Singh Anamika, Gebhart Mathias, Fritsch Reinhard, Sinnegger-Brauns Martina J, Poggiani Chiara, Hoda Jean-Charles, Engel Jutta, Romanin Christoph, Striessnig Jörg, Koschak Alexandra
Institute of Pharmacy, Pharmacology, and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, Peter-Mayr-Strasse 1/I, Innsbruck, Austria.
J Biol Chem. 2008 Jul 25;283(30):20733-44. doi: 10.1074/jbc.M802254200. Epub 2008 May 15.
Low voltage activation of Ca(V)1.3 L-type Ca(2+) channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Ca(V)1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in Ca(V)1.4 L-type Ca(2+) channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Ca(V)1.3 alpha1 subunit C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), we investigated if a C-terminal modulatory mechanism also controls Ca(V)1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with beta3 and alpha2delta1 subunits in HEK-293 cells. Activation of calcium current through Ca(V)1.3(42A) channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Ca(V)1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Ca(V)1.3(DeltaC116) channels showed gating properties similar to Ca(V)1.3(42A) that were reverted by co-expression of the corresponding C-terminal peptide C(116). Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Ca(V)1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Ca(V)1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Ca(V)1.3 channel activation at lower voltages expected to favor Ca(V)1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.
Ca(V)1.3 L型钙通道的低电压激活控制着感觉细胞、中枢神经元以及窦房结起搏的兴奋性。Ca(V)1.3介导的起搏决定了帕金森病中受影响的多巴胺能纹状体神经元的神经元易损性。我们之前发现，在Ca(V)1.4 L型钙通道中，激活、电压和钙依赖性失活受一个内在的远端C末端调节剂控制。由于Ca(V)1.3 α1亚基C末端的可变剪接产生了一种长形式（Ca(V)1.3(42)）和一种短形式（Ca(V)1.3(42A)），我们研究了C末端调节机制是否也控制Ca(V)1.3的门控。在HEK-293细胞中与β3和α2δ1亚基一起异源表达后，比较了两种剪接变体的生物物理特性。通过Ca(V)1.3(42A)通道的钙电流激活在负电压下更明显，并且由于增强的钙依赖性失活，失活更快。通过研究几种Ca(V)1.3通道截短体，我们将调节剂活性限制在C末端的最后116个氨基酸。所得的Ca(V)1.3(ΔC116)通道显示出门控特性类似于Ca(V)1.3(42A)，通过共表达相应的C末端肽C(116)可使其恢复。荧光共振能量转移实验证实了Ca(V)1.3通道C末端存在分子内蛋白质相互作用，该相互作用也调节钙调蛋白结合。这些实验揭示了一种新的通道调节机制，使细胞能够通过可变剪接紧密控制Ca(V)1.3通道活性。短剪接形式中C末端调节剂的缺失有助于Ca(V)1.3通道在较低电压下激活，预期这有利于在调节神经元放电行为和窦房结起搏所需的阈值电压下Ca(V)1.3的活性。
