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)初级传入神经元中的电活动影响基因转录。将电活动与生化反应联系起来的一个可能的信号级联始于细胞内钙离子([Ca2+]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这一级联反应的功能。然而，对于生理温度下背根神经节神经元内[Ca~(2+)]i的控制，以及生化效应器的特性和它们是如何工作的，人们知之甚少。为了了解依赖活性的基因表达调控，我们提出了一些实验，这些实验将研究动作电位刺激下[Ca~(2+)]i的生理变化，控制这些变化的机制，以及Ca~(2+)/钙调蛋白依赖的蛋白激酶II(CaMKII)对Ca~(2+)内流的反应。我们将调查三个假说。(1)生理温度下激活的不动钙缓冲作用，将电压门控钙通道引起的钙内流限制在质膜附近的体积内。(2)在生理温度下，CICR不能增加钙离子内流，提高钙离子浓度。(3)生理钙离子瞬变是由CaMKII编码的。如果前两个假设是正确的，那么通过电压门控钙离子通道进入的钙离子不太可能增加细胞核内的游离钙离子。在这种情况下，钙离子必须与质膜附近的钙结合蛋白相互作用，这种复合体必须影响基因转录。我们将使用电生理测量(钙电流的动作电位和电压钳测量)与[Ca~(2+)]i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+)内流和钙缓冲。我们将使用活性分析来研究CaMKII在静息和活性依赖性钙内流时自主活性的增加。

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.