Researchers from the University of Illinois have discovered a key factor responsible for facilitating signal transduction in Cys-loop receptors, providing insight into a longstanding question in the field of ion-channel physiology and contributing to rational drug design. Their findings appear in Science Advances.
Cys-loop receptors are ligand-gated ion channels with two domains connected by a short linker. The top domain is located extracellularly, while the bottom domain sits in the cell membrane. When an external signal such as a small molecule binds to the top domain, the bottom domain is triggered to open, allowing ions to move across the membrane.
These domains are well-connected and can communicate with one another. When the top is probed, the bottom can feel this probing effect, stimulating the ion channel to open.
But the mechanism behind this domain–domain communication remained unclear, with various ideas suggested by ion-channel physiologists. Simply put: How can the bottom domain feel that the top domain is bound to a ligand?
Members of Claudio Grosman’s lab in the Department of Molecular & Integrative Physiology in the School of Molecular & Cellular Biology set out to answer this question, starting with a focused approach. Many ion-channel physiologists study this phenomenon from the perspective of the interfacial region, or the area intersecting the two domains. The Illinois researchers began here; by testing loss-of-function mutations, they discovered that the physical distance between two domains is critical for proper signal transduction.
Historically, ion-channel physiologists have believed that top and bottom domain communication can be interrupted through a series of substitution mutations in this interfacial region. But researchers from the Grosman lab found that signals can still transduce, even when this area of the protein is extensively mutated. Postdoctoral researcher Nicole Godellas, who earned a BS in molecular and cellular biology and PhD in molecular and integrative physiology from the University of Illinois, then dissected a handful of mutations in this region that rendered the channel “electrically silent”.
“When a channel is electrically silent, we know that it is expressing on the cell, but when we look for the ions to go through the channel, we don’t see them,” Godellas said. “The channels are there, but they appear to be non-functional.”
By using various linker lengthening mutations in this interfacial region of the protein, Godellas and her colleagues noted a loss of communication between the domains. Physically separating the two domains by adding an amino acid to the linker between them interrupted the bottom domain’s ability to feel the binding activity of the top domain.
Like the sandy contents inside a child’s magnetic toy table moving in response to a magnet, the top and bottom domains respond to one another. But if a thick textbook is inserted between the sand table and its magnet, the connection between the magnet and the sand above will be lost. This physical buffer is similar to the insertion mutation creating distance between the top and bottom Cys-loop-receptor domains, rendering the channel electrically silent.
By probing canonical binding sites with a competition ligand-binding assay, Godellas discovered that the linker region does not tolerate insertions or deletions of amino acids, which provides an evolutionary insight into the narrowly defined length of this region of the protein.
The researchers’ next step is gaining insight into the structural consequences of linker mutations, which may answer outstanding questions about what limits signal transduction.
In the meantime, the Grosman lab will continue studying Cys-loop receptors, which are important for therapeutic drug design in inflammatory, neurological, motor, and psychiatric disorders.
“We’re studying the very mechanistic and molecular functions of these membrane receptors,” Godellas said. “We’re providing molecular insights so medicinal chemists can design their targeted drugs to be safer and more efficacious. We’re conducting the very basic science towards rational drug design.”
