Activation of a Ca2+-dependent K+ current by the oncogenic receptor protein tyrosine kinase v-Fms in mouse fibroblasts

We investigated the effects of the receptor-coupled protein tyrosine kinase (RTK) v-Fms on the membrane current properties of NIH3T3 mouse fibroblasts. We found that v-Fms, the oncogenic variant of the macrophage colony-stimulating factor receptor c-Fms, activates a K⁺ current that is absent in control cells. The activation of the K⁺ current was Ca²⁺-dependent, voltage-independent, and was completely blocked by the K⁺ channel blockers charybdotoxin, margatoxin and iberiotoxin with IC₅₀ values of 3nM, 18 nM and 76nM, respectively. To identify signalling components that mediate the activation of this K⁺ current, NIH3T3 cells that express different mutants of the wildtype v-Fms receptor were examined. Mutation of the binding site for the Ras-GTPase-activating protein led to a complete abolishment of the K⁺ current. A reduction of 76% and 63%, respectively, was observed upon mutation of either of the two binding sites for the growth factor receptor binding protein 2. Mutation of the ATP binding lobe, which disrupts the protein tyrosine kinase activity of v-Fms, led to a 55% reduction of the K⁺ current. Treatment of wild-type v-Fms cells with Clostridium sordellii lethal toxin or a farnesyl protein transferase inhibitor, both known to inhibit the biological function of Ras, reduced the K⁺ current amplitude to 17% and 6% of the control value, respectively. This is the first report showing that an oncogenic RTK can modulate K⁺ channel activity. Our results indicate that this effect is dependent on the binding of certain Ras-regulating proteins to the v-Fms receptor and is not abolished by disruption of its intrinsic protein tyrosine kinase activity. Furthermore, our data suggest that Ras plays a key role for K⁺ channel activation by the oncogenic RTK v-Fms. Received: 19 November 1997 / Accepted: 21 January 1998     

Fast,persistent, Ca2+-dependent K+ current controls graded electrical activity in crayfish muscle

The early outward current in opener muscle fibres of crayfish (Procambarus clarkii) was studied using the two-electrode voltage-clamp technique. This current was abolished in Ca²⁺-free and 5 mM Cd²⁺ solutions, and was blocked by extra- or intracellular tetraethylammonium, indicating that it was a Ca²⁺-dependent K⁺ current [I _K(Ca)]. I _K(Ca) was voltage dependent, apamin insensitive and sensitive to charybdotoxin (CTX), which, in addition to its tetraethylammonium sensitivity, suggests that the channels mediating I _K(Ca) behave in a BK type manner. I _K(Ca) activation was extremely fast, reaching a maximum within 5 ms, and the inactivation was incomplete, stabilizing at a persistent steady-state. I _K(Ca) was insensitive to intracellular ethylenebis(oxonitrilo)tetraacetate (EGTA), but was abolished by injection of the faster Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid (BAPTA), suggesting that voltage-dependent Ca²⁺ channels and those mediating I _K(Ca) should be clustered closely on the membrane. Under two-electrode current-clamp recording mode, low amplitude, graded responses were evoked under control conditions, whereas repetitive all-or-none spikes were elicited by application of CTX or after loading the cells with BAPTA. We conclude that I _K(Ca) activates extremely quickly, is persistent and is responsible for the generation and control of the low amplitude, graded, active responses of opener muscle fibres.     

A small-conductance charybdotoxin-sensitive,apamin-resistant Ca2+-activated K+ channel in aortic smooth muscle cells (A7r5 line and primary culture)

A small conductance K⁺ channel was identified in smooth muscle cells of the rat aortic cell line A7r5 and also in rat aortic smooth muscle cells in primary culture, using conventional single-channel recording techniques. The single-channel conductance shows no rectification, either in the range –70 to +40 mV under asymmetrical conditions (9.1 pS), or in the range –100 to +50 mV in symmetrical 150 mM K⁺ (37 pS). Channel activity is reversibly inhibited by extracellular application of charybdotoxin, with a concentration of 8 nM producing half-maximal inhibition. It is unaffected by apamin or scyllatoxin. Channel activity depends on the presence of free Ca²⁺ on the cytosolic face of the membrane, with an activation zone between 0.1 and 1 M. This small-conductance, charybdotoxin-sensitive, Ca²⁺-regulated K⁺ channel is activated by vasoconstrictors such as vasopressin and endothelin.     

