Lin Jen-Wei, Fu Qinghao, Allana Tariq
Department of Biology, Boston University, 5 Cummington St., Boston, Massachuetts 02215, USA.
J Neurophysiol. 2005 Jul;94(1):377-86. doi: 10.1152/jn.00617.2004.
Ca2+ indicators of varying affinity and mobility were pressure injected into the presynaptic axon of the inhibitor of the crayfish neuromuscular junction (NMJ). Fluorescence transients recorded at a 2-kHz resolution were used to probe physiological parameters governing the decay of fluorescence transients within 100 ms after an action potential (early decay). Blocking Ca2+ extrusion or Ca2+ sequestration processes did not significantly alter early decay, arguing against a role for either mechanism. Fluorescence transients recorded with low mobility or fixed indicators exhibited early decay similar to that recorded with indicators of comparable affinity but high mobility, suggesting that early decay was not due to the rate of Ca2+-indicator diffusion. The extent of early decay correlated closely with the affinity, but not mobility, of the Ca2+ sensitive dyes tested. These results implicate intrinsic buffers with slow Ca2+ binding kinetics as the most likely determinants of early decay. However, computer simulations showed that intrinsic buffers with a slow binding rate are unlikely to be the only ones present in the system because the slow kinetics would be unable to buffer incoming Ca2+ during an action potential and would result in momentary indicator saturation. In fact, experimental data show that the peak amplitude of an action potential activated Ca+ transient is about 20% of the maximal fluorescence intensity activated by prolonged Ca2+ influx. We conclude that endogenous buffering at the crayfish NMJ includes both fast and slow components, the former being fast enough to compete with fast Ca2+ indicators, and the latter dictating the early decay.
将具有不同亲和力和迁移率的钙离子指示剂压力注入小龙虾神经肌肉接头（NMJ）抑制剂的突触前轴突中。以2千赫兹分辨率记录的荧光瞬变用于探测控制动作电位后100毫秒内荧光瞬变衰减（早期衰减）的生理参数。阻断钙离子外流或钙离子螯合过程并不会显著改变早期衰减，这表明这两种机制都不起作用。用低迁移率或固定指示剂记录的荧光瞬变显示出的早期衰减与用具有相当亲和力但高迁移率的指示剂记录的相似，这表明早期衰减并非由于钙离子指示剂的扩散速率。早期衰减的程度与所测试的钙离子敏感染料的亲和力密切相关，但与迁移率无关。这些结果表明，具有缓慢钙离子结合动力学的内在缓冲剂最有可能是早期衰减的决定因素。然而，计算机模拟表明，结合速率缓慢的内在缓冲剂不太可能是系统中唯一存在的缓冲剂，因为缓慢的动力学无法在动作电位期间缓冲进入的钙离子，会导致指示剂瞬间饱和。事实上，实验数据表明，动作电位激活的钙离子瞬变的峰值幅度约为长时间钙离子内流激活的最大荧光强度的20%。我们得出结论，小龙虾神经肌肉接头处的内源性缓冲包括快速和缓慢成分，前者速度足够快，能够与快速钙离子指示剂竞争，后者则决定早期衰减。
