Sensations and thoughts arise from the transmission of electrochemical signals through the nervous system. Neurons release molecules called neurotransmitters that dock onto receptor proteins on the surfaces of neighboring cells. Often a receptor is made from a group of identical protein parts. The way the group assembles determines whether and how a cell responds to a signal. Andrew Plested's laboratory at the FMP has now exposed some of the physical and chemical properties that determine how components of an important type of receptor bind to each other. The project helps explain how nerve cells respond to signals and yields important insights into the behavior of other receptors. The study appears in the recent issue of PNAS.
 
Andrew and colleagues at the National Institutes of Health in the U.S.
(Utpal Das, Janesh Kumar and Mark Mayer) have been studying glutamate receptor ion channels, or iGluRs. In this receptor, four identical protein components combine on the surface of a nerve cell to create and control a gate-like channel through the cell membrane. When activated by the neurotransmitter glutamate, the receptors open the channel and permit charged particles called ions to enter the cell. Their passage changes the balance of electrical charges between the cell interior and exterior, exciting the nerve into firing an impulse. This impulse can drive the cell to release neurotransmitters that affect the next cell. But the system shouldn't be switched on all the time, so the gate needs to close again.
"The purpose of the project was to obtain a detailed picture of how the four protein parts are bound to each other in the intact receptor," Andrew says. "We hoped this would reveal the physical and chemical features that allow them to open and close the ion channel."
 
Researchers had already obtained a structural picture that showed how pairs of iGluR subunits can connect to each other. The iGluR was said to be formed from two connected pairs, but scientists had little idea how this happened. The iGluR has to stretch to open the channel – a bit like loosening the drawstring of a bag to put something inside – and then tighten again to shut off a signal. But to get a clear idea of the operation of the ion channel, they needed to see an assembly of all four molecules. This required obtaining crystals of the complex of four protein parts, in which millions of copies of the molecules are stacked into tight arrays. Exposing the crystal to X-rays gave the researchers a precise map of the positions of each component in the cluster. This picture revealed how the regions of each protein binds to its partners.
 
iGluRs and other proteins are made of strings of amino acids that fold into small modules called domains. Folding puts some parts of the molecule inside and leaves other regions exposed on the outside, where they can form chemical bonds to other proteins. A module called the ligand binding domain (LBD) is responsible for latching onto glutamate; iGluRs also have a domain called the amino terminal domain (ATD) that plays a role in linking copies together.
Andrew’s study concerned a particular iGluR called the kainate receptor. Recently another laboratory discovered that another receptor in the iGluR family, called the AMPA receptor, assembles in a surprising way. The ATD domains of two molecules join to create the pairs, and then their LBD modules link to those of the other pair, forming a twisted arrangement. Andrew and his colleagues wondered whether kainate receptors such as iGluR behaved the same way.
 
The experiments revealed that the structure and behavior of the kainate receptor complex closely resembles that of the AMPA receptor. In additional experiments, the scientists produced mutant forms of iGluR, making single "spelling changes" in the amino acid recipes of the molecule. Mutations alter a protein's chemistry, and these mutations added a kind of molecular glue to the surfaces. "Gluing together the proteins generally disturbed the operation of the channel," Andrew says. "But the effects varied dramatically depending on the position where the glue was added, which suggests that the four copies may have different roles in sensitizing the cell to signals."
This study also shows that while there are differences in the amino acid makeup of iGluR receptor proteins, they fold in similar ways. The mutation studies show that even subtle differences may affect their functions.
 
Opening and closing ion channels requires changes in the positions of the modules of the four proteins – a sort of breathing motion that requires some of the contact surfaces to break, and later reform. Where are these breakages likely to happen? "The links between the ATD modules are the strongest," Andrew says, "which suggests that they play the dominant role in assembling the complex in the first place. Some of the links we found between LBD regions are probably very weak, and this makes sense, because we already know these parts have to move to open the channel- they are meant to break.”
The study suggests that the proteins contain a hierarchy of contact points that cooperate to maneuver the parts into the correct positions – just as with a door, some parts are hinges, and others are just free to swing, but there must also be a frame. "The connections between one region of the LBD are strong enough to stay intact when the receptor is activated to open the channel," Andrew says, "but they can break to turn it off. At that point, other surfaces hold the complex together. Other movements appear to be the key in the changes that occur when the channel gets opened." Further work, he hopes, will show precisely how this happens.
- Russ Hodge

A. Plested is a member of the Cluster of Excellence NeuroCure at the Charité, Berlin (DFG: EXC 257)

 
Reference:
Das U, Kumar J, Mayer ML, Plested AJ. Domain organization and function in GluK2 subtype kainate receptors. Proc Natl Acad Sci U S A. 2010 May 4;107(18):8463-8.


Links:
Link to the full paper:
www.pnas.org/content/107/18/8463.long

Link to the homepage of the Plested group:
here