The role of trace metals and T-channels in pain

描述（由申请人提供）：顽固性疼痛对生活质量指标有显著的不良影响，现有的治疗方法都不能完全逆转这些症状。最近的研究已经证实，CaV 3.2 T型电压门控钙通道（T通道）在外周伤害感受器的细胞兴奋性控制中起关键作用 因此代表了用于开发疼痛疗法的有希望的新细胞靶点。在我们以前的工作中，我们确定了几种内源性金属螯合剂，包括氨基酸，如L-组氨酸和L-半胱氨酸，可以选择性地调节T通道的CaV3.2亚型的功能，从而影响背根神经节（DRG）的伤害性感觉神经元的神经元兴奋性。重要的是，我们的初步数据揭示了CaV3.2 T通道在支持脊髓背角（DH）浅层伤害性神经元中自发兴奋性突触传递中的新作用。此外，我们发现了一种新的内源性血清因子，暂命名为“物质T”，这是约100倍更有效的天然和重组CaV3.2 T-电流的调制器比以前描述的化合物已知的调节这些通道的功能。基于尺寸排阻透析，已知Cav3.2通道通过H191对金属螯合剂的敏感性，以及H191 Q突变体中“物质T”效应的丧失，我们假设T物质是一种低分子量肽，可作为微量金属的螯合剂，其可增加伤害感受性DRG和DH神经元的细胞兴奋性，并因此通过增强CaV3.2的功能来调节疼痛感觉渠道我们计划测试我们的中心假设，并通过追求以下具体目标来实现本申请的目的：目的1：鉴定作为CaV3.2电流的内源性调节剂发挥作用的推定血清因子。具体的实验最初将集中在使用固定化金属亲和柱，反相高效液相色谱法（HPLC），质谱和重组CaV3.2 T-电流的膜片钳记录的这种分子的识别。目标二：探讨内源性微量金属在脊髓背角和背根神经节痛觉传递中的作用。我们将使用膜片钳记录从本地DH和DRG神经元和体内疼痛的研究，旨在研究“T物质”，微量金属和金属螯合剂在调节细胞的兴奋性和疼痛传递在这些神经元的生理作用。本文的创新之处在于提出了一种新的渠道监管机制。这在医学上具有重要意义，因为了解这一调控途径的细节将有助于开发针对这一途径中可能用于治疗顽固性神经性疼痛的步骤的新型疼痛疗法。

DESCRIPTION (provided by applicant): Intractable pain has significant adverse effects on quality of life measures, and none of the available treatments can completely reverse these symptoms. Recent studies have established that CaV3.2 T- type voltage-gated calcium channels (T-channels) play a key role in control of cellular excitability of peripheral nociceptors and thus represent promising new cellular targets for development of pain therapies. In our previous work, we identified several endogenous metal chelators, including amino acids such as L-histidine and L-cysteine, which can selectively modulate function of the CaV3.2 isoform of T-channels and consequently influence neuronal excitability of nociceptive sensory neurons of dorsal root ganglia (DRG). Importantly, our Preliminary Data reveal a new role of CaV3.2 T-channels in supporting spontaneous excitatory synaptic transmission in nociceptive neurons of superficial lamina of the spinal cord dorsal horn (DH). Furthermore, we discovered a novel endogenous serum factor, tentatively named "Substance T" that is about 100-fold more potent a modulator of native and recombinant CaV3.2 T-currents than previously described compounds known to modulate function of these channels. Based on size exclusion dialysis, known sensitivity of Cav3.2 channels to metal chelators via H191, and loss of the "Substance T" effect in H191Q mutants, we hypothesize that "Substance T" is a low molecular weight peptide that can act as a chelator of trace metals that can increase cellular excitability of nociceptive DRG and DH neurons and consequently modulate pain sensation by potentiating function of CaV3.2 channels. We plan to test our central hypothesis and accomplish the objective of this application by pursuing the following specific aims: Aim 1: To identify the putative serum factor that functions as an endogenous modulator of CaV3.2 currents. Specific experiments will focus initially on the identification of this molecule using immobilized metal-affinity columns, reverse-phase high performance liquid chromatography (HPLC), mass spectrometry and patch-clamp recordings of recombinant CaV3.2 T-currents. Aim 2: To investigate the role of endogenous trace metals in modulation of pain transmission in the dorsal horn of the spinal cord and in dorsal root ganglion. We will use patch-clamp recordings from native DH and DRG neurons and in vivo pain studies designed to examine the physiological roles of "Substance T", trace metals and metal chelators in regulating cellular excitability and pain transmission in these neurons. The proposed work is innovative in that a new mechanism for channel regulation will be characterized. It is medically significant because understanding the details of this regulatory pathway will facilitate development of novel pain therapies targeting steps in this pathway that could be useful for treatment of intractable neuropathic pain.