Smith S M, Piedras-Renterà E S, Namkung Y, Shin H S, Tsien R W
Department of Molecular and Cellular Physiology, Beckman Center, Stanford University Medical Center, California 94305, USA.
Ann N Y Acad Sci. 1999 Apr 30;868:175-98. doi: 10.1111/j.1749-6632.1999.tb11286.x.
Many neurons of the central and peripheral nervous systems display multiple high voltage-activated (HVA) Ca2+ currents, often classified as L-, N-, P-, Q, and R-type. The heterogeneous properties of these channels have been attributed to diversity in their pore-forming alpha 1, subunits, in association with various beta subunits. However, there are large gaps in understanding how individual subunits contribute to Ca2+ channel diversity. Here we describe experiments to investigate the roles of alpha 1E and beta 3 subunits in mammalian neurons. The alpha 1E subunit is the leading candidate to account for the R-type channel, the least understood of the various types of high voltage-activated Ca2+ channels. Incubation with alpha 1E antisense oligonucleotide caused a 53% decrease in the peak R-type current density, while no significant changes in the current expression were seen in sense oligonucleotide-treated cells. The specificity of the alpha 1E antisense oligonucleotides was supported by the lack of change in the amplitude of P/Q current. These results upheld the hypothesis that members of the E class of alpha 1 subunits support the high voltage-activated R-type current in cerebellar granule cells. We studied the role of the Ca2+ channel beta 3 subunit using a gene targeting strategy. In sympathetic beta 3-/- neurons, the L-type current was significantly reduced relative to wild type (wt). In addition, N-type Ca2+ channels made up a smaller proportion of the total Ca2+ current than in wt due to a lower N-type current density in a group of neurons with small total currents. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence. The absence of the beta 3 subunit was associated with a shift in the more depolarized component of the activation along the voltage axis toward more negative potentials. The overall conclusion is that deletion of the beta 3 subunit affects at least three distinct types of HVA Ca2+ channel, but no single type of channel is solely dependent on beta 3.
中枢神经系统和周围神经系统的许多神经元都表现出多种高电压激活（HVA）Ca2+电流，通常分为L型、N型、P型、Q型和R型。这些通道的异质性归因于其孔形成α1亚基与各种β亚基结合后的多样性。然而，在理解单个亚基如何促成Ca2+通道多样性方面仍存在很大差距。在此，我们描述了研究α1E和β3亚基在哺乳动物神经元中作用的实验。α1E亚基是解释R型通道的主要候选者，R型通道是各类高电压激活Ca2+通道中了解最少的。用α1E反义寡核苷酸孵育导致R型电流峰值密度降低53%，而在正义寡核苷酸处理的细胞中未观察到电流表达的显著变化。P/Q电流幅度没有变化，这支持了α1E反义寡核苷酸的特异性。这些结果支持了以下假设：α1亚基E类成员支持小脑颗粒细胞中的高电压激活R型电流。我们使用基因靶向策略研究了Ca2+通道β3亚基的作用。在交感神经β3-/-神经元中，L型电流相对于野生型（wt）显著降低。此外，由于一组总电流较小的神经元中N型电流密度较低，N型Ca2+通道在总Ca2+电流中所占比例比wt小。P/Q型Ca2+通道的电压依赖性激活由两个具有不同电压依赖性的玻尔兹曼成分描述。β3亚基的缺失与激活的更去极化成分沿电压轴朝着更负电位的偏移有关。总体结论是，β3亚基的缺失影响至少三种不同类型的HVA Ca2+通道，但没有单一类型的通道完全依赖于β3。
