The role of trace metals and T-channels in pain

描述(申请人提供)：顽固性疼痛对生活质量测量有显著的不利影响，现有的治疗方法都不能完全扭转这些症状。最近的研究证实，CaV3.2T型电压门控钙通道(T通道)在外周伤害性感受器的细胞兴奋性控制中起关键作用 从而为疼痛疗法的发展提供了新的细胞靶点。在我们以前的工作中，我们发现了几种内源性金属螯合剂，包括L-组氨酸和L-半胱氨酸等氨基酸，它们可以选择性地调节CaV3.2亚型T通道的功能，从而影响背根神经节伤害性感觉神经元的兴奋性。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浅层伤害性神经元自发兴奋性突触传递中的新作用。此外，我们还发现了一种新的内源性血清因子，暂定为T物质，它对天然和重组CaV3.2 T电流的调节剂比先前描述的调节这些通道功能的化合物强约100倍。基于尺寸排斥透析、已知的Cav3.2通道通过H191对金属螯合剂的敏感性以及H191Q突变体中“T物质”效应的丧失，我们假设“T物质”是一种小分子多肽，可以作为微量金属的螯合剂，可以增加伤害性DRG和DH神经元的细胞兴奋性，从而通过增强CaV3.2通道的功能来调节痛觉。我们计划验证我们的中心假设，并通过追求以下特定目标来实现这一应用的目标：目标1：确定可能的血清因素，作为CaV3.2电流的内源性调节器。具体的实验将首先集中于使用固定化金属亲和色谱柱、反相高效液相色谱(HPLC)、质谱仪和膜片钳记录重组CaV3.2 T电流来鉴定该分子。目的：探讨内源性微量金属在脊髓背角和背根神经节痛传递调控中的作用。我们将使用膜片钳记录来自天然的DH和DRG神经元，以及活体疼痛研究，旨在研究“T物质”、微量金属和金属螯合剂在调节这些神经元的细胞兴奋性和疼痛传递中的生理作用。拟议的工作是创新的，因为将表征一种新的渠道监管机制。这具有重要的医学意义，因为了解这一调控通路的细节将促进针对这一通路中的步骤的新疼痛疗法的开发，这可能有助于治疗顽固性神经性疼痛。

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.