Loyola University Chicago, Maywood, IL, USA.
Am J Physiol Cell Physiol. 2010 Jun;298(6):C1353-62. doi: 10.1152/ajpcell.00235.2009. Epub 2010 Feb 10.
We report a novel form of modulation of T-type calcium currents carried out by the neuronal actin-binding protein (ABP) Kelch-like 1 (KLHL1). KLHL1 is a constitutive neuronal ABP localized to the soma and dendritic arbors; its genetic elimination in Purkinje neurons leads to dendritic atrophy and motor insufficiency. KLHL1 participates in neurite outgrowth and upregulates voltage-gated P/Q-type calcium channel function; here we investigated KLHL1's role as a modulator of low-voltage-gated calcium channels and determined the molecular mechanism of this modulation with electrophysiology and biochemistry. Coexpression of KLHL1 with Ca(V)3.1 or Ca(V)3.2 (alpha(1G) or alpha(1H) subunits) caused increases in T-type current density (35%) and calcium influx (75-83%) when carried out by alpha(1H) but not by alpha(1G). The association between KLHL1 and alpha(1H) was determined by immunoprecipitation and immunolocalization in brain membrane fractions and in vitro in HEK-293 cells. Noise analysis showed that neither alpha(1H) single-channel conductance nor open probability was altered by KLHL1, yet a significant increase in channel number was detected and further corroborated by Western blot analysis. KLHL1 also induced an increase in alpha(1H) current deactivation time (tau(deactivation)). Interestingly, the majority of KLHL1's effects were eliminated when the actin-binding motif (kelch) was removed, with the exception of the calcium influx increase during action potentials, indicating that KLHL1 interacts with alpha(1H) and actin and selectively regulates alpha(1H) function by increasing the number of alpha(1H) channels. This constitutes a novel regulatory mechanism of T-type calcium currents and supports the role of KLHL1 in the modulation of cellular excitability.
我们报告了一种新型的 T 型钙电流调制形式，由神经元肌动蛋白结合蛋白 (ABP) Kelch-like 1 (KLHL1) 执行。KLHL1 是一种组成型神经元 ABP，定位于胞体和树突树突；其在浦肯野神经元中的遗传消除导致树突萎缩和运动功能不足。KLHL1 参与轴突生长，并上调电压门控 P/Q 型钙通道功能；在这里，我们研究了 KLHL1 作为低电压门控钙通道调节剂的作用，并通过电生理学和生物化学确定了这种调节的分子机制。KLHL1 与 Ca(V)3.1 或 Ca(V)3.2（alpha(1G) 或 alpha(1H) 亚基）共表达时，当由 alpha(1H)而不是 alpha(1G)进行时，会导致 T 型电流密度增加（35%）和钙内流增加（75-83%）。KLHL1 与 alpha(1H)的结合通过脑膜部分的免疫沉淀和免疫定位以及在体外的 HEK-293 细胞中确定。噪声分析表明，KLHL1 既不改变 alpha(1H) 单通道电导也不改变 alpha(1H) 开放概率，但检测到通道数量显著增加，并通过 Western blot 分析进一步证实。KLHL1 还诱导 alpha(1H) 电流失活时间（tau(deactivation)）增加。有趣的是，当肌动蛋白结合基序（kelch）被去除时，KLHL1 的大部分作用都被消除，除了动作电位期间的钙内流增加，表明 KLHL1 与 alpha(1H)和肌动蛋白相互作用，并通过增加 alpha(1H) 通道的数量选择性地调节 alpha(1H) 功能。这构成了 T 型钙电流的一种新型调节机制，并支持 KLHL1 在细胞兴奋性调节中的作用。
