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.