Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous cAMP-dependent protein kinase

 Single Ca²⁺-activated K⁺ channels of human erythrocytes were studied with the patch-clamp technique, to identify the mechanisms of their modulation by phosphorylation. In the cell-attached configuration, the openings of these channels were infrequent, as expected by the low cell Ca²⁺ content. After patch excision, the activity increased to levels determined by the Ca²⁺ concentration (0.5–10 μM) in the bath solution, then the channel activity ran down within a few minutes, to reach values of open probability lower than 0.10. The perfusion of the patch with MgATP increased the channel activity, with delayed and variable effects. Furthermore, the application of a mixture of cAMP (1 mM), MgATP (1 mM) and theophylline (1 mM) to the cytoplasmic side of excised patches led to dramatic enhancement of channel activity, which appeared within 20–120 s and decayed in tens of seconds after wash-out. The activation of the channel by the mixture was reversibly blocked by PKI_5–24, a peptide inhibitor specific to cAMP-dependent protein kinase (PKA). The level of activation promoted by cAMP and ATP was dependent on the Ca²⁺ concentration in the bathing solution. These results provide direct evidence that an endogenous PKA modulates the calcium sensitivity of Ca²⁺-activated K⁺ channels of human erythrocytes. Received: 19 February 1998 / Received after revision: 14 April 1998 / Accepted: 20 April 1998     

Protein kinase C regulation of cardiac calcium channels expressed in Xenopus oocytes

L-Type cardiac Ca²⁺ channels expressed in Xenopus oocyte were studied following rat heart ribonucleic acid, messenger (mRNA) injection. We demonstrate that exogenous Ca²⁺ channels are sensitive to intracellular regulation by protein kinase C (PKC). This was performed by using two types of PKC activators [phorbol esters and a structural analogue of diacylglycerol (DAG)] and a specific peptidic inhibitor. Ca²⁺ channel modulation resulted in an initial increase of the inward current, without any modification of the voltage-dependent properties, and a second delayed phase, specifically observed with phorbol esters, characterized by a progressive decrease in current amplitude. Concomitantly, a reduction of membrane capacitance, reflecting a reduction of the total membrane surface area, was observed. We suggest that this phenomenon underlies the irreversible decrease of the expressed Ba²⁺ current via sequestration of Ca²⁺ channels and/or PKC. We also demonstrate that regulation of cardiac mRNA-directed Ca²⁺ channels by PKC activators was strictly dependent on intracellular Ca²⁺ concentration, and was partially additive with cyclic-adenosine-monophosphate-(cAMP) dependent regulation.     

Small (SKCa) Ca2+-activated K+ channels in cultured rat hippocampal pyramidal neurones

 Small (SK_Ca) Ca²⁺-activated K⁺ channels were identified in membrane patches excised from cultured CA1-CA3 pyramidal neurones of the neonatal rat hippocampus. When recorded in low-K⁺ extracellular solution ([K⁺]_o=2.5 mM), SK_Ca channels had a low conductance (@3 pS at 0 mV), were activated by ≥175 nM Ca²⁺ (P _o=0.54 at 500 nM Ca²⁺) and there were two open-time components (2.1 and @70 ms) to their activity. These properties of single SK_Ca channels are similar to those of slow after-hyperpolarization channels (sAHP) previously inferred from fluctuation analysis of the sAHP current. It is concluded that the SK_Ca channel reported here may be the channel that generates the sAHP in hippocampal pyramidal neurones. Received: 9 July 1998 / Received after revision: 5 October 1998 / Accepted: 7 October 1998     

Effects of temperature on human L-type cardiac Ca2+ channels expressed in Xenopus oocytes

