*Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6BT, U.K.
†Dulbecco Telethon Institute c/o Department of Neuroscience, IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy.
Biochem J. 2014 Mar 1;458(2):365-74. doi: 10.1042/BJ20131405.
It has been shown recently that PrP (prion protein) and the calcium channel auxiliary α2δ subunits interact in neurons and expression systems [Senatore, Colleoni, Verderio, Restelli, Morini, Condliffe, Bertani, Mantovani, Canovi, Micotti, Forloni, Dolphin, Matteoli, Gobbi and Chiesa (2012) Neuron 74, 300-313]. In the present study we examined whether there was an effect of PrP on calcium currents. We have shown that when PrP is co-expressed with calcium channels formed from CaV2.1/β and α2δ-1 or α2δ-2, there is a consistent decrease in calcium current density. This reduction was absent when a PrP construct was used lacking its GPI (glycosylphosphatidylinositol) anchor. We have reported previously that α2δ subunits are able to form GPI-anchored proteins [Davies, Kadurin, Alvarez-Laviada, Douglas, Nieto-Rostro, Bauer, Pratt and Dolphin (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 1654-1659] and show further evidence in the present paper. We have characterized recently a C-terminally truncated α2δ-1 construct, α2δ-1ΔC, and found that, despite loss of its membrane anchor, it still shows a partial ability to increase calcium currents [Kadurin, Alvarez-Laviada, Ng, Walker-Gray, D'Arco, Fadel, Pratt and Dolphin (2012) J. Biol. Chem. 1287, 33554-33566]. We now find that PrP does not inhibit CaV2.1/β currents formed with α2δ-1ΔC, rather than α2δ-1. It is possible that PrP and α2δ-1 compete for GPI-anchor intermediates or trafficking pathways, or that interaction between PrP and α2δ-1 requires association in cholesterol-rich membrane microdomains. Our additional finding that CaV2.1/β1b/α2δ-1 currents were inhibited by GPI-GFP, but not cytosolic GFP, indicates that competition for limited GPI-anchor intermediates or trafficking pathways may be involved in PrP suppression of α2δ subunit function.
最近有研究表明，朊病毒蛋白（PrP）和钙通道辅助α2δ亚基在神经元和表达系统中相互作用[Senatore, Colleoni, Verderio, Restelli, Morini, Condliffe, Bertani, Mantovani, Canovi, Micotti, Forloni, Dolphin, Matteoli, Gobbi and Chiesa (2012) Neuron 74, 300-313]。在本研究中，我们检查了 PrP 是否对钙电流有影响。我们已经表明，当 PrP 与由 CaV2.1/β 和 α2δ-1 或 α2δ-2 形成的钙通道共表达时，钙电流密度会持续下降。当使用缺乏其糖基磷脂酰肌醇（GPI）锚定的 PrP 构建体时，这种减少是不存在的。我们之前曾报道过，α2δ 亚基能够形成 GPI 锚定蛋白[Davies, Kadurin, Alvarez-Laviada, Douglas, Nieto-Rostro, Bauer, Pratt and Dolphin (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 1654-1659]，并在本文中进一步提供了证据。我们最近对截短的 C 端α2δ-1 构建体α2δ-1ΔC 进行了特征描述，并发现尽管其膜锚丢失，但它仍然具有增加钙电流的部分能力[Kadurin, Alvarez-Laviada, Ng, Walker-Gray, D'Arco, Fadel, Pratt and Dolphin (2012) J. Biol. Chem. 1287, 33554-33566]。我们现在发现 PrP 不会抑制与α2δ-1ΔC 形成的 CaV2.1/β 电流，而是抑制与α2δ-1 形成的 CaV2.1/β 电流。PrP 和α2δ-1 可能竞争 GPI-锚定中间体或运输途径，或者 PrP 与α2δ-1 的相互作用需要在富含胆固醇的膜微区中发生。我们的另一个发现是，CaV2.1/β1b/α2δ-1 电流被 GPI-GFP 抑制，但不是胞质 GFP 抑制，这表明对有限的 GPI-锚定中间体或运输途径的竞争可能参与了 PrP 对α2δ 亚基功能的抑制。
