Temperature Activation Mechanism of TRP Ion Channels

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2017

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Grandl, Jorg

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Abstract

Organisms need to sense temperature to avoid detrimental damage to cells and tissues. In mammals, this is thought to be mediated, at least in part, by several members of the transient receptor potential (TRP) superfamily of ion channels. TRP channels are outwardly rectifying channels with 6 transmembrane segments that assemble as a tetramer. Some TRP channels are activated by cold or heat, chemicals, and depolarizing voltages. Temperature sensitive TRP channels are expressed in a variety of cell types including keratinocytes and medium- to small-diameter nociceptors where they are involved in the detection of noxious chemicals, inflammatory mediators, and temperature. In response to noxious stimuli, TRP channels mediate the depolarization of nociceptors ultimately leading to the perception of pain. Due to their critical role in nociception they are excellent candidates for the development of analgesic drugs that can be used as treatments for different pain modalities. However, drugs that target these ion channels have many unwanted side effects that include hypo or hyperthermia. Clearly, a thorough understanding of the structures and mechanism that mediate temperature activation of TRP channels is needed for the clever development of novel drugs that do not evoked these side effects.

Although countless studies have tried to identify the structures and mechanisms that confer temperature sensitivity to TRP channels, no consensus about these have been attained. One recent proposed mechanism assumes that temperature activation is driven by the exposure of hydrophobic residues to solvent. This mechanism further predicts that residues are exposed to solvent in a coordinated way, but without necessarily being near each other. However, there is little experimental evidence supporting this mechanism in TRP channels. Here I tackle these questions using a variety of approaches: First, I tested the sufficiency of the pore domain of TRPV1 towards temperature sensitivity using minimal ‘pore-only’ channels, but found that my minimalistic approach does not yield functional channels. I then tested the sufficiency of the entire ankyrin repeat 6, or single-point mutation on the same repeat of drosophila TRPA1 for inverting the temperature directionality of the channel, but found that the structure was not sufficient to make heat-activated drosophila TRPA1 cold sensitive. Lastly, I took a combinatorial approach and used random mutagenesis, coupled to high-throughput screening and massive parallel sequencing to identify and characterize mutations from ~7,300 randomly mutated TRPV1 clones. I found that residues important for temperature activation are randomly spread throughout the entire sequence of the channel indicating that temperature does not activate the channel by acting on a single coherent domain, but rather in the entire protein. This implies that it will be very complicated to develop analgesic drugs that do not affect the temperature activation mechanism since residues throughout the protein are involved in temperature sensitivity. Additionally, I found that large decreases in hydrophobicity of amino acids are better tolerated for activation by capsaicin than for activation by hot temperature, suggesting that strong hydrophobicity might be specifically required for temperature activation. This provides initial support for a previously hypothesized temperature activation mechanism involving amino acid hydrophobicity in TRP ion channels.

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Sosa Pagan, Jason Omar (2017). Temperature Activation Mechanism of TRP Ion Channels. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/14474.

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