 Temperature normally affects peak L-type Ca²⁺ channel (CaCh) current with a temperature coefficient (Q ₁₀) of between 1.8 and 3.5; in cardiomyocytes attenuating protein kinase A activity increases Q ₁₀ whilst activating it lowers Q ₁₀. We examine temperature effects using cloned human cardiac CaChs expressed in Xenopus oocytes. Peak inward currents (I _Ba) through expressed CaChs (i.e. α_1Cα₂/δ_aβ_1b) exhibited a Q ₁₀ of 5.8±0.4 when examined between 15 and 25°C. The nifedipine-sensitive I _Ba exhibited a higher Q ₁₀ of 8.7±0.5, whilst the nifedipine-insensitive I _Ba exhibited Q ₁₀ of 3.7±0.3. Current/voltage (I/V) relationships shifted to negative potentials on warming. Using instead a different CaCh β subunit isoform, β_2c, gave rise to an I _Ba similar to those expressed using β_1b. We utilized a carboxyl deletion mutant, α_1C-Δ1633, to determine the temperature sensitivity of the pore moiety in the absence of auxiliary subunits; I _Ba through this channel exhibited a Q ₁₀ of 9.3±0.3. However, the Q ₁₀ for macroscopic conductance was reduced compared to that of heteromeric channels; decreasing from 5.0 (i.e. α_1Cα₂/δ_aβ_1b) and 3.9 (i.e. α_1Cα₂/δ_aβ_2c) to 2.4 (α_1C-Δ1633). These observations differ markedly from those made in studies of cardiomyocytes, and suggest that enhanced sensitivity may depend on the membrane environment, channel assembly or other regulatory factors. Received: 16 December 1997 / Accepted: 23 February 1998     

血管紧张素(1-7)通过抑制中电导钙激活钾离子通道蛋白表达参与肾脏纤维化

目的 探讨血管紧张素（Ang）（1-7）在肾纤维化过程中的保护作用与中电导钙激活钾离子通道（KCa3.1）的关系。 方法 60只雄性小鼠随机分为5组：对照组 (WT)；血管紧张素Ⅱ（Ang Ⅱ）组：皮下注射 Ang Ⅱ ［1.4 mg/(kg.d)］；注射Ang Ⅱ 的同时给予以下药物干预： Ang Ⅱ阻断剂 洛沙坦（Losartan）组：皮下注射 Losartan［40 mg/(kg.d)］； Ang（1-7）组：皮下注射 Ang（1-7）［0.14 mg/(kg.d)］；血管紧张素转化酶2（ACE2）激动剂重氮氨苯脒乙酰甘氨酸盐（DIZE）组：皮下注射DIZE［10 mg/(kg.d)］。连续给药4周后对相关指标进行检测。Masson染色法检测肾组织胶原沉积变化；Western blotting法检测肾组织 Ⅰ 型胶原、Ⅲ 型胶原和 KCa3.1通道蛋白表达的变化。 结果 与对照组相比，Ang Ⅱ组小鼠肾组织内胶原沉积量明显增加（n=12，P<0.01），表明肾纤维化模型复制成功。Ang Ⅱ使肾组织Ⅰ、Ⅲ型胶原合成显著增多（n=6，P<0.01），同时促进了肾组织KCa3.1通道蛋白的表达（P<0.01），而Ang （1-7）及ACE2激活剂 DIZE 的应用抑制了肾组织内胶原沉积量、Ⅰ/Ⅲ型胶原合成及KCa3.1通道蛋白的表达（n=12或6，P<0.01）。结论 Ang （1-7）在肾纤维化过程中发挥保护作用，这一作用可能与其下调肾组织中KCa3.1通道蛋白表达有关。     

A high-conductance Ca2+-activated K+ channel in cultured human eccrine sweat gland cells

Recordings have been made of a potassium-selective ion channel in primary cultures of cells derived from expiants of human eccrine sweat glands obtained from normal subjects and from subjects suffering from cystic fibrosis. There appears to be no functional difference between potassium channels derived from normal subjects and those from cystic fibrosis subjects. The channel falls into the group generally known as Maxi-K channels, and has a slope conductance, with symmetrical solutions in bath and pipette containing 140 mM K⁺, of 230 pS. It is calcium-activated, pH-sensitive and can be blocked by barium and quinine. The channel appears only very rarely in patches derived from confluent cells (in approximately 1 in 30 patches containing channels), but it is more frequently observed in younger cultures of dividing cells derived from recently explanted (within the previous 48–72 h) glands. It is possible that this channel is normally located on the basolateral membrane of the cell, and is responsible for the calcium-dependent secretory and absorptive events seen in the intact sweat gland.     

