Regulation of Intracellular Calcium in Sensory Neurons

描述（由申请人提供）：各种数据表明背根神经节（DRG）初级传入神经元的电活动影响基因转录。将电活动与生化反应联系起来的可能的信号级联始于细胞内Ca 2+（[Ca 2 +]i）的变化。此外，很明显，[Ca 2 +]i振荡的频率、动力学和亚细胞位置可以决定该级联的功能。然而，很少有人知道在生理温度下的DRG神经元中的[Ca 2 +]i的控制或有关的生化效应器的身份，以及它们如何工作。为了了解活性依赖的基因表达控制，我们提出的实验，将调查生理变化[Ca 2 +]i响应动作电位刺激，控制它们的机制，和Ca 2 +/钙调蛋白依赖性蛋白激酶II（CaMKII）的反应Ca 2+流入。我们将研究三个假设。(1)固定的Ca 2+缓冲，在生理温度下活跃，限制由Ca 2+通过电压门控Ca 2+通道流入质膜附近的体积引起的[Ca 2 +]i的增加。(2)在生理温度下，CICR不增加Ca ~（2+）内流和增加[Ca ~（2+）]i。(3)生理Ca 2+瞬变由CaMKII编码。如果前两个假设是正确的，那么通过电压门控Ca 2+通道进入的Ca 2+不太可能升高细胞核中的游离Ca 2+。在这种情况下，Ca 2+必须与质膜附近的Ca 2+结合蛋白相互作用，这种复合物必须影响基因转录。我们将使用电生理学测量（动作电位和电压钳测量钙电流）与[Ca 2 +]i的荧光测量相结合的实验。这些实验将研究生理温度下的Ca 2+内流和Ca 2+缓冲。我们将使用活性测定来研究CaMKII响应静息和活性依赖性Ca 2+内流的自主活性的增加。

DESCRIPTION (provided by applicant): A variety of data indicate that electrical activity in primary afferent neurons of the dorsal root ganglia (DRG) influences gene transcription. A likely signal cascade linking electrical activity to biochemical responses starts with a change in intracellular Ca2+ ([Ca2+]i). Further, it is clear that the frequency, kinetics, and subcellular location of oscillations in [Ca2+]i can determine the function of this cascade. However, little is known about the control of [Ca2+]i in DRG neurons at physiological temperature or about the identity of the biochemical effectors and how they work. In order to understand activity-dependent control of gene expression, we are proposing experiments that will investigate physiological changes in [Ca2+]i in response to action potential stimulation, the mechanisms that control them, and the response of Ca2+/calmodulin-dependent protein kinase II (CaMKII) to Ca2+ influx. We will investigate three hypotheses. (1) Immobile Ca2+ buffering, active at physiological temperature, restricts the increase in [Ca2+]i caused by Ca2+ influx via voltage-gated Ca2+ channels to the volume near the plasma membrane. (2) At physiological temperature, CICR does not amplify Ca2+ influx and raise bulk [Ca2+]i. (3) Physiological Ca2+ transients are encoded by CaMKII. If the first two hypotheses are correct, then Ca2+ entering via voltage-gated Ca2+ channels is unlikely to raise free Ca2+ in the nucleus. In this case, Ca2+ must interact with a Ca2+-binding protein near the plasma membrane and this complex must influence gene transcription. We will use experiments combining electrophysiological measurements (action potentials and voltage-clamp measurement of Ca2+ currents) with fluorescence measurements of [Ca2+]i. These experiments will investigate Ca2+ influx and Ca2+ buffering at physiological temperature. We will use activity assays to study the increase in autonomous activity of CaMKII in response to resting and activity-dependent Ca2+ influx.