The effect of racemic ketamine on the large conductance Ca-activated potassium (BK) channels in GH3 cells

Recently, inhalation anesthetics have been reported to block BK channels in adrenal chromaffin cells. To determine if BK block was characteristic only of inhalation anesthetics or was also a property of other general anesthetics we examined the effects of ketamine, an intravenous general anesthetic which is structurally different than inhalation anesthetics. Cell-attached and excised patch single channel and standard whole cell recording techniques were used to examine the effect of racemic ketamine on the BK channel activity in GH₃ cells. When solutions containing 150 mM KCl are used in both the pipette and bath, the BK channels are characterized as a voltage-dependent channel with a unit conductance of 150–300 pS. Racemic ketamine (at clinically relevant concentrations; 2–500 μM) selectively blocked BK channels in a dose-dependent, reversible manner as evidenced by decreases in NP_o (number of channels × open probability). This decrease was due to both a decrease in mean open time and an increase in the mean closed time but without a decrease in single-channel current amplitude. Ketamine shifts the P_o vs voltage curve to higher potentials without a change in the slope of the voltage dependence. Ketamine also shifts the P_o vs [Ca⁺²] relationship to higher Ca⁺² concentrations. The IC₅₀ for the single-channel block by ketamine is 20.3 ± 15.9 μM. In an effort to confirm that the effect of ketamine was predominantly due to a block of the BK channels, standard whole cell techniques were utilized. As with the single-channel experiments, ketamine (2–500 μM) produced a dose-dependent, voltage-independent and reversible decrease in outward current with an IC₅₀ of 23.7 ± 11.5 μM. Addition of 100 μM ketamine to cells pretreated with the BK channel blocker, charybdotoxin (ChTX), did not result in a further decrease in outward current. These results demonstrate a selective effect of ketamine at clinically relevant concentrations which is consistent with results reported for inhalation anesthetics.     

Immunomodulatory effects of diclofenac in leukocytes through the targeting of Kv1.3 voltage-dependent potassium channels

Kv1.3 plays a crucial role in the activation and proliferation of T-lymphocytes and macrophages. While Kv1.3 is responsible for the voltage-dependent potassium current in T-cells, in macrophages this K⁺ current is generated by the association of Kv1.3 and Kv1.5. Patients with autoimmune diseases show a high number of effector memory T cells that are characterized by a high expression of Kv1.3 and Kv1.3 antagonists ameliorate autoimmune disorders in vivo. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) used in patients who suffer from painful autoimmune diseases such as rheumatoid arthritis. In this study, we show that diclofenac impairs immune response via a mechanism that involves Kv1.3. While diclofenac inhibited Kv1.3 expression in activated macrophages and T-lymphocytes, Kv1.5 remained unaffected. Diclofenac also decreased iNOS levels in Raw 264.7 cells, impairing their activation in response to lipopolysaccharide (LPS). LPS-induced macrophage migration and IL-2 production in stimulated Jurkat T-cells were also blocked by pharmacological doses of diclofenac. These effects were mimicked by Margatoxin, a specific Kv1.3 inhibitor, and Charybdotoxin, which blocks both Kv1.3 and Ca²⁺-activated K⁺ channels (K_Ca3.1). Because Kv1.3 is a very good target for autoimmune therapies, the effects of diclofenac on Kv1.3 are of high pharmacological relevance.     

Immature properties of large-conductance calcium-activated potassium channels in rat neuroepithelium

The pharmacological and biophysical properties of large-conductance Ca-activated K (BK) channels from embryonic rat telencephalic neuroepithelium were investigated with in situ patch-clamp techniques. A fraction of these channels exhibited properties characteristic of BK channels recorded in well differentiated cells, including normal gating mode (BK_N channels). The vast majority of BK channels expressed distinctive properties, the most conspicuous being their buzz gating mode (BK_B channels). BK_B channels were insensitive to a concentration of charybdotoxin that completely and reversibly blocked BK_N channels. In contrast with the strict dependence of BK_N channel activation on cytoplasmic Ca, BK_B channels displayed substantially high open probability (P _o) after inside-out patch excision in a Ca-free medium. Intracellular trypsin down-regulated the P _o of BK_B channels, which then exhibited a greater sensitivity to cytoplasmic Ca, mainly in the positive direction (increased P _o with increased Ca). This suggested a modulatory role for Ca as opposed to its gating role in BK_N channels. Ca ions also reduced current amplitude of both types of channels. BK_B channels were less voltage sensitive than BK_N channels, but this was not correlated with their lower Ca sensitivity. We speculate that BK_B channels may represent immature forms in the developmental expression of BK channels. Received: 1 August 1995 /Received after revision: 24 October 1995 /Accepted: 25 October 1995     