Complete reversal of run-down in rabbit cardiac Ca2+ channels by patch-cramming in Xenopus oocytes; partial reversal by protein kinase A

 The rabbit cardiac Ca²⁺ channel (α_1C) expressed in Xenopus oocytes exhibited a complete run-down of ionic currents when cell-attached patches were excised. The α_1C channel was expressed alone or was coexpressed with the accessory β_2a or β_1b subunit. The catalytic subunit of protein kinase A (PKAc) and MgATP were capable of delaying the run-down of single-channel currents. In 33% of the α_1C patches, and 26% of the α_1C+β_2a patches, inclusion of PKAc in the bath solution delayed the run-down for a maximum of 20 min. In experiments where PKAc in the bath was not sufficient to delay the run-down of channel activity, insertion of the patch back into the oocyte (patch-cramming) could restore channel activity. Gating currents were also measured in the α_1C+β_1b channel and were not subject to any run-down, even after the complete run-down of ionic currents. The results presented here reveal that PKAc is capable of delaying the run-down of currents in a subset of patches. The patch-cramming results suggest that a cytoplasmic factor, in addition to phosphorylation of the channel (by PKAc), may be involved in the maintenance of channel activity. Received: 29 October 1998 / Accepted: 4 January 1999     

Modulation by protein kinase A of a cloned rat brain potassium channel expressed in Xenopus oocytes

A potassium channel from rat brain was expressed in Xenopus oocytes in order to study modulation of channel function by phosphorylation via protein kinase A. Application of 8-Br-cAMP to oocytes expressing the drk1 channel (with the first 139 amino acids of the N terminus delected, Ndrk1) caused a voltage-independent elevation of current amplitude, which was not seen for endogenous currents or for wild-type full-length drk1 channel. This effect on Ndrk1 was blocked by pre-injection of oocytes with Walsh-peptide protein kinase A inhibitor, suggesting mediation via protein kinase A. The protein kinase inhibitor also reduced both Ndrk1 and full-length drk1 currents. Substitution of the serine residues by alanine at one or both of the two consensus protein kinase A phosphorylation sites on the C terminus (residues 440 and 492) of Ndrk1 resulted in a loss of function of the expressed channels. These results indicate that phosphorylation via protein kinase A modulates drk1 channel function and that both consensus phosphorylation sites seems to be essential for channels to function.     

Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: Evidence for tetrameric channel formation

Unitary currents were recorded from insideout membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent K _d of 80 M at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent K _d at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80%, 55%, 25% and 10%. These values agreed well with those expected for inhibition by TEA of currents through channels containing 3, 2, 1 and 0 tyrosine residues at the channel mouth, assuming that a tyrosine residue from each of four subunits contributes equally to the binding of the TEA ion. This indicates that Slowpoke channels form as tetramers.     

Blockade of HERG channels expressed in Xenopus oocytes by external H+

 We have investigated the effect of external H⁺ concentration ([H⁺]_o)on the human-ether-a-go-go-related gene (HERG) current (I _HERG), the molecular equivalent of the cardiac delayed rectifier potassium current (I _Kr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H⁺]_o was increased, the amplitude of the I _HERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pH_o) values (the potential required for half-maximum activation, V _1/2, was: –41.8 mV, –38.0 mV, –33.7 mV, –26.7 mV in pH_o 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (g _max) was also affected by [H⁺]_o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pH_o from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca²⁺ concentration, which is also known to block HERG channels. When the extracellular Ca²⁺ concentration was increased from 0.5 mM to 5 mM, the shift in V _1/2 caused by increasing [H⁺]_o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in g _max caused by increasing [H⁺]_o was not significantly affected by changing external Ca²⁺ levels. The results indicate that HERG channels are inhibited by [H⁺]_o by two different mechanisms: voltage-dependent blockade (shift of V _1/2) and the decrease in g _max. With respect to the voltage-dependent blockade, the interaction between H⁺ and Ca²⁺ is competitive, whereas for the decreasing g _max, their interaction is non-competitive. Received: 12 January 1999 / Received after revision: 15 February 1999 / Accepted: 16 February 1999     

Cyclic AMP-dependent regulation of P-type calcium channels expressed in Xenopus oocytes

