Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
J Neurosci. 2010 Jun 9;30(23):7863-77. doi: 10.1523/JNEUROSCI.4574-09.2010.
The norepinephrine transporter (NET) is a presynaptic plasma membrane protein that mediates reuptake of synaptically released norepinephrine. NET is also a major target for medications used for the treatment of depression, attention deficit/hyperactivity disorder, narcolepsy, and obesity. NET is regulated by numerous mechanisms, including catalytic activation and membrane trafficking. Amphetamine (AMPH), a psychostimulant and NET substrate, has also been shown to induce NET trafficking. However, neither the molecular basis nor the nature of the relevant membrane compartments of AMPH-modulated NET trafficking has been defined. Indeed, direct visualization of drug-modulated NET trafficking in neurons has yet to be demonstrated. In this study, we used a recently developed NET antibody and the presence of large presynaptic boutons in sympathetic neurons to examine basal and AMPH-modulated NET trafficking. Specifically, we establish a role for Rab11 in AMPH-induced NET trafficking. First, we found that, in cortical slices, AMPH induces a reduction in surface NET. Next, we observed AMPH-induced accumulation and colocalization of NET with Rab11a and Rab4 in presynaptic boutons of cultured neurons. Using tagged proteins, we demonstrated that NET and a truncated Rab11 effector (FIP2DeltaC2) do not redistribute in synchrony, whereas NET and wild-type Rab11a do. Analysis of various Rab11a/b mutants further demonstrates that Rab11 regulates NET trafficking. Expression of the truncated Rab11a effector (FIP2DeltaC2) attenuates endogenous Rab11 function and prevented AMPH-induced NET internalization as does GDP-locked Rab4 S22N. Our data demonstrate that AMPH leads to an increase of NET in endosomes of single boutons and varicosities in a Rab11-dependent manner.
去甲肾上腺素转运体（NET）是一种突触前质膜蛋白，介导突触释放的去甲肾上腺素的再摄取。NET 也是用于治疗抑郁症、注意力缺陷多动障碍、嗜睡症和肥胖症的药物的主要靶点。NET 受许多机制的调节，包括催化激活和膜运输。安非他命（AMPH），一种精神兴奋剂和 NET 底物，也已被证明能诱导 NET 转运。然而，AMPH 调节的 NET 转运的分子基础和相关膜隔室的性质尚未确定。事实上，药物调节的 NET 转运在神经元中的直接可视化尚未得到证明。在这项研究中，我们使用了一种新开发的 NET 抗体和交感神经元中存在的大突触末梢，来研究基础和 AMPH 调节的 NET 转运。具体来说，我们确定 Rab11 在 AMPH 诱导的 NET 转运中的作用。首先，我们发现，在皮质切片中，AMPH 诱导表面 NET 的减少。接下来，我们观察到 AMPH 诱导 NET 在培养神经元的突触末梢中与 Rab11a 和 Rab4 积累和共定位。使用标记蛋白，我们证明 NET 和截断的 Rab11 效应物（FIP2DeltaC2）不能同步重新分布，而 NET 和野生型 Rab11a 可以。对各种 Rab11a/b 突变体的分析进一步表明，Rab11 调节 NET 转运。截断的 Rab11a 效应物（FIP2DeltaC2）的表达减弱了内源性 Rab11 功能，并阻止了 AMPH 诱导的 NET 内化，正如 GDP 锁定的 Rab4 S22N 所阻止的那样。我们的数据表明，AMPH 导致单个末梢和小球中的 NET 在 Rab11 依赖性方式下增加内体。
