pigmented ciliary epithelial cells using the single channel patch clamp technique. In symmetrical NaGluconate, the channel had a maximum conductance of 285 pS. The channel was characterized by frequent flickery transitions between the fully open and closed levels. The channel did not discriminate very clearly between anions and cations; when the cytoplasmic face of excised patches was bathed in a dilute NaCl solution, the channel had a chloride-to-sodium permeability ratio (P _Cl/P _Na) of 1.3. However, the channel showed a small anion selectivity (P _Cl/P _Na= 3.7) when bathed in a concentrated NaCl solution. Gd³⁺ blocked the channel reversibly. Channel kinetics were characterised by slow (≈ min) voltage-dependent activation and inactivation rate constants. The channel was most active in the range −60 to −140 mV and showed a peak at −120 mV. A similar time- and voltage-dependent activation was also observed in cell-attached recordings. In conclusion, hyperpolarization of pigmented ciliary epithelial cell membrane patches activated a large conductance, nonselective ion channel. This combination of nonselectivity and hyperpolarizing activation is consistent with the involvement of this channel in ion loading from the blood into pigmented ciliary epithelial cells—the first phase in the secretion of aqueous humor. Received: 30 June 1995/Revised: 16 November 1995     

Whole-Cell Mechanosensitive Currents in Rat Ventricular Myocytes Activated by Direct Stimulation

Mechanosensitive channels may have a significant role in the development of cardiac arrhythmia following infarction, but the data on mechanical responses at the cellular level are limited. Mechanosensitivity is a ubiquitous property of cells, and although the structure of bacteriological mechanosensitive ion channels is becoming known by cloning, the structure and force transduction pathway in eukaryotes remains elusive. Isolated adult rat ventricular myocytes were voltage clamped and stimulated with a mechanical probe. The probe was set in sinusoidal motion (either in, or normal to, the plane of the cell membrane), and then slowly lowered onto the cell. The sinusoidal frequency was held constant at 1 Hz but the stimulation amplitude was increased and the probe gradually lowered until a mechanically sensitive whole cell current was seen, which usually followed several minutes of stimulation. The whole cell mechanosensitive current in rat cells had two components: (i) a brief large inward current spike current; (ii) a more sustained smaller inward current. The presence of the initial sharp inward current suggests that some structure within the cell either relaxes or is broken, exposing the mechanosensitive element(s) to stress. Metabolic changes induced by continued stress prior to the mechanosensitive response may weaken the elements that break producing the spike, or simple stress-induced fracture of the cytoskeleton itself may occur.     

ClC-2 Activation Modulates Regulatory Volume Decrease

ClC-2 belongs to a large family of chloride channels and its expression in certain cell types is associated with the appearance of swelling-activated chloride (Cl⁻) currents. In the present report, we examined the hypothesis that ClC-2 plays a role in regulatory volume decrease by expressing ClC-2 in Sf9 cells using the baculovirus system. First, we showed that ClC-2 protein expression is associated with appearance of a Cl⁻ conductance which is activated by hypo-osmotic shock and can be distinguished from swelling-activated chloride currents endogenous to Sf9 cells on the basis of its pharmacology and specific inhibition by an anti-ClC-2 antibody. Second, we show that the rate of regulatory volume decrease is significantly enhanced in Sf9 cells expressing ClC-2 protein. Hence, our data support the hypothesis that ClC-2 is capable of mediating regulatory volume decrease. Received: 12 August/Revised: 23 October 1998     

Resistivity of Red Blood Cells Against High-Intensity, Short-Duration Electric Field Pulses Induced by Chelating Agents

The interaction of human red blood cells (RBCs) with diethylenetriamine-pentaacetic acid (DTPA) or its Gd-complex (Magnevist, a widely used clinical magnetic resonance contrast agent containing free DTPA ligands) led to the following, obviously interrelated phenomena. (i) Both compounds protected erythrocytes against electrohemolysis in isotonic solutions caused by a high-intensity DC electric field pulse. (ii) The inhibition of electrohemolysis was observed only when cells were electropulsed in low-conductivity solutions. (iii) The uptake of Gd-DTPA by electropulsed RBCs was relatively low. (iv) (Gd-) DTPA reduced markedly deformability of erythrocytes, as revealed by the electrodeformation experiments using high-frequency electric fields. Taken together, the results indicate that (Gd-) DTPA produce stiffer erythrocytes that are more resistant to electric field exposure. The observed effects of the chelating agents on the mechanical properties and the electropermeabilization of RBCs must have an origin in molecular changes of the bilayer or membrane-coupled cytoskeleton, which, in turn, appear to result from an alteration of the ionic equilibrium (e.g., Ca²⁺ sequestration) in the vicinity of the cell membrane. Received: 19 January 1999/Revised: 1 April 1999     

Regulatory Volume Decrease by SV40-transformed Rabbit Corneal Epithelial Cells Requires Ryanodine-sensitive Ca2+-induced Ca2+ Release

The relationship between relative cell volume and time-dependent changes in intracellular Ca²⁺ concentration ([Ca²⁺] _i ) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (τ _vr ) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca²⁺] _i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca²⁺] _i transients were inhibited by either: (a) Ca²⁺-free medium; (b) 5 mm Ni²⁺ (inhibitor of plasmalemma Ca²⁺ influx); (c) 10 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca²⁺ content); or (d) 100 μm ryanodine (inhibitor of Ca²⁺ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca²⁺ influx and the emptying of intracellular Ca²⁺ stores during the [Ca²⁺] _i transients, Mn²⁺ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn²⁺ permeability 6-fold. However, Mn²⁺ permeability remained unchanged during exposure to either: 1.100 μm ryanodine; 2.10 μm CPA and 100 μm ryanodine. This report for the first time documents the time dependence of the components of the [Ca²⁺] _i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca²⁺ release from an intracellular store leads to a subsequent increase in plasmalemma Ca²⁺ influx, and that both are required for cells to undergo RVD. Received: 7 November 1996/Revised: 6 January 1997     