Xenopus oocytes injected with rat cerebellum mRNA, express voltage-dependent calcium channels (VDCC). These were identified as P-type Ca²⁺ channels by their insensitivity to dihydropyridines and -conotoxin and by their blockade by Agelenopsis aperta venom (containing the funnel-web spider toxins: FTX and -Aga-IV-A). Coinjection of cerebellar mRNA and antisense oligonucleotide complementary to the dihydropyridine-resistant brain Ca²⁺ channel, named BI [Mori Y. et al. (1991) Nature 350:398–402] or rbA [Starr T. V. B. et al. (1991) Proc Natl Acad Sci USA 88:5621–5625], strongly reduced the expressed Ba²⁺ current suggesting that these clones encode a P-type VDCC. The macroscopic Ca²⁺ channel activity was increased by direct intraoocyte injection of cAMP. This increase in current amplitude was concomitant with a slowing of current inactivation, and was attributed to activation of protein kinase A, since it could be antagonized by a peptidic inhibitor of this enzyme. Positive regulation of P-type VDCC could be of importance in Purkinje neurons and motor nerve terminals where this channel is predominant.     

Single Ca2+-activated K+ channels in excised membrane patches of hamster oocytes

The properties of the Ca²⁺-activated K⁺ channel in unfertilized hamster oocytes were investigated at the single-channel level using inside-out excised membrane patches. The results indicate a new type of Ca²⁺-activated K⁺ channel which has the following characteristics: (1) single-channel conductance of 40–85 pS for outward currents in symmetrical K⁺ (150 mM) solutions, (2) inward currents of smaller conductance (10–50 pS) than outward currents, i.e. the channel is outwardly rectified in symmetrical K⁺ solutions, (3) channel activity dependent on the internal concentration of free Ca⁺ and the membrane potential, (4) modification of the channel activity by internal adenosine 5 diphosphate (0.1 mM) producing a high open probability regardless of membrane potential.     

cAMP-activation of amiloride-sensitive Na+ channels from guinea-pig colon expressed in Xenopus oocytes

Guinea-pig distal colonic mRNA injection into Xenopus laevis oocytes resulted in expression of functional active epithelial Na⁺ channels in the oocyte plasma membrane. Poly(A)⁺ RNA was extracted from distal colonic mucosa of animals fed either a high-salt (HS) or a low-salt (LS) diet. The electrophysiological properties of the expressed amiloride-sensitive Na⁺ conductances were investigated by conventional two-electrode voltage-clamp and patch-clamp measurements. Injection of poly(A)⁺ RNA from HS-fed animals [from hereon referred to as HS-poly(A)⁺ RNA] into oocytes induced the expression of amiloride-sensitive Na⁺ conductances. On the other hand, oocytes injected with poly(A)⁺ RNA from LS-fed animals [LS-poly(A)⁺ RNA] expressed a markedly larger amount of amiloride-blockable Na⁺ conductances. LS-poly(A)⁺ RNA-induced conductances were completely inhibitable by amiloride with a K _i of 77 nM, and were also blocked by benzamil with a K _i of 1.8 nM. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA), even in high doses (25 μM), had no detectable effect on the Na⁺ conductances. Expressed amiloride-sensitive Na⁺ channels could be further activated by cAMP leading to nearly doubled clamp currents. When Na⁺ was replaced by K⁺, amiloride (1 μM) showed no effect on the clamp current. Single-channel analysis revealed slow gating behaviour, open probabilities (P _o) between 0.4 and 0.9, and slope conductances of 3.8 pS for Na⁺ and 5.6 pS for Li⁺. The expressed channels showed to be highly selective for Na⁺ over K⁺ with a permeability ratio P _Na/P _K > 20. Amiloride (500 nM) reduced channel P _o to values < 0.05. All these features make the guinea-pig distal colon of LS-fed animals an interesting mRNA source for the expression of highly amiloride-sensitive Na⁺ channels in Xenopus oocytes, which could provide new insights in the regulatory mechanism of these channels. Received: 16 October 1995/Received after revision: 30 November 1995/Accepted: 12 December 1995     

Ca2+ channels,ryanodine receptors and Ca2+-activated K+ channels: a functional unit for regulating arterial tone

Local calcium transients (‘Ca²⁺ sparks’) are thought to be elementary Ca²⁺ signals in heart, skeletal and smooth muscle cells. Ca²⁺ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca²⁺ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately ?40 mV, an elevation of arterial wall [Ca²⁺]_i and contraction (‘myogenic tone’) of arteries. Ca²⁺ sparks activate calcium-sensitive K⁺ (K_Ca) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca²⁺ sparks by ryanodine, or of K_Ca channels by iberiotoxin, leads to membrane depolarization, activation of L -type voltage-gated Ca²⁺ channels, and vasoconstriction. Conversely, activation of Ca²⁺ sparks can lead to vasodilation through activation of K_Ca channels. Our recent work is aimed at studying the properties and roles of Ca²⁺ sparks in the regulation of arterial smooth muscle function. The modulation of Ca²⁺ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca²⁺ entry through voltage-dependent Ca²⁺ channels in the regulation of Ca²⁺ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca²⁺ channels, RyR channels, and K_Ca channels function as a coupled unit, through Ca²⁺ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.     

