Nowhere is that more critical to know than in the brain, where interactions governing channel protein activity can alter an organism's behavior. A team of biologists at the Salk Institute for Biological Studies has recently deciphered a molecular code that regulates availability of a brain channel that modulates neuronal excitability, a discovery that might aid efforts to treat drug addiction and mental disorders.
In the this week's Online Early Edition of the Proceedings of the National Academy of Sciences, Paul Slesinger, Ph.D., Associate Professor in the Clayton Foundation Laboratories for Peptide Biology, and colleagues detail how a regulatory factor called SNX27 distinguishes a brain channel protein called GIRK (short for G-protein-coupled inwardly rectifying potassium channels) from structurally similar proteins and then targets it for destruction.
That work extends the group's 2007 study showing that when SNX27 proteins capture GIRK channels, they are reducing the number of channels at their rightful destination, the cell membrane. "We were curious about what determined the selectivity of this interaction," says Slesinger. "We knew that SNX27 interacted with a structural motif found on GIRK channels but many channel proteins display a similar motif. We wanted to know what allowed SNX27 to specifically choose GIRK channels."
Knowing this is critical because of the connection of GIRK channels to substance abuse. Slesinger and others have shown that alcohol or club drugs linked to sexual assault (GHB) affects GIRK channel function in the brain. Loss-of-inhibition behaviors associated with abuse of these substances result from the fact that GIRK channels allow potassium ions to leak out of a stimulated neuron, thereby dampening a cell's excitability.
In the new study Slesinger's team confirmed that SNX27 resides in neurons, just below the membrane where active GIRK channels sit. Additional experiments using brain cells manipulated to express abnormally high SNX27 levels showed that cells were less responsive to drugs that activate channels, suggesting that SNX27 waylays membrane-bound GIRKs and blocks their function.
The fact that SNX27 displays a common protein-interaction signature called PDZ domain suggested how SNX27 grabs its partner: GIRKs contain a short, 4-residue sequence that binds to PDZ domains, a recognition motif Slesinger likens to a zip code. But channels similar to GIRKs, called IRKs, displayed an almost identical sequence but were impervious to destruction by SNX27. "We were puzzled by this similarity and swapped the 4-residue code in IRK with the corresponding sequence from GIRK," says Slesinger. Surprisingly, this IRK/GIRK hybrid did not bind SNX27, indicating that the IRK lacked other elements necessary for SNX27 recognition.
To define these new elements, Slesinger consulted with a long-standing collaborator, Senyon Choe, Ph.D., professor in Salk's Structural Biology Laboratory. Choe is an expert on a technique known as X-ray crystallography, used to determine the three-dimensional structure of proteins. The team scrutinized crystallized forms of SNX27 wrapped around the GIRK binding motif to try to visualize where the proteins made contact.
"We observed a binding cleft in the SNX27 PDZ domain and a region that formed another pocket with a lot of positive charges," says Slesinger. "The GIRK fragment lying there had a negative charge upstream of the 4-residue "zip code". That suggested that this second site allowed a previously unknown electrostatic interaction between these two proteins." Therefore, SNX27 may recognize a 6-residue motif, like the "zip plus 4' code.
More swap experiments targeting the GIRK negatively charged region confirmed the hypothesis. Synthetic forms of GIRK lacking the region no longer bound to SNX27. By contrast, an artificial version of IRK engineered to contain the GIRK negative charges homed to SNX27.
Most significant were experiments conducted by Bartosz Balana, Ph.D., a postdoctoral fellow in the Slesinger lab and the study's first author. Balana measured currents from cells engineered to carry GIRK channels lacking the charged region and found that GIRK currents were no longer dampened by SNX27, while cells expressing IRK channels displaying the false GIRK "address" now responded to SNX27. "This functional assay pin-pointed residues that dictate SNX27 binding beyond the normal PDZ recognition sequence," says Bartosz. "This supports a two-site binding model and emphasizes that second site can overrule binding at the classical site."