Differential actions of charybdotoxin on central and daughter branch arteries of the rabbit isolated ear

1. By use of rabbit isolated perfused intact ears and isolated perfused segments of central and first generation daughter branch ear arteries, we investigated the actions of charybdotoxin (ChTX), a blocker of calcium-activated K⁺ channels (K_Ca channels), and N^ω-nitro-L-arginine methyl ester (L-NAME) on pressure-flow and diameter-flow relationships.
2. ChTX (1 nM) induced an upwards shift in the pressure-flow curve in the rabbit intact isolated ear preconstricted with 5-hydroxytryptamine (5-HT; 100 nM) with subsequent administration of L-NAME (100 μM) inducing a further upwards shift. L-NAME itself induced an upwards shift in the pressure-flow curve, but subsequent administration of ChTX was without significant effect.
3. Microangiographic analysis revealed a tendency of ChTX (1 nM) to decrease vessel diameter in the central ear artery (G₀) with little effect on the first two generations of daughter branch arteries (G₁ and G₂) in the intact ear. Subsequent addition of L-NAME (100 μM) did not significantly further decrease vessel diameter in G₀, but did decrease vessel diameter in G₁ and G₂. L-NAME itself showed a tendency to decrease vessel diameter in G₀, G₁ and G₂ vessels with subsequent addition of ChTX being without significant effect.
4. In an isolated G₀ preparation which was preconstricted with 5-HT (100 nM), ChTX (1 nM) caused an upwards shift in the pressure-flow curve which was augmented by subsequent addition of L-NAME (100 μM). L-NAME (100 μM) itself caused an upwards shift in the pressure-flow curve but subsequent addition of ChTX (1 nM) had no significant effect.
5. In comparison, in an isolated G₁ preparation which was preconstricted with 5-HT (100 nM), ChTX (1 nM) had no significant effect on the pressure-flow curve relative to control, but subsequent addition of L-NAME (100 μM) caused an upwards shift. L-NAME (100 μM) itself induced an upwards shift in the pressure-flow curve with subsequent addition of ChTX (1 nM) being without significant effect.
6. ChTX (10 pM–10 nM) caused a concentration-dependent increase in perfusion pressure in isolated G₀ and G₁ preparations at fixed flow rates of 2 ml min⁻¹ and 0.5 ml min⁻¹, respectively. These responses were enhanced in the presence of L-NAME (100 μM) in G₁ but not G₀ preparations.
7. We conclude that at 1 nM, ChTX exhibits differential actions on central and daughter branch arteries of the intact ear of the rabbit, which are also apparent in the corresponding arteries when studied in isolation. The action of 1 nM ChTX in G₀ vessels may reflect inhibition of either the release or action of nitric oxide as it was blocked in the presence of L-NAME. At higher concentrations of ChTX, there would appear to be a direct constrictor effect on vascular smooth muscle which is apparent in both G₀ and G₁ vessels. This observed heterogeneity could reflect different distributions of K_Ca channels between central and daughter branch arteries at either the endothelial or smooth muscle levels, or both.

     

Migration of transformed renal epithelial cells is regulated by K+ channel modulation of actin cytoskeleton and cell volume

