Kindzelskii Andrei L, Petty Howard R
Department of Ophthalmology and Visual Sciences, The University of Michigan Medical School, 1000 Wall Street, Ann Arbor, MI 48105, USA.
Eur Biophys J. 2005 Dec;35(1):1-26. doi: 10.1007/s00249-005-0001-2. Epub 2005 Jul 26.
We have tested Galvanovskis and Sandblom's prediction that ion channel clustering enhances weak electric field detection by cells as well as how the elicited signals couple to metabolic alterations. Electric field application was timed to coincide with certain known intracellular chemical oscillators (phase-matched conditions). Polarized, but not spherical, neutrophils labeled with anti-K(v)1.3, FL-DHP, and anti-TRP1, but not anti-T-type Ca(2+) channels, displayed clusters at the lamellipodium. Resonance energy transfer experiments showed that these channel pairs were in close proximity. Dose-field sensitivity studies of channel blockers suggested that K(+) and Ca(2+) channels participate in field detection, as judged by enhanced oscillatory NAD(P)H amplitudes. Further studies suggested that K(+) channel blockers act by reducing the neutrophil's membrane potential. Mibefradil and SKF93635, which block T-type Ca(2+) channels and SOCs, respectively, affected field detection at appropriate doses. Microfluorometry and high-speed imaging of indo-1-labeled neutrophils was used to examine Ca(2+) signaling. Electric fields enhanced Ca(2+) spike amplitude and triggered formation of a second traveling Ca(2+) wave. Mibefradil blocked Ca(2+) spikes and waves. Although 10 microM SKF96365 mimicked mibefradil, 7 microM SKF96365 specifically inhibited electric field-induced Ca(2+) signals, suggesting that one SKF96365-senstive site is influenced by electric fields. Although cells remained morphologically polarized, ion channel clusters at the lamellipodium and electric field sensitivity were inhibited by methyl-beta-cyclodextrin. As a result of phase-matched electric field application in the presence of ion channel clusters, myeloperoxidase (MPO) was found to traffic to the cell surface. As MPO participates in high amplitude metabolic oscillations, this suggests a link between the signaling apparatus and metabolic changes. Furthermore, electric field effects could be blocked by MPO inhibition or removal while certain electric field effects were mimicked by the addition of MPO to untreated cells. Therefore, channel clustering plays an important role in electric field detection and downstream responses of morphologically polarized neutrophils. In addition to providing new mechanistic insights concerning electric field interactions with cells, our work suggests novel methods to remotely manipulate physiological pathways.
我们已经验证了加尔瓦诺夫斯基斯和桑德布洛姆的预测，即离子通道聚集可增强细胞对弱电场的检测能力，以及所引发的信号如何与代谢变化相耦合。施加电场的时间设定为与某些已知的细胞内化学振荡器同步（相位匹配条件）。用抗K（v）1.3、荧光二氢吡啶（FL-DHP）和抗TRP1标记的极化嗜中性粒细胞（而非球形嗜中性粒细胞），以及用抗T型钙通道标记的嗜中性粒细胞，在片足处显示出簇状结构。共振能量转移实验表明，这些通道对彼此靠近。通道阻滞剂的剂量-场敏感性研究表明，钾离子（K⁺）和钙离子（Ca²⁺）通道参与电场检测，这可通过振荡的烟酰胺腺嘌呤二核苷酸磷酸（NAD(P)H）振幅增强来判断。进一步的研究表明，钾离子通道阻滞剂通过降低嗜中性粒细胞的膜电位起作用。分别阻断T型钙通道和储存式钙通道（SOCs）的米贝拉地尔和SKF93635，在适当剂量下会影响电场检测。使用indo-1标记的嗜中性粒细胞的显微荧光测定法和高速成像来检测钙离子信号。电场增强了钙离子尖峰的振幅，并触发了第二个传播性钙离子波的形成。米贝拉地尔阻断了钙离子尖峰和波。虽然10微摩尔的SKF96365模拟了米贝拉地尔的作用，但7微摩尔的SKF96365特异性地抑制了电场诱导的钙离子信号，这表明一个对SKF96365敏感的位点受到电场的影响。尽管细胞在形态上仍保持极化，但片足处的离子通道簇和电场敏感性受到甲基-β-环糊精的抑制。由于在存在离子通道簇的情况下施加了相位匹配的电场，发现髓过氧化物酶（MPO）转运到细胞表面。由于MPO参与高振幅的代谢振荡，这表明信号传导装置与代谢变化之间存在联系。此外，电场效应可被MPO抑制或去除所阻断，而某些电场效应可通过向未处理的细胞中添加MPO来模拟。因此，通道聚集在形态极化的嗜中性粒细胞的电场检测和下游反应中起重要作用。除了提供有关电场与细胞相互作用的新机制见解外，我们的工作还提出了远程操纵生理途径的新方法。
