Dove L S, Abbott L C, Griffith W H
Department of Medical Pharmacology and Toxicology, College of Medicine, Texas A&M University Health Science Center, College Station, Texas 77843-1114, USA.
J Neurosci. 1998 Oct 1;18(19):7687-99. doi: 10.1523/JNEUROSCI.18-19-07687.1998.
The leaner (tgla) mutation in mice results in severe ataxia and an overt neurodegeneration of the cerebellum. Positional cloning has revealed that the tgla mutation occurs in a gene encoding the voltage-activated calcium channel alpha1A subunit. The alpha1A subunit is highly expressed in the cerebellum and is thought to be the pore-forming subunit of P- and Q-type calcium channels. In this study we used both whole-cell and single-channel patch-clamp recordings to examine the functional consequences of the tgla mutation on P-type calcium currents. High-voltage-activated (HVA) calcium currents were recorded from acutely dissociated cerebellar Purkinje cells of homozygous leaner (tgla/tgla) and age-matched wild-type (+/+) mice. In whole cell recordings, we observed a marked reduction of peak current density in tgla/tgla Purkinje cells (-35.0 +/- 1.8 pA/pF) relative to that in +/+ (-103.1 +/- 5.9 pA/pF). The reduced whole-cell current in tgla/tgla cells was accompanied by little to no alteration in the voltage dependence of channel gating. In both genotypes, HVA currents were predominantly of the omega-agatoxin-IVA-sensitive P-type. Cell-attached patch-clamp recordings revealed no differences in single-channel conductance between the two genotypes and confirmed the presence of three distinct conductance levels (9, 13-14, and 17-18 pS) in cerebellar Purkinje cells. Analysis of patch open-probability (NPo) revealed a threefold reduction in the open-probability of channels in tgla/tgla patches (0.04 +/- 0.01) relative to that in +/+ (0.13 +/- 0.02), which may account for the reduced whole-cell current in tgla/tgla Purkinje cells. These results suggest that the tgla mutation can alter native P-type calcium channels at the single-channel level and that these alterations may contribute to the neuropathology of the leaner phenotype.
小鼠中的瘦素（tgla）突变会导致严重共济失调和小脑明显的神经退行性变。定位克隆显示，tgla突变发生在一个编码电压激活钙通道α1A亚基的基因中。α1A亚基在小脑中高度表达，被认为是P型和Q型钙通道的孔形成亚基。在本研究中，我们使用全细胞和单通道膜片钳记录来研究tgla突变对P型钙电流的功能影响。从纯合瘦素（tgla/tgla）和年龄匹配的野生型（+/+）小鼠急性分离的小脑浦肯野细胞中记录高压激活（HVA）钙电流。在全细胞记录中，我们观察到tgla/tgla浦肯野细胞的峰值电流密度（-35.0±1.8 pA/pF）相对于+/+小鼠（-103.1±5.9 pA/pF）显著降低。tgla/tgla细胞中全细胞电流的降低伴随着通道门控电压依赖性的几乎没有改变。在两种基因型中，HVA电流主要是对ω-芋螺毒素-IVA敏感的P型。细胞贴附式膜片钳记录显示两种基因型之间单通道电导没有差异，并证实小脑浦肯野细胞中存在三种不同的电导水平（9、13 - 14和17 - 18 pS）。膜片开放概率（NPo）分析显示，tgla/tgla膜片中通道的开放概率（0.04±0.01）相对于+/+（0.13±0.02）降低了三倍，这可能解释了tgla/tgla浦肯野细胞中全细胞电流的降低。这些结果表明，tgla突变可以在单通道水平改变天然P型钙通道，并且这些改变可能导致瘦素表型的神经病理学。