Uptake of L-leucine by Trout Red Blood Cells and Peripheral Lymphocytes

The uptake of l-leucine by trout red blood cells and peripheral lymphocytes has been analyzed. The present study shows two functionally different Na⁺-independent systems for apolar branched-chain amino acids. They are designated as L systems because they share some properties with the mammalian L system. The carrier present in red blood cells has low K _m values, is trans-stimulable and not stereospecific for leucine uptake; on the other hand, the system present in lymphocytes is stereospecific for leucine uptake and trans-inhibitable. Both carriers are pH sensitive in a similar fashion at low pHs, but there are important differences at higher pH values (above neutrality). These properties are compared with these of the asc systems previously reported in these cells. Received: 2 June 1995/Revised: 7 March 1996     

Regulation of Cation-selective Channels in Liver Cells

In liver cells, cation-selective channels are permeable to Ca²⁺ and have been postulated to represent a pathway for receptor-mediated Ca²⁺ influx. This study examines the mechanisms involved in the regulation of these channels in a model liver cell line. Using patch-clamp recording techniques, it is shown that channel open probability is a saturable function of cytosolic [Ca²⁺], with half-maximal opening at 660 nm. By contrast, channel opening is not affected by membrane voltage or cytosolic pH. In intact cells, reduction of cytosolic [Cl⁻], a physiological response to Ca²⁺-mobilizing hormones and cell swelling, is also associated with an increase in channel opening. Finally, channel opening is inhibited by intracellular ATP through a mechanism that does not involve ATP hydrolysis. These findings suggest that opening of cation-selective channels is coupled to the metabolic state of the cell and provides a positive feedback mechanism for regulation of receptor-mediated Na⁺ and Ca²⁺ influx. Received: 8 October 1996     

Effects of Na+ on the Predominant K+ Channel in the Tonoplast of Chara: Decrease of Conductance by Blocks in 100 Nanosecond Range and Induction of Oligo- or Poly-subconductance Gating Modes

We present three mechanisms by which Na⁺ inhibits the open channel currents of the predominant K⁺ channel in the tonoplast of Chara corallina: (i) Fast block, i.e., short (100 ns range) interruptions of the open channel current which are determined by open channel noise analysis, (ii): Oligo-subconductance mode, i.e., a gating mode which occurs preferentially in the presence of Na⁺; this mode comprises a discrete number (here 3) of open states with smaller conductances than normal, and (iii): Polysubconductance mode, i.e., a gating mode with a nondiscrete, large number (>30) of states with smaller conductances than the main open channel conductance. This novel mode has also been observed only in the presence of Na⁺. Received: 16 November 1999/Revised: 8 February 2000     

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     

Modulation of I A Potassium Current in Adrenal Cortical Cells by a Series of Ten Lanthanide Elements

The modulation of I _A K⁺ current by ten trivalent lanthanide (Ln³⁺) cations spanning the series with ionic radii ranging from 0.99 ? to 1.14 ? was characterized by the whole-cell patch clamp technique in bovine adrenal zona fasciculata (AZF) cells. Each of the ten Ln³⁺s reduced I _A amplitude measured at +20 mV in a concentration-dependent manner. Smaller Ln³⁺s were the most potent and half-maximally effective concentrations (EC₅₀s) varied inversely with ionic radius for the larger elements. Estimation of EC₅₀s yielded the following potency sequence: Lu³⁺ (EC₅₀= 3.0 μm) ≈ Yb³⁺ (EC₅₀= 2.7 μm) > Er³⁺ (EC₅₀= 3.7 μm) ≥ Dy³⁺ (EC₅₀= 4.7 μm) > Gd³⁺ (EC₅₀= 6.7 μm) ≈ Sm³⁺ (EC₅₀= 6.9 μm) > Nd³⁺ (EC₅₀= 11.2 μm) > Pr³⁺ (EC₅₀= 22.3 μm) > Ce³⁺ (EC₅₀= 28.0 μm) > La³⁺ (EC₅₀= 33.7 μm). Ln³⁺s altered selected voltage-dependent gating and kinetic parameters of I _A with a potency and order of effectiveness that paralleled the reduction of I _A amplitude. Ln³⁺s markedly slowed activation kinetics and shifted the voltage-dependence of I _A gating such that activation and steady-state inactivation occurred at more depolarized potentials. In contrast, Ln³⁺s did not measurably alter inactivation or deactivation kinetics and only slightly slowed kinetics of inactivated channels returning to the closed state. Replacement of external Ca²⁺ with Mg²⁺ had no effect on the concentration-dependent inhibition of I _A by Ln³⁺s. In contrast to their action on I _A K⁺ current, Ln³⁺s inhibited T-type Ca²⁺ currents in AZF cells without slowing activation kinetics. These results indicate that Ln³⁺ modulate I _A K⁺ channels through binding to a site on I _A channels located within the electric field but which is not specific for Ca²⁺. They are consistent with a model where Ln³⁺ binding to negative charges on the gating apparatus alters the voltage-dependence and kinetics of channel opening. Ln³⁺s modulate transient K⁺ and Ca²⁺ currents by two fundamentally different mechanisms. Received: 21 January 1997/Revised: 3 April 1998     

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

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