Pain begins with transduction at peripheral nerve terminals of specialized sensory neurons called nociceptors. Understanding how these nociceptors respond to pain-producing stimuli is a key step towards the understanding of the biology of pain. This research concerns ion channels underlying nociception, in particular, the cloned VR 1 receptor, which detects and integrates multiple physical and chemical stimuli including heat, vanilloids and acids. Our goal is to understand, at the molecular level, how the receptor functions as a versatile noxious detector in response to various stimuli. Our approach involves patch-clamp recordings from recombinant channels in heterologous expression systems, combined with kinetic analysis to unravel the molecular events occurring during activation, along with mutagenesis to identify functional domains of the receptor. Our first objective is to understand how heat activates the channel. We will determine the energetic landscape of heat activation and explore the existence of possible heat sensors in the receptor and their structural basis. Our second objective is to investigate how capsaicin, the pungent ingredient in hot peppers, activates the channel. We will study the biophysical properties of the activation process regarding capsaicin binding, unbinding and channel gating, and determine their molecular basis. We will correlate heat and capsaicin activation pathways and examine whether different mechanisms are used for physical and chemical stimulus detection. The third objective is to understand how hyperalgesic mediators like acids sensitizes the channel. We will probe possible allosteric mechanisms by which proton binding promotes heat and capsaicin activation. The fourth objective is to investigate the voltage dependence of the channel. We will determine how the activation kinetics is altered by voltage and where the voltage sensitivity originates. The proposed research will improve our knowledge of nociceptive sensory transduction and will benefit clinical advances in pain therapy, in particular, the search for analgesic drugs that have an entirely new mode of action and an unprecedented selectivity for nociceptors.
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