TRP Channel Mediated Pain Circuitry

This Program Project seeks to understand how pain signals are generated andprocessed and to develop new strategies for treating pain. The focus is Transient Receptor Potential (TRP)channels, key elements in the pain system. In four intertwined projects, we will map the distribution andfunction of TRP channels in skin and nerve, characterize their function in signaling different modalities ofpain and itch, and exploit their large-pore nature to selectively introduce charged molecules into pain- anditch-sensing neurons and thereby block their function. Project 1 will characterize the function of TRPV3channels in skin keratinocytes and peripheral nerve endings to process pain from tissue damage and heat,using a combination of physiology, immunocytochemistry, pharmacology, and behavioral assays. Project 2will use conditional gene knockout and cell ablation techniques to identify the function of specificpopulations of sensory neurons in mediating transmission of specific pain and itch information. Project 3will use targeted delivery of an impermeant cationic sodium channel blocker (QX-314) through TRPchannels to electrically silence distinct subsets of a different mediating pain and itch. Project 4 will developcharged calcium channel blockers that can be targeted via TRP channels to block synaptic transmissionfrom specific populations of pain-sensing neurons. The work will be greatly enhanced by thecomplementary expertise of the four investigators in pain, TRP channels, genetic analysis of circuits, andion channel pharmacology. The work will involve extensive co-development and use of powerfulexperimental tools, including generation of novel mouse lines to identify and ablate specific nociceptorpopulations and development of novel pharmacological agents to silence populations of nociceptors. Thegoals of all the projects will be greatly facilitated by a common Core facility to enable rapid and high qualitybehavioral assays for multiple types of pain signaling. Clapham project aims are:

Aim 1. Determine and quantify the ability of TRPV3 activation to release confirmed itch and nociceptive mediators from skin. Using pharmacological and genetic tools, we will determine whether specific TRPV3 ion channel activation releases pruritic and nociceptive mediators from skin and compare this release to that by other TRPV channels present in keratinocytes.

Aim 2. Distinguish skin TRPV3-mediated pain or itch responses from those in nociceptor dorsal root ganglion neurons innervating skin. We will generate mice in which the gene encoding TrpV3 is deleted (TRPV3 global KO), conditional mice in which TrpV3 is deleted only in skin keratinocytes (TRPV3 KO-skin), and a new conditional mouse in which TrpV3 is deleted only in dorsal root ganglion neurons (TRPV3 KODRG). Using additional transgenic and pharmacological tools, we will determine whether activation of TRPV3 releases algesic or pruritic agents from skin to underlying nerve terminals, and whether in the absence of TRPV3 in skin, TRPV3 in nerve can relay pain and itch signals.

Aim 3. Determine whether TRPV3 can be employed as an entry portal for cell-specific drug delivery. Upon activation, TRPV3 becomes a large pore entry pathway for molecules. We will deliver sodium (Nav) and calcium (Cav) channel blockers via TRPV3 into keratinocytes to block ATP release, thereby blocking nociceptors expressing purinergic receptors. This same strategy will be employed to deliver agents blocking Nav and Cav channels in peripheral nerve terminals of fibers innervating skin.