Mechanism of the inhibition of Ca2+-activated Cl- currents by phosphorylation in pulmonary arterial smooth muscle cells.
Mechanism of the inhibition of Ca2+-activated Cl- currents by phosphorylation in pulmonary arterial smooth muscle cells.
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DOI：
10.1085/jgp.200609507
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发表时间：
2006-07
期刊：
影响因子：
--
通讯作者：
Greenwood IA
中科院分区：
文献类型：
--
作者：
Angermann JE;Sanguinetti AR;Kenyon JL;Leblanc N;Greenwood IA
本研究的目的是提供一个关于磷酸酶活性如何影响兔肺动脉肌细胞钙激活氯离子通道的机械性见解。用含20~1000 nM Ca~(2+)的吸管溶液诱发Ca~(2+)依赖的Cl-−电流(IClCa)，并测定其钙和电压依赖性。在含有三磷酸腺苷和500nM钙离子的吸管溶液的对照条件下，膜破裂时立即诱发IClCa，但随后显示明显下降到初始值的20%的∼。相反，当使用含有腺苷5‘-(β，γ-亚胺)-三磷酸的吸管溶液或省略三磷酸腺苷的吸管溶液抑制磷酸化时，细胞的降解受到严重损害，在透析20min后，IClCa达到初始水平的∼100%。用含有AMP-PNP的吸管溶液记录的IClCa明显大于对照电流，并且在正电位下具有较快的动力学，而在负电位下具有较慢的失活动力学。IClCa的显著增加是由于激活的电压依赖性的负移，而不是由于对钙的表观结合亲和力的增加。数学模拟是基于涉及三个钙离子的电压无关结合的门控方案进行的，每个结合步骤导致以固定的钙离子但逐渐增大的“开启”速率打开通道，以及电压依赖的关闭步骤(“关闭”速率)。我们的模型很好地再现了IClCa的钙离子和电压依赖性及其动力学特性。整体磷酸化的影响可以通过改变通道闭合率的大小、电压依赖和门控变量的状态来很好地模拟。这些数据表明，钙激活的氯−通道复合体的磷酸化状态通过一个或多个关键的电压依赖步骤显著影响电流产生。
The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl− currents (IClCa) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, IClCa was evoked immediately upon membrane rupture but then exhibited marked rundown to ∼20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5′-(β,γ-imido)-triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, IClCa was ∼100% of initial levels. IClCa recorded with AMP-PNP–containing pipette solutions were significantly larger than control currents and had faster kinetics at positive potentials and slower deactivation kinetics at negative potentials. The marked increase in IClCa was due to a negative shift in the voltage dependence of activation and not due to an increase in the apparent binding affinity for Ca2+. Mathematical simulations were carried out based on gating schemes involving voltage-independent binding of three Ca2+, each binding step resulting in channel opening at fixed calcium but progressively greater “on” rates, and voltage-dependent closing steps (“off” rates). Our model reproduced well the Ca2+ and voltage dependence of IClCa as well as its kinetic properties. The impact of global phosphorylation could be well mimicked by alterations in the magnitude, voltage dependence, and state of the gating variable of the channel closure rates. These data reveal that the phosphorylation status of the Ca2+-activated Cl− channel complex influences current generation dramatically through one or more critical voltage-dependent steps.
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DOI：
10.1085/jgp.109.5.647
发表时间：
1997-05
期刊：
The Journal of general physiology
影响因子：
--
作者：
Cui J;Cox DH;Aldrich RW
通讯作者：
Aldrich RW
影响因子：
3.8
作者：
Zhuge, Ronghua;Fogarty, Kevin E;Tuft, Richard A;Walsh, John V Jr
通讯作者：
Walsh, John V Jr
影响因子：
4.5
作者：
BOTON, R;SINGER, D;DASCAL, N
通讯作者：
DASCAL, N
影响因子：
5.5
作者：
Xie, WW;Solomons, KRH;Shears, SB
通讯作者：
Shears, SB
影响因子：
5.5
作者：
Greenwood, IA;Ledoux, J;Leblanc, N
通讯作者：
Leblanc, N