The probe binds to a voltage-activated potassium ion channel subtype, lighting up when the channel is turned off and dimming when it is activated.
This is the first time researchers have been able to visually observe these electrical signaling proteins turn on without genetic modification. These visualization tools are prototypes of probes that could some day help researchers better understand the ion channel dysfunctions that lead to epilepsy, cardiac arrhythmias and other conditions. The study appears in the Proceedings of the National Academy of Sciences (PNAS) on October 20.
The UC Davis research team includes (from left to right) Vladimir Yarov-Yarovoy, Daniel Austin, Sebastian Fletcher-Taylor, Jon Sack and Kenneth S. Eum, who passed away earlier this year. The team has dedicated the work to his memory. “Ken was a talented postdoctoral student, a driven and caring soul who brought joy to the lives of those who knew him,” they said.
“Ion channels have been called life’s transistors because they act like switches, generating electrical feedback” said senior author Jon Sack, assistant professor of physiology and membrane biology at UC Davis. “To understand how neural systems or the heart works, we need to know which switches are activated. These probes tell us when certain switches turn on.”
Voltage-gated channels are proteins that allow specific ions, such as potassium or calcium, to flow in and out of cells. They perform a critical function, generating an electrical current in neurons, muscles and other cells. There are many different types, including more than 40 potassium channels. Though other methods can very precisely measure electrical activity in a cell, it has been difficult to differentiate which specific channels are turning on.
“There are about 40 voltage-gated potassium channel genes that are basically doing the same thing, and it’s been shockingly hard to figure out which ones are doing something that’s physiologically relevant,” Sack said.
The video shows the imaging capabilities of the fluorescent tarantula toxin probe to observe the Kv2 potassium ion channels “turning on” in live nerve cells. The cell on the left was kept at a constant resting voltage while the cell on the right was electrically stimulated to specific voltages. Because both cellular membranes contain Kv2 potassium ion channels, a specific type of voltage-activated protein associated with epilepsy when dysfunctional, they shine brightly as the probe binds to the channel protein. But when the cell on the right is activated by increasing voltage, the signal dims as the tarantula toxins fall off. When the cells are returned to the resting voltage, the tatarantula toxins find the channels again. The video was produced by the late Kenneth S. Eum, Lillian Patrón and Christophe Dupré, all of UC Davis.
The tarantula toxin, guangxitoxin-1E, was an ideal choice because it naturally binds to the Kv2 channels. These channels are expressed in most, if not all, neurons, yet their regulation and activity are complex and actively debated. Sack and his laboratory worked closely with Bruce Cohen, a scientist in the Lawrence Berkeley Lab’s Molecular Foundry, who has been studying how fluorescent molecules and nanoparticles can be used to image live cells.s
To study the channels, the team engineered variants of tarantula toxin that could be fluorescently labeled and retain function. These probes were designed to bind to the potassium channels when they were at rest and let go when they became active. The researchers then tested them on living cells. To their surprise, the probes worked right away.
“A lot of times you see ambiguous results, but when we added the probes to living cells there was a very clear signal,” Sack said. “When we added potassium to stimulate the cells, the probes fell right off.”
While this is just a first step towards imaging the activity of potassium and possibly other ion channels, this approach holds vast potential to help scientists understand the underlying mechanisms behind cardiac arrhythmias, muscle defects and other channelopathies.
“There are dozens of known channelopathies, and more being uncovered at an increasing pace” Sack said. “If you have electrical signaling, you have to have a potassium channel, and when that channel goes bad, the cell doesn’t work the same anymore. For example, the Kv2.1 channel that this probe binds to leads to epilepsy when it’s not functioning properly.”
In addition, the ability to better observe electrical signaling could help researchers map the brain at its most basic levels.
“Understanding the molecular mechanisms of neuronal firing is a fundamental problem in unraveling the complexities of brain function,” Cohen said.
While creating a probe that can read whether the Kv2.1 channel is firing or at rest is an important proof-of-concept, there’s still a lot of work to be done. Sack and Cohen will continue to collaborate, testing other types of spider venoms that bind to different potassium channels.
“The beauty of this is the potential,” Sack said. “This is a toehold into a new way of visualizing electrical activity, and there’s a huge family of spider toxins that target different ion channels. We’ve tagged a Ford, we should be able to tag a Chevy.”
The researchers who conducted this study include: Drew C. Tilley, Kenneth S. Eum, Sebastian Fletcher-Taylor, Daniel C. Austin and Vladimir Yarov-Yarovoy of the Department of Physiology and Membrane Biology at UC Davis; Christophe Dupré and Lilian Patrón, Neurobiology Course, Marine Biological Laboratory, Woods Hole; Rita L. Garcia, Molecular Foundry, Lawrence Berkeley National Laboratory; and Kit Lam, Department of Biochemistry and Molecular Medicine at UC Davis.
This project was funded by: NIH grants 5P30GM092328-02, R01NS042225-09S1, R25NS063307, T32HL086350; AHA grant 10SDG4220047; and the Milton L. Shifman Endowed Scholarship for the Neurobiology Course at Woods Hole. Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract DE-AC02-05CH11231.