Neurons are the cells of our brain, spinal cord, and overall nervous system. They form complex networks to communicate with each other through electrical signals that are generated by chemicals. These chemicals bind to structures on the surface of neurons that are called neuroreceptors, opening or closing electrical pathways that allow transmission of the signal from neuron to neuron. One neuroreceptor, called 5HT3-R, is involved in conditions like chemotherapy-induced nausea, anxiety, and various neurological disorders such as schizophrenia. Despite its clinical importance, the exact way that 5HT3-R works has been elusive because its complexity has prevented scientists from determining its three-dimensional structure. Publishing in Nature, researchers at EPFL in Switzerland have now uncovered for the first time the 3-D structure of 5HT3-R, opening the way to understanding other neuroreceptors as well.
Neuroreceptors: structure and function
Communication between the neurons of our body is mediated by neuroreceptors that are embedded across the cell membrane of each neuron. Neuronal communication begins when a neuron releases a small molecule, called a ‘neurotransmitter’, onto a neighboring neuron, where it is identified by its specific neuroreceptor and binds to it. The neurotransmitter causes the neuroreceptor to open an electrically conducting channel, which allows the passage of electrical charge across the neuron’s membrane. The membrane then becomes electrically conducting for a fraction of a millisecond, generating an electrical pulse that travels across the neuron. The family of neuroreceptors that work in this way is widespread across the nervous system, and is referred to as the “ligand-gated channel” family.
The mystery is how the binding of the neurotransmitter can induce the opening of an electrical channel to transport a signal into the neuron. The understanding of these molecular machines is of great medical importance, especially since neuroreceptors are involved in many neurological diseases. Currently, none of the mammalian ligand-gated channel neuroreceptors have been structurally described, which significantly limits our understanding of their function on a molecular level.
Uncovering the structure of 5HT3-R
The team of Horst Vogel at EPFL has used x-ray crystallography to determine the 3D structure of a representative ligand-gated channel neuroreceptor, the type-3 serotonin receptor (5HT3-R). This neuroreceptor recognizes the neurotransmitter serotonin and opens a transmembrane channel that allows electrical signals to enter certain neurons. The 5HT3 receptor was grown in and then isolated from human cell cultures, and finally crystallized.
But before obtaining the 5HT3-R crystals, the EPFL team had to overcome a number of challenges. First, the relatively large size of the membrane-embedded 5HT3-R, like that of other similar channel neuroreceptors, makes it notoriously difficult to purify in sufficient quality and quantity. After years of painstaking work, the EPFL scientists succeeded in obtaining a few milligrams of 5HT3-R, which was still not enough to grow crystals using conventional methods.
Still, the crystal quality was insufficient. To address this, Vogel’s team used small antibodies, so-called nanobodies, which were obtained from llamas after the animals were injected with purified 5HT3-R. From a large library of isolated nanobodies, a particular one was found to form a stable complex with the 5HT3-R, and this complex eventually yielded crystals of exceptional quality.
After this, the procedure was straightforward: The crystals for x-ray crystallography were investigated at the synchrotron facilities at the Paul Scherrer Institut in Villigen and the European facilities in Grenoble. In this well-established technique, the crystals diffract x-rays in a characteristic pattern from which the 3-D structure can be reconstructed.
The X-ray diffraction experiments yielded the 3-D structure of 5HT3-R at an unprecedented resolution of 3.5 Ångstroms (3.5 millionths of a millimeter). The resulting 3-D image shows a bullet-shaped 5HT3 receptor with its five subunits symmetrically arranged around a central water-filled channel that traverses the neuron’s cell membrane. The channel can adopt two states: a closed, electrically non-conducting state or, after binding a neurotransmitter, an open, electrically conducting state that allows the flow of electrical charges in and out of the neuron to generate an electrical signal.
“We have now elucidated the molecular anatomy of a receptor that plays a central role in neuronal transmission,” says Horst Vogel. “It is the first 3-D structure of its kind and may serve as a blueprint for the other receptors of this family. In the next step, we have to improve the resolution of the structure, which might give us information on how to design novel medicines that influence this neuroreceptor’s function.”
This work is supported by the Swiss National Science Foundation (www.snf.ch) and represents a collaboration between EPFL’s Laboratory of Physical Chemistry of Polymers and Membranes; the Center for Cellular Imaging and NanoAnalytics of the University of Basel; the Swiss Light Source and the Laboratory of Biomolecular Research of the Paul Scherrer Institute; the Architecture et Fonction des Macromolécules Biologiques of the Centre National de la Recherche Scientifique; the Université Grenoble Alpes; and the Unité de Dynamique Structurale des Macromolécules of the Institut Pasteur.