高血压大鼠主动脉平滑肌BK通道表达增强

 目的 研究在自发性高血压大鼠(Spontaneous hypertensive rats,SHR)和正常对照组大鼠(wistar-kyoto,WKY)主动脉平滑肌上大电导钙激活钾通道(BK)的功能改变及其机制。方法 用实时定量PCR反应和免疫印迹对SHR和WKY大鼠的主动脉组织的BK通道组成亚基α和β1表达量的检测。结果 SHR大鼠α和β1亚基的mRNA的表达量明显高于WKY大鼠（n=6,p<0.05）,SHR大鼠的α亚基的蛋白表达量与WKY没有显著性差异（n=6,p>0.05）,β1亚基的蛋白表达量SHR大鼠却明显高于WKY大鼠（n=6,p<0.05）。结论 SHR大鼠BK通道在mRNA水平和蛋白水平的表达都比WKY增强,可能是在高血压状况下机体负反馈抑制血管张力的一种调节机制。     

Facilitation of Ca2+-activated K+ channels (IKCa1) by mibefradil in B lymphocytes

K⁺ channels play critical roles in the proliferation and activation of lymphocytes. Mouse B cells express large-conductance background K⁺ channel (LK_bg) in addition to the voltage-gated K⁺ channel (Kv) and Ca²⁺-activated K⁺ channel current (IKCa1). Mibefradil, a blocker of T-type Ca²⁺ channels, has been reported to affect the proliferation of immune cells. In this study, we investigated the effects of mibefradil on the membrane potential and ion channels in murine B cell lines, WEHI-231 and Bal-17. In the whole-cell patch clamp experiments, mibefradil blocked Kv and LK_bg current with half inhibitory concentration (IC₅₀), 1.9 and 2.3 μM, respectively. Interestingly, IKCa1 current was increased by mibefradil. In the inside-out patch clamp study with cloned murine IKCa1 (mIKCa1) in HEK-293, mibefradil increased both Ca²⁺ sensitivity and maximum activity of mIKCa1. At high concentrations (>10 μM), mibefradil inhibited mIKCa1 in a voltage-dependent manner. Application of anti-IgM antibody to stimulate B cell receptors (BCR-ligation) induced transient hyperpolarization of Bal-17 and WEHI-231 cells, which became persistent with 1 μM mibefradil. The hyperpolarizing response was abolished by charybdotoxin, a selective blocker for SK4/IKCa1. In summary, our study firstly reports the ion channel-activating effects of mibefradil. The selective potent activation of IKCa1 suggests that mibefradil-derived drugs might be useful in the control of cell responses related with IKCa1. HY Yoo, H Zheng, and JH Nam contributed equally to this study.     

Inactivation of calcium-activated chloride conductance in Xenopus oocytes: roles of calcium and protein kinase C

Inactivation of Ca²⁺ -induced Cl^– currents was studied in Xenopus oocytes using the two-electrode voltage-clamp technique. In oocytes permeabilized to Ca²⁺ by treatment with the ionophore A23187, Ca²⁺ influx caused by the addition of 2.5–5 mM Ca²⁺ to the extracellular solution elicited Cl^– currents consisting of two components: a fast, transient one (I _fast) and a slow one (I _slow). In response to a subsequent application of the same dose of Ca²⁺, I _fast and I _slow were reduced (inactivation phenomenon). The inactivation did not depend on the direction of current flow, but did depend on the duration of the first exposure to Ca²⁺. The extent of inactivation of I _fast was more significant than that to I _slow. Both I _fast and I _slow fully recovered from inactivation in less than 30 min. Intracellular injections of 100–400 pmol CaCl₂ evoked large inward currents but did not reduce the amplitude of currents evoked by Ca²⁺ influx. The activator of protein kinase C, -phorbol dibutyrate, caused full inhibition of I _fast without any change in I _slow. H-7 (1,5-isoquinolinesulfonyl-1,2 methylpiperazine), an inhibitor of protein kinases, strongly reduced the extent of inactivation. Our results suggest that elevation of intracellular Ca²⁺ by Ca²⁺ influx through the plasma membrane causes inactivation of the Ca²⁺ -dependent Cl^– conductance via activation of a Ca²⁺ -dependent protein kinase, possibly protein kinase C, whereas Ca²⁺ arriving at the membrane from inside the cell does not initiate the processes leading to inactivation.     

