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NEUROBIOLOGY First Domino Falls in Touch ResearchCharacterization of Ion Channel May Speed Discovery of Touch-sensing Complex If sensory research were a station wagon, work on the sense of touch would have spent the last 20 years riding in the way back. During this time, molecular biology investigations on vision, taste, and smell have shared the driver's seat. Yet recent findings suggest that touch research may be ready to take a turn at the wheel.
A flow analysis of ion channel protein BNaC1-alpha shows that it takes a novel one-way route from the cell body to peripheral sensory terminals. To track the path of BNaC1-alpha, the researchers tied the dorsal root and sciatic nerve on either side of the dorsal root ganglion; the dorsal root extends from cell bodies in the ganglion to the spinal cord, and the sciatic nerve runs from the ganglion to peripheral sensory terminals. The accumulation of channel protein on the ganglion side of the sciatic nerve ligature, as shown above by the fluorescent cluster in the left-hand image, demonstrates a disruption of the protein's one-way route to the periphery. Courtesy of David Corey A paper in the April 15 Journal of Neuroscience characterizes an ion channel in mammals that appears to mediate the sense of touch. It is the first molecule to be identified in the macromolecular complex that converts touch stimuli to a neural signal. And it belongs to a new family of ion channels, dubbed brain sodium channels, or BNaCs, which may be mechanically gated. Co-authors are Jaime García-Añoveros, Howard Hughes associate and HMS research fellow in neurobiology; David Corey, Howard Hughes investigator and HMS professor of neurobiology; Clifford Woolf, the Richard J. Kitz professor of anesthesia research, and colleagues, all at Massachusetts General Hospital. An early collaborator of Corey's, Jim Hudspeth, now a Howard Hughes investigator and head of the Laboratory of Sensory Neuroscience at Rockefeller University, said of the work: "Despite the importance of our mechanical senses, we still do not know how mechanical forces are converted into electrical responses in the nervous system. The paper by García-Añoveros, Corey, and their colleagues provides an important step toward such an understanding. The researchers have for the first time demonstrated that a particular ion channel, termed BNaC1-alpha, is produced by mechanically sensitive nerve cells and shipped to their responsive nerve endings." Unlike the other four senses, touch is ubiquitous, involving sensory terminals dispersed over the outside and on the inside of the body. This somatosensory system encodes a variety of sensations in addition to touch, such as pain, vibration, pressure, stretch, itch, texture, and temperature. The system is sensitive to certain chemical states like painful tissue acidity, the result of inflammation or infection. Touch also underlies proprioception, the brain's sense of where parts of the body are positioned at any given moment, crucial to motor control. Since touch complexes are spread throughout the body instead of clustered in a single sensory epithelium, purifying the proteins is relatively difficult. According to Corey, this is one reason that research on touch was left by the wayside when cloning of genes involved in other senses began taking off around 1980.
David Corey (right), Jaime García-Añoveros, and colleagues have presented strong evidence that a certain member of the BNaC family of ion channels mediates the sense of touch. Photo by Justin Knight Touch does have something in common with hearing, of course—a transduction pathway that converts mechanical stimuli to electrical impulses. It stands to reason that ion channels involved in these senses would be directly gated by mechanical means. But to date, there is no direct evidence for a mechanically gated ion channel in eukaryotes. The current paper, however, which is supported by Howard Hughes and the NIH—together with related research—makes a strong case that BNaC1-alpha is operated by mechanical force. Corey began working on mechanosensation in hair cells, the auditory receptors, in the mid-1970s. In the early '80s, he developed a model of the process with Jim Hudspeth. They proposed that a mechanosensory complex would be composed of a mechanically gated ion channel in the hair cell membrane, attached to the cytoskeleton by an elastic linkage. Closing In on the GateHoping to find a mechanically gated channel that mediates the hair cells' sensitivity to sound, Corey turned to the mechanosensitive neurons—and the degenerin genes linked to mechanosensitivity—that had been identified in C. elegans. García-Añoveros joined the lab around this time, the mid-1990s, because he had done extensive work on nematode degenerins as a graduate student. In the June 1998 Neuron, García-Añoveros, Corey, and colleagues demonstrated that the degenerins—proteins