UT Southwestern Medical Center researchers have published a 3-D atomic structure of the ion channel found in mammals that is implicated in a rare, inherited neurodegenerative disease in humans. The work marks the first such structure determined using the university’s $17 million cryo-electron microscopy (cryo-EM) facility.
In cryo-EM, samples are rapidly frozen to prevent the formation of damaging ice crystals and then viewed at -321°F. This enables atomic-level views of proteins that resist the crystallization necessary for traditional X-ray crystallography. The UT Southwestern facility operates round-the-clock and is one of the top facilities for cryo-EM structural biology.
In this study, the UT team solved the structure of the mouse TRPML1 (transient receptor potential mucolipin 1) ion channel, the one that regulates the flow of calcium ions in every mammal. The channel sits in the membrane of organelles inside cells called lysosomes, which contain enzymes that aid in cellular recycling by breaking down large molecules.
“Functioning ion channels are needed for the proper movement of electrically charged particles—ions—in and out of cells and organelles to run cellular processes,” says Dr. Youxing Jiang, a professor of physiology and biophysics and co-author of the study.
About 50 lysosomal storage diseases (LSDs) have been identified in humans, according to Jiang, including one class of LSDs caused by loss-of-function mutations in genes governing the TRPML1 channels. This LSD, called mucolipidosis type IV, is marked by delayed development of mental and motor skills and vision impairment, according to the National Institutes of Health.
Determination of TRPML1’s structure could aid in the search for treatments for mucolipidosis type IV, according to Jiang. “Due to its link to that class of lysosomal storage diseases, TRPML1 has been a potential target for small-molecule therapeutics and several potential agonists (channel openers) have been developed,” he says.
A distinction in this study, according to the researchers, is the successful use of a relatively new sample preparation technique that involves embedding the protein of interest in a nanodisc structure made from lipids and other biological materials.
“For a long time, detergent has been used to extract proteins from membranes for study. People have suggested that detergent might change the protein structure from its native state,” says Dr. Xiaochen Bai, an assistant professor of biophysics and cell biology and co-author of the study. “Membrane proteins, such as those we studied, are usually wrapped in lipids. Nanodiscs are used to provide a native environment for the protein sample.”
UT Southwestern’s cryo-EM facility houses three high-tech instruments, including a nearly 13-foot-tall, 2-ton Titan Krios that shoots a high-powered beam through each sample while a special camera captures images of the scattered electrons that result. Each of the three microscopes has its own room in the facility with many automated elements. A robotic arm inside the Titan Krios machine, for example, can hold and precisely move a dozen flash-frozen samples in an automated manner so that thousands of images can be recorded, processed via computers and interpreted to generate 3-D images for study. Additionally, all researchers remain in control rooms outside the microscope rooms during operation since temperature changes as small as those from a human body could disrupt the precision machinery.