Lacinová L
Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava 37, Slovakia.
Gen Physiol Biophys. 2005 Jun;24 Suppl 1:1-78.
Voltage-activated calcium channels can be divided into two subgroups based on their activation threshold, low-voltage-activated (LVA) and high-voltage-activated (HVA). Auxiliary subunits of the HVA calcium channels contribute significantly to biophysical properties of the channels. We have cloned and characterized members of two families of auxiliary subunits: alpha2delta and gamma. Two new alpha2delta subunits, alpha2delta-2 and alpha2delta-3, regulate all classes of HVA calcium channels. While the ubiquitous alpha2delta-2 modulates both neuronal and non-neuronal channels with similar efficiency, the alpha2delta-3 subunit regulates Ca(v)2.3 channels more effectively. Furthermore, alpha2delta-2 may modulate the LVA Ca(v)3.1 channel. Four new gamma subunits, gamma-2, gamma-3, gamma-4 and gamma-5, were characterized. The gamma-2 subunit modulated both the non-neuronal Ca(v)1.2 channel and the neuronal Ca(v)2.1 channel. The gamma-4 subunit affected only the Ca(v)2.1 channel. The gamma-5 subunit may be a regulatory subunit of the LVA Ca(v)3.1 channel. The Ca(v)1.2 channel is a major target for treatment of cardiovascular diseases. We have mapped the interaction site for clinically important channel blockers - dihydropyridines (DHPs) - and analysed the underlying inhibition mechanism. High-affinity inhibition is characterized by interaction with inactivated state of the channel. Its structural determinants are amino acids of the IVS6 segment, with smaller contribution of the IS6 segment, which contributes to voltage-dependence of DHP inhibition. Removal of amino acids responsible for the high-affinity inhibition revealed a low-affinity open channel block, in which amino acids of the IIIS5 and IIIS6 segments take part. Experiments with a permanently charged DHP suggested that there is another low-affinity interaction site on the alpha(1) subunit. We have cloned and characterized murine neuronal LVA Ca(v)3.1 channel. The channel has high sensitivity to the organic blocker mibefradil, moderate sensitivity to phenytoin, and low sensitivity to ethosuximide, amiloride and valproat. The channel is insensitive to tetrodotoxin and DHPs. The inorganic blockers Ni2+ and Cd2+ are moderately effective compared to La3+. The current through the Ca(v)3.1 channel inactivates faster with Ba2+ compared to Ca2+. Molecular determinants of fast inactivation are located in amino side of the intracellular carboxy terminus. The voltage dependence of charge movement is very shallow compared to the voltage dependence of current activation. Transfer of 30 % of charge correlates with activation of 70 % of measurable macroscopic current. Prolonged depolarization does not immobilize charge movement of the Ca(v)3.1 channel.
电压门控性钙通道可根据其激活阈值分为两个亚组，即低电压激活（LVA）通道和高电压激活（HVA）通道。HVA钙通道的辅助亚基对通道的生物物理特性有重要影响。我们已克隆并鉴定了两个辅助亚基家族的成员：α2δ和γ。两个新的α2δ亚基，α2δ-2和α2δ-3，可调节所有类型的HVA钙通道。普遍存在的α2δ-2以相似的效率调节神经元和非神经元通道，而α2δ-3亚基更有效地调节Ca(v)2.3通道。此外，α2δ-2可能调节LVA Ca(v)3.1通道。鉴定了四个新的γ亚基，γ-2、γ-3、γ-4和γ-5。γ-2亚基调节非神经元Ca(v)1.2通道和神经元Ca(v)2.1通道。γ-4亚基仅影响Ca(v)2.1通道。γ-5亚基可能是LVA Ca(v)3.1通道的调节亚基。Ca(v)1.2通道是治疗心血管疾病的主要靶点。我们已确定了临床上重要的通道阻滞剂——二氢吡啶（DHP）的相互作用位点，并分析了其潜在的抑制机制。高亲和力抑制的特征是与通道的失活状态相互作用。其结构决定因素是IVS6片段的氨基酸，IS6片段的贡献较小，IS6片段有助于DHP抑制的电压依赖性。去除负责高亲和力抑制的氨基酸后，揭示了一种低亲和力的开放通道阻滞，其中IIIS5和IIIS6片段的氨基酸参与其中。使用永久带电的DHP进行的实验表明，α(1)亚基上还有另一个低亲和力相互作用位点。我们已克隆并鉴定了小鼠神经元LVA Ca(v)3.1通道。该通道对有机阻滞剂米贝地尔高度敏感，对苯妥英中度敏感，对乙琥胺、阿米洛利和丙戊酸低度敏感。该通道对河豚毒素和DHP不敏感。与La3+相比，无机阻滞剂Ni2+和Cd2+的效果中等。与Ca2+相比，通过Ca(v)3.1通道的电流在Ba2+存在时失活更快。快速失活的分子决定因素位于细胞内羧基末端的氨基侧。与电流激活的电压依赖性相比，电荷移动的电压依赖性非常浅。30%的电荷转移与70%可测量的宏观电流的激活相关。长时间去极化不会使Ca(v)3.1通道的电荷移动固定。