that García-Añoveros and others had linked to touch sensitivity—do indeed form ion channels. "When it was clear that a couple of the proteins needed for mechanosensation were ion channels, we began to look for homologues of those channels in vertebrates," Corey said.The researchers found a novel expressed sequence tag in humans that was homologous to the worm degenerins. With this probe, they cloned BNaC-1 and -2 from human brain, but discovered that the proteins were not produced by receptor cells of the inner ear. The biophysical properties of the clones also were different from those of the hearing channels (which had not been cloned but studied directly in cells). "It was discouraging in a way," Corey said, "and I pretty much decided to drop the whole project. But Jaime had made a good antibody and stuck with it. He started looking elsewhere and came up with another, equally interesting localization." This was in the dorsal root ganglia, groups of sensory neurons adjacent to the spinal cord. A 1998 paper by a team of French researchers had reported that in situ hybridization, not a highly sensitive technique, had failed to find the BNaC1-alpha isoform in these ganglia. They had discovered that other channels in the BNaC family were sensitive to acid and hypothesized that these were involved in pain sensation. Yet with genes cloned from mice, García-Añoveros and Corey did find BNaC1-alpha expressed in the ganglia's large-diameter neurons; these respond to low-threshold stimuli like touch but are pain insensitive. The protein also was present in just a few small-diameter neurons, some of which sense high-threshold mechanical pain like a pinch or pin prick. This suggests that BNaC1-alpha is not the only mechanotransducer responsive to this sort of pain. The researchers confirmed their findings with polymerase chain reaction and the antibody García-Añoveros had developed and purified. One-way TrafficWithin the large neurons, BNaC1-alpha showed a novel unidirectional pattern of transport. Through antibody labeling, García-Añoveros, Corey, and Woolf detected the protein in the cytoplasm, where it was particularly clustered in the axon hillocks, as if queued up for delivery. Where was it going? The researchers did not find it in the spinal cord, the central termination of the neurons. But they found that "it definitely goes to the sensory terminals in the skin," García-Añoveros said. "We found it concentrated in all the classically defined mechanosensory terminals we examined."To elucidate the distribution system, the three researchers first tied the middle of the dorsal roots, the nerve bundles extending from the ganglia to the spinal cord. In a separate experiment, they tied peripheral axons in the sciatic nerve, which run from lumbar ganglia to the periphery. And they looked for BNaC1-alpha antibody reactivity on either side of the ligatures. The protein was absent from both sides of the ligated dorsal roots, suggesting that it is not transported toward the central presynaptic terminals in the spinal cord. But the researchers did detect substantial immunoreactivity on the dorsal root ganglia side of the bound sciatic nerve (see figure above), demonstrating that BNaC1-alpha is transported only from the cell bodies toward the peripheral sensory terminals. Further evidence for the role of BNaC1-alpha in the cell has come from a recent knockout study. "Michael Welsh's group at the University of Iowa made a knockout of this gene—encoding the same protein we've localized. They recorded from the touch-sensitive neurons and found that a subset of them—low-threshold, rapidly adapting mechanoreceptors—do not respond to touch as well," García-Añoveros explained. "While there was a defect in touch response, they found no other type of phenotypic change." Because the animal is still touch-sensitive to some degree, the nerve cells must have other ways to respond to touch, which may include substituting another member of the BNaC family for this isoform. There is no obvious clinical impact in the identification of BNaC1-alpha, but according to co-author and pain researcher Clifford Woolf (see Focus, April 6, 2001), "The discovery of this mechanotransducer may help in discovering what the high-threshold mechanotransducer is. Such a discovery may be very useful for the treatment of pain." BNaC1-alpha will be a primary tool for fully illuminating the touch-sensing system. "The good news is that now we have one molecule, and it's almost certainly part of a complex," García-Añoveros said. "We can use it to look for all of the other binding proteins. Until now there was no way of getting at one of those molecules." Corey's expectations are also pinned to this domino effect in molecular biology. "I'm hoping that with one protein, you can perhaps fish for others," he said. "I expect our whole understanding of touch to move much more rapidly in the next 10 years." —Robert Neal |
