UNC-13-Munc13s play a central function in synaptic vesicle priming through their MUN domains. However, it is unclear whether this function arises from the ability of the MUN domain to mediate the transition from the Munc18-1–closed syntaxin-1 complex to the SNARE complex in vitro. The crystal structure of rat Munc13-1 MUN domain now reveals an elongated, arch-shaped architecture formed by α-helical bundles, with a highly conserved hydrophobic pocket in the middle. Mutation of two residues (NF) in this pocket abolishes the stimulation caused by the Munc13-1 MUN domain on SNARE complex assembly and on SNARE-dependent proteoliposome fusion in vitro. Moreover, the same mutation in UNC-13 abrogates synaptic vesicle priming in C. elegans neuromuscular junctions. These results strongly support the notion that orchestration of syntaxin-1 opening and SNARE complex assembly underlies the central role of UNC-13-Munc13s in synaptic vesicle priming.
Summary
Mechanotransduction channels mediate several common sensory modalities such as hearing, touch, and proprioception; however, very little is known about the molecular identities of these channels. Many TRP family channels have been implicated in mechanosensation, but none of them has been demonstrated to form a mechanotransduction channel, raising the question of whether TRP proteins simply play indirect roles in mechanosensation. Using C. elegans as a model, here we have recorded a mechanosensitive conductance in a ciliated mechanosensory neuron in vivo. This conductance develops very rapidly upon mechanical stimulation with its latency and activation time constant reaching the range of micro-seconds, consistent with mechanical gating of the conductance. TRP-4, a TRPN (NOMPC) subfamily channel, is required for this conductance. Importantly, point mutations in the predicted pore region of TRP-4 alter the ion selectivity of the conductance. These results identify TRP-4 as the first TRP protein that functions as an essential pore-forming subunit of a native mechanotransduction channel.
The “eyeless” animal C. elegans possesses the sense of light and engages in phototaxis behavior mediated by photoreceptor cells. However, the molecular and cellular mechanisms underlying phototransduction in C. elegans remain largely unclear. By recording the photoreceptor neuron ASJ in wild-type and various mutant worms, here we show that phototransduction in ASJ is a G protein-mediated process and requires membrane-associated guanylate cyclases but not typical cGMP-cleaving phosphodiesterases (PDEs). In addition, we find that C. elegans phototransduction requires LITE-1, a candidate photoreceptor protein known to be a member of the invertebrate taste receptor family. Genetic, pharmacological and electrophysiological data suggest a model whereby LITE-1 transduces light signals in ASJ through G-protein signaling, which leads to up-regulation of the second messenger cGMP followed by opening of cGMP-sensitive CNG channels and thereby stimulation of photoreceptor cells. Our results identify a phototransduction cascade in C. elegans and implicate the function of a “taste receptor" in phototransduction.
Most animals can distinguish two distinct types of touch stimuli: gentle (innocuous) and harsh (noxious/painful) touch, but the underlying mechanisms are not well understood. C. elegans is a highly successful model for the study of gentle touch sensation. However, little is known about harsh touch sensation in this organism. Here we characterize harsh touch sensation in C. elegans. We show that C. elegans exhibits differential behavioral responses to harsh touch and gentle touch. Laser ablations identify distinct sets of sensory neurons and interneurons required for harsh touch sensation at different body segments. Optogenetic stimulation of the circuitry can drive behavior. Patch-clamp recordings reveal that TRP family and amiloride-sensitive Na+ channels mediate touch-evoked currents in different sensory neurons. Our work identifies the neural circuits and characterizes the sensory channels mediating harsh touch sensation in C. elegans, establishing it as a genetic model for studying this sensory modality.
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