Wakamori M, Yamazaki K, Matsunodaira H, Teramoto T, Tanaka I, Niidome T, Sawada K, Nishizawa Y, Sekiguchi N, Mori E, Mori Y, Imoto K
Department of Information Physiology, National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan.
J Biol Chem. 1998 Dec 25;273(52):34857-67. doi: 10.1074/jbc.273.52.34857.
Recent genetic and molecular biological analyses have revealed many forms of inherited channelopathies. Homozygous ataxic mice, tottering (tg) and leaner (tgla) mice, have mutations in the P/Q-type Ca2+ channel alpha1A subunit gene. Although their clinical phenotypes, histological changes, and locations of gene mutations are known, it remains unclear what phenotypes the mutant Ca2+ channels manifest, or whether the altered channel properties are the primary consequence of the mutations. To address these questions, we have characterized the electrophysiological properties of Ca2+ channels in cerebellar Purkinje cells, where the P-type is the dominant Ca2+ channel, dissociated from the normal, tg, and tgla mice, and compared them with the properties of the wild-type and mutant alpha1A channels recombinantly expressed with the alpha2 and beta subunits in baby hamster kidney cells. The most striking feature of Ca2+ channel currents of mutant Purkinje cells was a marked reduction in current density, being reduced to approximately 60 and approximately 40% of control in tg and tgla mice, respectively, without changes of cell size. The Ca2+ channel currents in the tg Purkinje cells showed a relative increase in non-inactivating component in voltage-dependent inactivation. Besides the same change, those of the tgla mice showed a more distinct change in voltage dependence of activation and inactivation, being shifted in the depolarizing direction by approximately 10 mV, with a broader voltage dependence of inactivation. In the recombinant expression system, the tg channel with a missense mutation (P601L) and one form of the two possible tgla aberrant splicing products, tgla (short) channel, showed a significant reduction in current density, while the other form of the tgla channels, tgla (long), had a current density comparable to the normal control. On the other hand, the shift in voltage dependence of activation and inactivation was observed only for the tgla (long) channel. Comparison of properties of the native and recombinant mutant channels suggests that single tottering mutations are directly responsible for the neuropathic phenotypes of reduction in current density and deviations in gating behavior, which lead to neuronal death and cerebellar atrophy.
最近的基因和分子生物学分析揭示了多种形式的遗传性离子通道病。纯合共济失调小鼠、蹒跚（tg）小鼠和瘦型（tgla）小鼠在P/Q型Ca2+通道α1A亚基基因中存在突变。尽管它们的临床表型、组织学变化和基因突变位置已知，但突变的Ca2+通道表现出何种表型，或者通道特性的改变是否是突变的主要后果仍不清楚。为了解决这些问题，我们对从小脑浦肯野细胞中分离出的Ca2+通道的电生理特性进行了表征，其中P型是主要的Ca2+通道，这些浦肯野细胞来自正常、tg和tgla小鼠，并将它们与在幼仓鼠肾细胞中与α-2和β亚基重组表达的野生型和突变型α1A通道的特性进行了比较。突变型浦肯野细胞Ca2+通道电流最显著的特征是电流密度显著降低，在tg和tgla小鼠中分别降至对照的约60%和约40%，而细胞大小没有变化。tg浦肯野细胞中的Ca2+通道电流在电压依赖性失活中显示出非失活成分的相对增加。除了相同的变化外，tgla小鼠的Ca2+通道电流在激活和失活的电压依赖性方面表现出更明显的变化，向去极化方向移动约10 mV，失活的电压依赖性更宽。在重组表达系统中，具有错义突变（P601L）的tg通道和两种可能的tgla异常剪接产物之一的tgla（短）通道显示电流密度显著降低，而另一种形式的tgla通道tgla（长）的电流密度与正常对照相当。另一方面，仅在tgla（长）通道中观察到激活和失活电压依赖性的移位。天然和重组突变通道特性的比较表明，单个蹒跚突变直接导致电流密度降低和门控行为偏差的神经病变表型，进而导致神经元死亡和小脑萎缩。
