INTERACTIONS BETWEEN H+ AND CA+ IN AXON

长期目标是更好地理解该法规。 细胞内游离钙(Ca~(2+))i和游离质子(H~+)i和 神经细胞中这两种离子浓度之间的相互依赖关系。 具体目标是研究分离的轴浆和完整的轴突。 在粘液巨型轴突中，有四个位点的(Ca~(2+))i调节机制： (A)质膜，(B)线粒体，(C)细胞器，如 内质网，通过三磷酸腺苷的水解来隔离钙，和(D)细胞 通过纯粹的物理化学过程结合钙的化学成分。这个 方法是从生理上区分不同类别的钙结合部位 通过原位研究琥珀酸依赖结合(线粒体)， 依赖于ATP的结合和能量不依赖的结合。对…的描述 分离的轴浆中的这些钙缓冲部位将由三种类型组成 测量范围：(A)轴浆的钙含量将测定如下 与之处于平衡状态的游离钙的函数。(B)同位素 分离的轴浆的ca40之间的自我交换(保存在聚乙烯中 管)和含有相同游离钙的相邻溶液的Ca45 量过了。已经迁移到轴浆中的Ca45的轮廓将是 通过冷冻样品，切开轴浆的圆柱形塞子来确定 放在25微米厚的圆盘中，并测定放射性。结果将是 用一种方法分析得出钙扩散系数和 钙结合反应的性质。(C)钙向内的净扩散 轴浆将用离子敏感微电极和 使用扩散-反应方程对行为进行分析。在完整的轴突中， 从注入的钙负荷中恢复的动力学将作为一个 负载相对于质膜的位置的函数。 将这些结果与分离的轴浆中的结果进行比较将提供 质膜在钙调节中的作用在其他 实验中，我们将研究Phi对Na-Ca交换的影响。而当 Phi是不同的，pCai将通过使用外源 向轴突内注入pH非依赖性钙缓冲系统。抑制力 H+I对钠氧依赖的钙外流和钙氧依赖的钠外流的影响 通过动力学检验来确定H+和H+之间的相互作用 Ca~(2+)或H~+和Na~+之间是竞争性的、非竞争性的或混合的。

Long-term objectives are to gain a better understanding of the regulation of both intracellular free calcium (Ca2+)i and free protons (H+)i and the interdependence between these two ion concentrations in nerve cells. Specific aims are to study, both in isolated axoplasm and in intact axons of the Myxicola giant axon, (Ca2+)i regulatory mechanisms at four loci: (a) the plasma membrane, (b) the mitochondrion, (c) organelles such as the endoplasmic reticulum that sequester Ca by ATP hydrolysis, and (d) the cell constituents that bind Ca by purely physio-chemical processes. The approach will be to physiologically distinguish classes of Ca binding sites in situ by studying succinate-dependent binding (mitochondria), ATP-dependent binding, and energy-independent binding. A description of these Ca buffering sites in isolated axoplasm will be made from three types of measurements: (a) The Ca content of the axoplasm will be determined as a function of the free Ca with which it is in equilibrium. (b) Isotopic self-exchange between Ca40 of isolated axoplasm (held in a polyethelene tube) and Ca45 of an adjacent solution containing the same free Ca will be measured. The profile of Ca45 that has migrated into the axoplasm will be determined by freezing the sample, slicing the cylindrical plug of axoplasm in 25 um thick disks, and assaying the radioactivity. Results will be analyzed by a method which yields the Ca diffusion coefficient and properties of the Ca binding reactions. (c) Net diffusion of Ca into axoplasm will be monitored with ion-sensitive microelectrodes and the behavior analyzed using the diffusion-reaction equation. In intact axons, the kinetics of recovery from injected Ca loads will be examined as a function of the location of the load with respect to the plasma membrane. Comparison of these results with results in isolated axoplasm will provide a measure for the role of the plasmalemma in Ca regulation. In other experiments, the effect of pHi on Na-Ca exchange will be studied. While pHi is varied, pCai will be held constant through the use of an exogenous pH-independent Ca buffering system injected into the axon. The inhibition of Na-o-dependent Ca efflux and Ca-o-dependent Na efflux by H+i will be examined kinetically to determine whether the interaction between H+ and Ca2+ or between H+ and Na+ is competitive, uncompetitive, or mixed.