An interesting corollary to GIRKs' involvement in drug-related behavior is that SNX27 levels reportedly increase in rodent models of addiction to stimulants like cocaine and methamphetamine. Selectively blocking this newly identified interaction between GIRK and SNX27 might thwart addiction. "Now we are able to better understand the role of these channels in responses to drugs of abuse. It is our hope that that this work will lead to new strategies to treat diseases such as alcoholism or even, diseases of excitability, such as epilepsy." says Slesinger.
Also contributing to the study were Kalyn Stern and Laia Bahima of the Slesinger Lab and Innokentiy Maslennikov, and Witek Kwiatkowski of Choe's Structural Biology Laboratory.
The study was funded by grants from the NIH and the National Alliance for Research on Schizophrenia and Depression.
About the Salk Institute for Biological Studies:
The Salk Institute for Biological Studies is one of the world's preeminent basic research institutions, where internationally renowned faculty probe fundamental life science questions in a unique, collaborative, and creative environment. Focused both on discovery and on mentoring future generations of researchers, Salk scientists make groundbreaking contributions to our understanding of cancer, aging, Alzheimer's, diabetes and infectious diseases by studying neuroscience, genetics, cell and plant biology and related disciplines.
Faculty achievements have been recognized with numerous honors, including Nobel Prizes and memberships in the National Academy of Sciences. Founded in 1960 by polio vaccine pioneer Jonas Salk, M.D., the Institute is an independent nonprofit organization and architectural landmark.
Gina Kirchweger | Newswise Science News
Cryo-electron microscopy achieves unprecedented resolution using new computational methods
24.03.2017 | DOE/Lawrence Berkeley National Laboratory
How cheetahs stay fit and healthy
24.03.2017 | Forschungsverbund Berlin e.V.
Astronomers from Bonn and Tautenburg in Thuringia (Germany) used the 100-m radio telescope at Effelsberg to observe several galaxy clusters. At the edges of these large accumulations of dark matter, stellar systems (galaxies), hot gas, and charged particles, they found magnetic fields that are exceptionally ordered over distances of many million light years. This makes them the most extended magnetic fields in the universe known so far.
The results will be published on March 22 in the journal „Astronomy & Astrophysics“.
Galaxy clusters are the largest gravitationally bound structures in the universe. With a typical extent of about 10 million light years, i.e. 100 times the...
Researchers at the Goethe University Frankfurt, together with partners from the University of Tübingen in Germany and Queen Mary University as well as Francis Crick Institute from London (UK) have developed a novel technology to decipher the secret ubiquitin code.
Ubiquitin is a small protein that can be linked to other cellular proteins, thereby controlling and modulating their functions. The attachment occurs in many...
In the eternal search for next generation high-efficiency solar cells and LEDs, scientists at Los Alamos National Laboratory and their partners are creating...
Silicon nanosheets are thin, two-dimensional layers with exceptional optoelectronic properties very similar to those of graphene. Albeit, the nanosheets are less stable. Now researchers at the Technical University of Munich (TUM) have, for the first time ever, produced a composite material combining silicon nanosheets and a polymer that is both UV-resistant and easy to process. This brings the scientists a significant step closer to industrial applications like flexible displays and photosensors.
Silicon nanosheets are thin, two-dimensional layers with exceptional optoelectronic properties very similar to those of graphene. Albeit, the nanosheets are...
Enzymes behave differently in a test tube compared with the molecular scrum of a living cell. Chemists from the University of Basel have now been able to simulate these confined natural conditions in artificial vesicles for the first time. As reported in the academic journal Small, the results are offering better insight into the development of nanoreactors and artificial organelles.
Enzymes behave differently in a test tube compared with the molecular scrum of a living cell. Chemists from the University of Basel have now been able to...
20.03.2017 | Event News
14.03.2017 | Event News
07.03.2017 | Event News
24.03.2017 | Materials Sciences
24.03.2017 | Physics and Astronomy
24.03.2017 | Physics and Astronomy