<p>For all organisms, the ability to sense and react to noxious environments is fundamental to their survival. For multi-celled organisms this process generally involves a nervous system and an extensive network of signal transduction pathways. TRPV ion channels have been shown to participate in signal transduction in response to noxious stimuli. At the cellular level these channels function in sensing of mechanical, thermal, and osmotic stimuli, and at the organismal level they function in homeostasis and nociception. TRPV ion channels participate in nociceptive signal transduction via cation influx, but exactly how these channels function at a mechanistic level and lead to activation of the cell or induction of a specific behavior is elusive. Previous research has shown that the pore-forming unit of an ion channel is critical for channel regulation, gating, ion selectivity, and ion permeation. Various regulatory domains have been identified to date in the pore-forming unit of TRP channels and a clearer picture of channel gating is beginning to emerge, but less is known about ion permeation. </p><p>To better understand the specific domains that are critical to ion capture, selectivity, and permeation in TRPV channels, we investigated the function of these regions using the <italic>C. elegans</italic> TRPV channel OSM-9 <italic>in vivo</italic>, and the mammalian TRPV channel TRPV4 in heterologous cell culture. OSM-9 is the functional ortholog of mammalian TRPV4 and it is likely that critical domains identified in OSM-9 are functionally conserved in TRPV4 and play a similar role in other TRPV channels. OSM-9 is expressed in the ASH neurons and is responsible for all of the behaviors initiated by that cell. The stereotypical avoidance behavior mediated by ASH, in response to noxious stimuli, serves as a model for nociception in vertebrates. As OSM-9 is necessary for all of these behavioral responses, activation of ASH acts as a read-out for OSM-9 function.</p><p>Through targeted mutagenesis of the OSM-9 loop domains and transgenic expression directed to the ASH head sensory neurons in an <italic>osm-9</italic> null background, we discovered a critical role for the amino acids both N- and C- terminal to the pore helix in osmotic avoidance behavior. We confirmed the existence of a selectivity filter C-terminal to the pore helix and revealed that the turret is critical for channel function, possibly as a component of the inactivation gate.</p><p>We first identified the boundaries of the selectivity filter to be M601-F<super>609</super>. We also determined what properties of those residues were critical to Ca<super>2+</super> and Na<super>+</super> selectivity. <italic>In vivo</italic> Ca<super>2+</super> imaging strongly suggested that residues Y<super>604</super>, D<super>605</super>, and F<super>609</super> are critical for Ca<super>2+</super> entry into the cell. Patch-clamp electrophysiology of a chimeric ion channel consisting largely of rat TRPV4, but encompassing transmembranes 5 through 6 of OSM-9, revealed that OSM-9 conducts both Ca<super>2+</super> and Na<super>+</super>. Mutation Y604G disrupted both Ca<super>2+</super> and Na<super>+</super> conductance, whereas mutations Y604F and Y606A increased or maintained Na+ conductance and severely reduced Ca<super>2+</super> conductance, while maintaining avoidance behavior. Homology modeling of OSM-9, based on an alignment of OSM-9 to Kv1.2, suggests that Y<super>604</super> and F<super>609</super> serve structural roles in maintaining filter constraints. Thus, aromatic and negative residues in the OSM-9 selectivity filter are critical to ion permeation and selectivity. </p><p>Our studies involving the selectivity filter support previous research that the selectivity filter is critical for TRP channel function. We also provide evidence that the selectivity filter is critical for nocifensive animal behavior. Fewer studies, however, have investigated the TM5-pore helix linker, known as the turret. The turret is believed to function in the binding of ligands and toxins in K<super>+</super> channels, and more recently was suggested to be critical for temperature sensing in TRPV1. We investigated the function of the turret residues in several sensory submodalities of the OSM-9 channel and found that all deletions tested result in channel defects, including gain- and loss-of-function phenotypes. Several charge reversal mutations in the OSM-9 turret also resulted in partial defects. The discovery of a gain-of-function mutation indicates that the turret functions in gating. When the turret is mutated in this way, the channel is unable to enter into the inactivated state, allowing continued ion influx after repeated stimulation. The loss-of-function phenotypes indicate that the secondary structure of the turret is critical to the function of the channel, and perhaps gating. These findings, combined with the observed charge-reversal defects, support the conclusion that the turret is necessary for transducing conformational changes in response to stimuli.</p><p>Our <italic>in vivo</italic> findings on the external pore forming structures increase the understanding of ion permeation in TRP channels and clarify mechanisms of activation in nociceptor neurons <italic>in vivo</italic>. Furthermore, these studies enhance our insights into evolution of mammalian nociception in view of the established functional orthology of OSM-9 and TRPV4.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/5643 |
| Date | January 2011 |
| Creators | Lindy, Amanda Sue |
| Contributors | Liedtke, Wolfgang B |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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