Migration of transformed renal epithelial (MDCK-F) cells depends on the polarized activity of a Ca²⁺-sensitive K⁺ channel (IK channel; Pflügers Arch 432:R87–R93, 1996). This study was aimed at elucidating the functional link between the IK channel and the actin cytoskeleton which is required for cell locomotion. We monitored migration of MDCK-F cells with video microscopy, quantified filamentous actin with phalloidin binding, and measured the intracellular Ca²⁺ concentration ([Ca²⁺]_i) with the fluorescent dye fura-2/AM. We compared the effects of IK channel activation or inhibition with those of hypotonic swelling or hypertonic shrinkage. IK channel inhibition with charybdotoxin (CTX) or cell swelling (omission of up to 50 mmol/l NaCl) as well as IK channel activation with 1-ethyl-2-benzimidazolinone (1-EBIO) or cell shrinkage (addition of up to 100 mmol/l mannitol) reduce the rate of migration dose-dependently by up to 80%, i.e., to the same extent as cytochalasin D. Inhibition of migration is accompanied either by actin depolymerization (CTX and cell swelling) or by actin polymerization (1-EBIO and cell shrinkage). Changes of migration and phalloidin binding induced by CTX and cell swelling or by 1-EBIO and cell shrinkage, respectively, are linearly correlated with each other. CTX and cell swelling elicit a rise of [Ca²⁺]_i whereas 1-EBIO and cell shrinkage induce a slight decrease of [Ca²⁺]_i in most MDCK-F cells. Taken together IK-channel-dependent perturbations of cell volume and anisotonicity elicit virtually identical effects on migration, actin filaments and [Ca²⁺]_i. We therefore suggest that cell volume – possibly via [Ca²⁺]_i– is the link between IK channel activity, actin filaments and migration. We propose a model for how temporal and local changes of cell volume can support the migration of MDCK-F cells. Received: 28 January 1999 / Received after revision: 15 April 1999 / Accepted: 16 April 1999     

Changes of the biophysical properties of calcium-activated potassium channels of rat skeletal muscle fibres during aging

 In the present work, we have investigated the effects of the aging process on Ca²⁺-activated K⁺ channels (K_Ca2+) of rat skeletal muscle fibres. K_Ca2+ channels of adult (5–7 months old) and aged (24–26 months old) rats were surveyed by the patch-clamp technique. In aged rats, K_Ca2+ channels were routinely detected on the surface membrane of the fibres in both cell-attached and inside-out configurations. Conversely, in adult rat fibres, K_Ca2+ channels were rarely detected. In the cell-attached configuration, the open probability of the aged rat K_Ca2+ channel, measured in the range of potentials from –60 mV to +20 mV, was about 1.5–2 times higher than that of the adult one. The number of functional channels was abnormally increased by aging. An average of three channels per patch/area was counted in the inside-out patches of aged rat fibres, whereas no more than one open channel per patch/area was detected in the adult rat fibres. The frequency of finding channels in the patches also increased with aging, i.e. 11.5% and 30.1% in the adult and in the aged rat fibres, respectively. However, no significant change in the single-channel conductance has been observed with aging: it was 227 pS and 231 pS for adult and aged rat channels, respectively. In detached patches, both the adult and aged rat channels showed a similar voltage dependence of open probability and a similar sensitivity to Ca²⁺ ions. The aging process did not alter the response of the single channel to charybdotoxin, or its modulation by nucleotides, MgATP and adenosine 5’-O-(3-thiotriphosphate) (ATP[γ-S]). On the other hand, charybdotoxin reduced the abnormally high resting macroscopic K⁺ conductance of the aged rat fibres, recorded using the two-intracellular-microelectrode technique. These findings indicate that, in skeletal muscle, the activity of K_Ca2+ channels increases with advancing age. Received: 10 April 1997 / Received after revision and accepted: 4 June 1997     

酶分离猪冠脉平滑肌细胞钙激活钾通道全细胞记录电学特性研究

目的 :观察急性酶分离猪冠脉平滑肌全细胞钙激活K 电流 (IKCa)特性及摸索可行的记录方法。方法 :采用常规膜片钳全细胞记录技术。结果 :分离的猪冠脉平滑肌细胞 (来自 15只猪的 4 7个细胞 ,膜电容 32 .3±8.8pF)上的主要K 电流为IKCa,此电流具电压依赖性和胞内游离Ca2 依赖性。IKCa选择性阻断剂Charybdotoxin(ChTX ,12 5～ 2 5 0× 10 - 9mol.L- 1 )可使外向电流降低 6 8± 8% (n =5 )。 10～ 2 0× 10 - 3mol.L- 1 TEA可阻断 74± 5 % (n =7)的外向电流。结论 :猪冠脉平滑肌细胞全细胞K 电流主要成分为IKCa,它与其它组织细胞报道的情况类似而又有独自的特点。我们的数据为猪冠脉平滑肌全细胞K 电流特性的认识提供了第一手资料 ,为进一步的研究工作奠定良好基础