Inhibition by calponin of isometric force in demembranated vascular smooth muscle strips: the critical role of serine-175.
Inhibition by calponin of isometric force in demembranated vascular smooth muscle strips: the critical role of serine-175.
复制标题
钙调蛋白对去膜血管平滑肌条等长力的抑制：丝氨酸 175 的关键作用。
作者：
Y. Uyama;Y. Imaizumi;M. Watanabe;M. Walsh
α-钙调蛋白是一种细丝相关蛋白，与平滑肌收缩的调节有关。对大鼠尾动脉平滑肌组织含量的定量显示，与鸡肫和其他平滑肌相比，相对于肌动蛋白，α-钙调蛋白的含量大约是鸡砂和其他平滑肌的一半，表明该组织特别适合研究外源性α-钙调蛋白对透化肌条收缩特性的影响。用 Triton X-100 脱膜的大鼠尾动脉条保留了大约 90% 的 α-钙调蛋白，而外源性鸡胗 α-钙调蛋白（其分子质量比大鼠动脉蛋白稍低）与透化肌肉结合，可能是通过其与肌动蛋白的高亲和力。当外源性α-钙调蛋白在次最大Ca(2+)诱导收缩的峰值时添加时，以浓度依赖性方式抑制去膜肌条中的力，在大约3μM α-钙调蛋白时具有半最大效应。用 α-钙调蛋白预处理去膜肌条可抑制激活范围内所有 Ca2+ 浓度下的后续力发展。 α-钙调蛋白的抑制作用被证明是不依赖于Ca(2+)的，因为在不含Ca2+的情况下，外源性α-钙调蛋白也会抑制含有硫代磷酸化肌球蛋白的脱膜肌条中的力。蛋白激酶 C 对 Ser-175 上的 α-钙调蛋白进行磷酸化可减轻 α-钙调蛋白对平滑肌收缩的抑制作用。为了检验这一假设，将磷酸化α-钙调蛋白和α-钙调蛋白的三个位点特异性突变体（其中Ser-175被Ala、Asp或Thr取代）对Ca(2+)诱导的和Ca(2+)独立的脱膜肌条收缩的影响与未磷酸化的组织纯化和重组野生型的影响进行了比较 α-钙调蛋白。重组野生型蛋白的行为与未磷酸化的组织纯化蛋白相同，S175T 突变体也是如此，已知它能以高亲和力结合肌动蛋白，并在体外抑制肌动蛋白激活的肌球蛋白 MgATP 酶。另一方面，磷酸化α-钙调蛋白和S175A和S175D突变体与肌动蛋白结合较弱，对体外肌动蛋白激活的肌球蛋白MgATP酶影响很小，未能对Ca2+或肌球蛋白硫代磷酸化诱导的力产生显着抑制。这些结果支持 α-钙调蛋白在平滑肌收缩调节中的作用，并表明 α-钙调蛋白的 Ser-175 作为磷酸化调节位点的功能重要性。
alpha-Calponin is a thin-filament-associated protein which has been implicated in the regulation of smooth muscle contraction. Quantification of the tissue content of rat tail arterial smooth muscle revealed approximately half the amount of alpha-calponin relative to actin compared with chicken gizzard and other smooth muscles, suggesting that this tissue would be particularly suitable for investigation of the effects of exogenous alpha-calponin on the contractile properties of permeabilized muscle strips. Rat tail arterial strips demembranated with Triton X-100 retained approximately 90% of their complement of alpha-calponin, and exogenous chicken gizzard alpha-calponin (which conveniently has a slightly lower molecular mass than the rat arterial protein) bound to the permeabilized muscle, presumably through its high affinity for actin. Exogenous alpha-calponin inhibited force in demembranated muscle strips in a concentration-dependent manner when added at the peak of a submaximal Ca(2+)-induced contraction, with a half-maximal effect at approximately 3 microM alpha-calponin. Pretreatment of demembranated muscle strips with alpha-calponin inhibited subsequent force development at all concentrations of Ca2+ examined over the activation range. The inhibitory effect of alpha-calponin was shown to be Ca(2+)-independent, since exogenous alpha-calponin also inhibited force in the absence of Ca2+ in demembranated muscle strips containing thiophosphorylated myosin. Phosphorylation of alpha-calponin on Ser-175 by protein kinase C has been suggested to alleviate the inhibitory effect of alpha-calponin on smooth muscle contraction. To test this hypothesis, the effects on Ca(2+)-induced and Ca(2+)-independent contractions of demembranated muscle strips of phosphorylated alpha-calponin and three site-specific mutants of alpha-calponin (in which Ser-175 was replaced by Ala, Asp or Thr) were compared with the effects of unphosphorylated tissue-purified and recombinant wild-type alpha-calponins. The recombinant wild-type protein behaved identically to the unphosphorylated tissue-purified protein, as did the S175T mutant, which is known to bind actin with high affinity and to inhibit the actin-activated myosin MgATPase in vitro. On the other hand, phosphorylated alpha-calponin and the S175A and S175D mutants, which bind weakly to actin and have little effect on the actin-activated myosin MgATPase in vitro, failed to cause significant inhibition of force induced by Ca2+ or myosin thiophosphorylation. These results support a role for alpha-calponin in the regulation of smooth muscle contraction and indicate the functional importance of Ser-175 of alpha-calponin as a regulatory site of phosphorylation.