Division of Cardiology, Department of Medicine, and the Ion Channel Research Unit, Duke University Medical Center, Durham, North Carolina, United States of America.
PLoS One. 2011;6(9):e25741. doi: 10.1371/journal.pone.0025741. Epub 2011 Sep 29.
Ca²⁺ influx through voltage-gated Ca²⁺ channels (VGCCs) plays important roles in neuronal cell development and function. Rem2 is a member of the RGK (Rad, Rem, Rem2, Gem/Kir) subfamily of small GTPases that confers potent inhibition upon VGCCs. The physiologic roles of RGK proteins, particularly in the brain, are poorly understood. Rem2 was implicated in synaptogenesis through an RNAi screen and proposed to regulate Ca²⁺ homeostasis in neurons. To test this hypothesis and uncover physiological roles for Rem2 in the brain, we investigated the molecular mechanisms by which Rem2 knockdown affected synaptogenesis and Ca²⁺ homeostasis in cultured rat hippocampal neurons. Expression of a cocktail of shRNAs targeting rat Rem2 (rRem2) reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) measured 10 d after transfection (14 d in vitro), but did not affect mEPSC amplitude. VGCC current amplitude after rRem2-targeted knockdown was not different from that in control cells, however, at either 4 or 10 d post transfection. Co-expression of a human Rem2 that was insensitive to the shRNAs targeting rRem2 was unable to prevent the reduction in mEPSC frequency after rRem2-targeted knockdown. Over-expression of rRem2 resulted in 50% reduction in VGCC current, but neither the mEPSC frequency nor amplitude was affected. Taken together, the observed effects upon synaptogenesis after shRNA treatment are more likely due to mechanisms other than modulation of VGCCs and Ca²⁺ homeostasis, and may be independent of Rem2. In addition, our results reveal a surprising lack of contribution of VGCCs to synaptogenesis during early development in cultured hippocampal neurons.
钙离子通过电压门控钙离子通道（VGCCs）内流在神经元细胞的发育和功能中发挥重要作用。Rem2 是小 GTP 酶 RGK（Rad、Rem、Rem2、Gem/Kir）亚家族的成员，对 VGCC 具有强大的抑制作用。RGK 蛋白的生理作用，特别是在大脑中的作用，知之甚少。Rem2 通过 RNAi 筛选被牵连到突触发生中，并被提议调节神经元中的钙离子稳态。为了验证这一假说并揭示 Rem2 在大脑中的生理作用，我们研究了 Rem2 敲低影响培养的大鼠海马神经元突触发生和钙离子稳态的分子机制。针对大鼠 Rem2（rRem2）的 shRNA 鸡尾酒的表达降低了转染后 10 天（体外 14 天）测量的微小兴奋性突触后电流（mEPSC）的频率，但不影响 mEPSC 幅度。然而，在 rRem2 靶向敲低后 4 天或 10 天，rRem2 靶向敲低后的 VGCC 电流幅度与对照细胞没有不同。共表达对靶向 rRem2 的 shRNA 不敏感的人 Rem2 不能防止 rRem2 靶向敲低后 mEPSC 频率的降低。rRem2 的过表达导致 VGCC 电流减少 50%，但 mEPSC 频率和幅度均不受影响。综上所述，shRNA 处理后突触发生的观察到的影响更可能是由于除了调制 VGCC 和钙离子稳态之外的机制，并且可能与 Rem2 无关。此外，我们的结果揭示了在培养的海马神经元中早期发育过程中 VGCC 对突触发生的贡献出人意料地缺乏。
