Otter T, Satir B H, Satir P
Cell Motil. 1984;4(4):249-67. doi: 10.1002/cm.970040404.
Trifluoperazine (TFP), a drug that binds to Ca2+-calmodulin (CaM) complexes, altered swimming behavior not only in living paramecia, but also in reactivated, Triton-extracted "models" of the ciliate. By comparing the responses of living cells and models, we have ascertained that two sites of drug action exist in paramecium cilia. Swimming movements were recorded in darkfield stroboscopic flash photomicrographs; this permitted accurate quantitation of velocities and body-shape parameters. When living paramecia were incubated in a standard buffer containing 10 microM TFP, their speed of forward swimming fell over several minutes and their bodies shortened. Untreated paramecia backed up repeatedly and frequently upon transfer to a solution containing barium ions (the "barium dance"), but cells preincubated in TFP did not "dance." Instead they swam forward slowly for long periods of time without reversing and occasionally then exhibited abnormally prolonged reversals. W7 effects on swimming mimicked low doses of TFP, and the analog W5 did not visibly alter normal swimming patterns. These results suggest that TFP induces a decrease in the intracellular pCa of living paramecia, perhaps by reducing the efficiency of a calmodulin-activated calcium pump in the cell membrane. Paramecia extracted with Triton X-100 and reactivated to swim forward (7 greater than or equal to pCa greater than or equal to 6) were not affected by addition of up to 40 microM TFP to the reactivation medium. We conclude that the main drug effect in living cells is probably not at the axoneme. However, at low pCa, TFP directly affected the ciliary axoneme to shift its behavior to one characteristic of a higher pCa: TFP inhibited backward swimming in models reactivated at pCa less than 6; instead they swam forward or rocked in place. The mechanism of ciliary reversal in paramecium may therefore depend on an axonemal Ca2+-sensor, possibly bound CaM, which is affected by TFP only at low pCa, as has been postulated for other types of cilia.
三氟拉嗪（TFP）是一种能与钙调蛋白（CaM）复合物结合的药物，它不仅改变了活草履虫的游动行为，还改变了经激活的、用曲拉通提取的纤毛虫“模型”的游动行为。通过比较活细胞和模型的反应，我们确定草履虫纤毛中存在两个药物作用位点。在暗视野频闪闪光显微照片中记录游动运动；这使得能够准确量化速度和身体形状参数。当活草履虫在含有10微摩尔TFP的标准缓冲液中孵育时，它们向前游动的速度在几分钟内下降，身体缩短。未经处理的草履虫转移到含有钡离子的溶液中（“钡之舞”）时会反复频繁地后退，但预先在TFP中孵育的细胞不会“跳舞”。相反，它们长时间缓慢向前游动而不反转，偶尔会出现异常延长的反转。W7对游动的影响模拟了低剂量的TFP，而类似物W5没有明显改变正常的游动模式。这些结果表明，TFP可能通过降低细胞膜中钙调蛋白激活的钙泵的效率，导致活草履虫细胞内游离钙离子浓度（pCa）降低。用曲拉通X - 100提取并重新激活以向前游动（7≥pCa≥6）的草履虫，在重新激活培养基中加入高达40微摩尔TFP时不受影响。我们得出结论，在活细胞中药物的主要作用可能不在轴丝。然而，在低pCa时，TFP直接影响纤毛轴丝，使其行为转变为更高pCa时的特征：TFP抑制在pCa小于6时重新激活的模型中的向后游动；相反，它们向前游动或原地摆动。因此，草履虫纤毛反转的机制可能取决于轴丝钙离子传感器，可能是结合的CaM，它仅在低pCa时受TFP影响，正如对其他类型纤毛所假设的那样。