中电导钙激活钾通道调节表皮生长因子诱导的人脐静脉内皮细胞体外增殖

目的观察表皮生长因子(EGF)对人脐静脉内皮细胞(HUVECs)离子通道中电导钙激活钾通道(KCa 3.1)表达的影响,以及KCa 3.1在EGF诱导HUVECs体外增殖过程中的作用。方法 MTT法筛选EGF最佳实验浓度;免疫荧光和Western blotting技术检测EGF对HUVECs细胞KCa 3.1的表达;经KCa 3.1阻断剂TRAM-34处理,MTT法分析EGF诱导的HUVECs增殖情况;流式细胞技术检测细胞周期,RT-PCR法分析细胞周期蛋白D1(cyclin D1)及周期蛋白依赖性蛋白激酶4(CDK4)表达的水平。结果 EGF处理细胞48h后,MTT实验证实25μg/L浓度的EGF促细胞增殖效应最强。免疫荧光染色显示,EGF明显促进KCa 3.1蛋白表达,且Western blotting提示KCa 3.1的表达上调1.4倍。进一步研究显示,高选择性阻断剂TRAM-34阻断KCa 3.1通道48h后EGF的促细胞增殖作用显著下降,并呈时间和剂量依赖性;细胞周期G1期细胞百分比明显增加;CDK4的mRNA表达下调,而cyclin D1表达无明显变化。结论 KCa 3.1可能通过影响细胞周期进程调节EGF诱导的HUVECs增殖过程。     

Characterization of Ca2+-activated K+ channels in excised patches of human T lymphocytes

Ca²⁺-activated K⁺ [K(Ca)] channels were studied in excised patches of resting and activated human peripheral blood T lymphocytes. The K(Ca) channel had a single-channel conductance of 50±6 pS in symmetrical high-K⁺ solutions in the potential range of –100 to –10 mV and was inwardly rectifying at more depolarized potentials. The channel was sensitive to block by charybdotoxin (10 nM) and insensitive to apamin (3 nM). Half-maximum activation occurred at an internal free Ca²⁺ concentration of 360±110 nM. The concentration-effect curve had a slope factor of 0.83±0.12, suggesting a 11 interaction of Ca²⁺ ions with the channel. Ca²⁺ affects the open time probability of the K(Ca) channels, mainly by modulating the frequency of channel opening. The open probability did not show voltage dependence. The kinetics of the channel could be described assuming one open state and two closed states. The time constant of the exponential describing the open time distribution amounted to 2.8±1.2 ms, whereas the closed time distribution could be described with two exponentials with time constants of 0.2±0.05 ms and 8.0±2.1 ms, respectively. Resting T lymphocytes expressed a low number of channels but the density of channels increased dramatically during chronic phytohaemagglutinin stimulation.     

Neuropeptide Y inhibits Ca2+-activated K+ channels in vascular smooth muscle cells from the rat tail artery

The effects of neuropeptide Y (NPY) on the Ca²⁺-activated K⁺ channel in smooth muscle cells from the rat tail artery were studied by whole-cell and single-channel patch-clamp recording techniques. In the presence of nifedipine (1 M), whole-cell outward currents through Ca²⁺-activated K⁺ channels were inhibited by NPY in a dose-dependent manner from 20 to 200 nM. A maximum inhibition to about 48% of the control current could be achieved. Recordings from outside-out patches showed that the open probability of Ca²⁺-activated K⁺ channels were similarly inhibited by NPY. At 200 nM NPY, the open probability was reduced to about 36% of the control value. NPY did not affect the open times or current amplitude, but increased significantly the short (from 0.49 to 0.58 ms) and long (from 441 to 728 ms) closed times. Inhibition of Ca²⁺-activated K⁺ channels by NPY may contribute to its excitatory action on vascular smooth muscle cells.