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Scientists take a close-up of key pain-sensing molecule

For release: Friday, May 16, 2014

    PRPV1 Image
Caught on camera: microscopic flying saucers reveal the workings of pain

These shapes, reminiscent of alien spaceships, are cryo-EM snapshots of individual molecules of TRPV1 with a pain inducer attached to them. Scientists used these pictures to decipher what the TRPV1 protein looks like and how it works. From Liao et al. Nature 504: 107-112, 2013.

A revolutionary microscopy technique could help design better treatments for chronic pain

Pain is something that touches a nerve with all of us, but what does it actually mean to feel pain? For nearly 100 million Americans living with chronic pain this question goes to the root of their daily suffering. A landmark study may offer an answer and new hope for relief. Using a unique approach, NIH-supported scientists at University of California, San Francisco (UCSF) captured detailed pictures of a key pain-sensing molecule in the act of decoding painful stimuli. These images may serve as a blueprint for understanding pain and designing new treatments.

“The study may be a key piece to unlocking the pain puzzle,” says Linda Porter, Ph.D., a leader of the Pain Consortium and a Pain Policy Advisor at the National Institute of Neurological Disorders and Stroke (NINDS), which is a part of the National Institutes of Health (NIH).

The study, reported in the journal Nature, was led by David Julius, Ph.D., Professor and Chair of the Department of Physiology and Yifan Cheng, Ph.D., Associate Professor in the Department of Biochemistry and Biophysics, both at UCSF.

A cool trick gets researchers closer to the heat 

The researchers were interested in the protein called TRPV1. It is found in specialized pain-sensing cells and is responsible for their ability to respond to thermal heat, spiciness of chili peppers, and other painful stimuli. TRPV1 is an ion channel - a valve on the surface of the cell. When open, the ion channel lets ions - small, charged molecules - into the cell, producing an electrical signal.

The researchers wanted to understand how TRPV1 opens in response to pain-causing agents. Since TRPV1 is organism’s first responder to pain, these images may help design more effective drugs that tackle pain at its origins, before its signal spreads along the nerves.

“You have a better chance of success of treating pain if you block it at an early stage,” explains Dr. Julius.

To obtain a close up image of a biological molecule, researchers would typically use a method called X-ray crystallography. It requires the molecule to be crystallized, i.e., precipitated out of solution into the form of a crystal, a highly ordered solid arrangement of many identically shaped particles. However TRPV1, along with many other interesting molecules, does not easily form crystals and thus cannot be studied by this method.

The researchers therefore tried another technique, called single particle cryo-EM. First they froze TRPV1 molecules by dipping them in an extremely cold liquid. Then they placed them under the electron microscope and took highly detailed pictures of each individual molecule.

This microscope shoots a beam of electrons at the molecule. Biological molecules are made up of large numbers of atoms, connected in elaborate cage-like structures. The electrons change paths as they bounce off the scaffold of the cage. These path changes help scientists understand approximately where, within the molecule, individual atoms are located and how they are connected with each other. However, the pictures obtained with this method are usually too grainy to see the precise positions of individual atoms.

Dr. Cheng, working alongside with other researchers in the field, solved this problem by building a new high-resolution camera that can capture many more individual electron paths than was possible before. He also wrote a new computer program that corrects for molecules that are wobbling within the frozen sample.

These modifications greatly reduced graininess, yielding pictures, which, according to Dr. Julius, “approached for the first time the resolution of single atoms.” The average size of detail that they could discern in these images was 3.4 angstrom (by comparison, human hair is approximately half a million angstroms thick).

Closing the gate on chronic pain

From these pictures researchers were able to see TRPV1’s overall shape, an inverted teepee, which looked similar to that of other ion channels. But they also saw that the molecule works in a novel way: TRPV1’s central chute, along which calcium ions dive into the cell, has two independent narrowings (upper and lower gates), both of which have to be open for ions to pass through.

Various pain-causing molecules attach themselves to TRPV1 in the vicinity of one or the other gate: capsaicin, the chemical that gives chilies their heat, and resiniferatoxin, a plant toxin, assume positions near the lower gate, whereas spider venom toxin DkTx sits near the upper one. The docking of these entities to either TRPV1 gate causes both gates to open, relaying the painful signal.

The fact that a pain inducer binding or attached near one gate can cause the other gate to open more suggests that the two gates are coupled, or talk to each other. This coupling is likely conveyed by the segment of the TRPV1 molecule called the pore helix - a long rigid handle protruding deep into TRPV1 molecule’s top opening. It is no surprise that spider venom toxin, an especially powerful pain inducer, positions itself on top of the pore helix: by turning the handle sideways, it pries the top part of the channel wide open.

The discovery that TRPV1 has two gates may be exploited to design better pain drugs.

Existing TRPV1 blockers are plagued by side effects, such as hyperthermia (elevated body temperature) and loss of sensitivity to harmful heat or cold. By selectively modulating one gate or the other, the benefits of reducing pain could potentially be parsed from these adverse effects.

“The problem is that if you completely close the channel,” explains Dr. Porter, “you also lose the protective sensing of heat. Now researchers can look for drugs that leave heat sensing intact.”

A better way to take close-ups of shy biomolecules

Insights into TRPV1 structure were made possible by single particle cryo-EM - authors’ new technique, which allowed them to obtain images of the TRPV1 molecule with resolution that was previously attainable only with X-ray crystallography.

Overall, the new technique has several advantages over the traditional method. It does not require the molecule to be crystallized, thus solving the challenge of determining structures of proteins, such as TRPV1, that are difficult to crystallize. Crystallization conditions often require painstaking tweaking and sometimes force the molecule to assume an unnatural shape. Cryo-EM saves researchers time and allows them to see the molecule in its natural state.

According to the authors, the usefulness of single particle cryo-EM may extend far beyond the studies of TRPV1. “It is a more powerful, if not revolutionary, tool in protein structural biology,” said Dr. Julius.

While there are clear short-term benefits of solving the TRPV1 structure for the design of better therapies for pain, it is authors’ new method of taking intimate close-ups of shy biomolecules that is this study’s biggest long-term payoff.

Links to original research articles

TRPV1 structures in distinct conformations reveal activation mechanisms. Cao E, Liao M, Cheng Y, Julius D. Nature. 2013 Dec 5;504(7478):113-118.

Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Liao M, Cao E, Julius D, Cheng Y. Nature. 2013 Dec 5;504(7478):107-112.

Last Modified May 17, 2014