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April 16, 2004
RESEARCHERS SOLVE A NAGGING QUESTION ABOUT HOW CELLS REGULATE CALCIUM
In the April 16 issue of Science, Hopkins researchers reported a significant discovery about how cells regulate the passage of calcium.
In the heart and brain, calcium represents a ubiquitous cellular signal, critical to the organs’ proper functions. Understanding how cells control calcium’s entrance and exit may reveal new strategies for correcting problems with brain function or heart rhythms.
In experiments with human kidney cells, the researchers determined that about 25 copies of a key calcium-binding protein called calmodulin are poised near the doors of each channel through which calcium enters the cell. Despite the large number of calmodulin molecules available, just one is needed to bind calcium and close the channel, the scientists discovered.
“For 15 years, researchers have been unable to gauge how many calmodulin molecules are within ‘earshot’ of each calcium channel,” says David Yue, M.D., Ph.D., professor of biomedical engineering. “Our discovery will help us create better models of how cells regulate calcium.”
To count the number of calmodulin molecules, Yue and co-authors Masayuki Mori, Ph.D., and Michael Erickson, Ph.D., used patch-clamp electrophysiology and genetically engineered calcium channels to estimate how many calmodulin molecules are affiliated with each channel, as they exist in living cells. Also important for the experiments, the authors used a relatively new and patented computational algorithm for quantifying fluorescence resonance energy transfer (FRET), a means of monitoring molecular proximity in living cells.
In FRET, if two fluorescently labeled proteins bind each other, there is a detectable increase in light intensity. The scientists attached one fluorescent tag to calmodulin and another to the channel, and observed an increase in light when produced in live cells.
“By analyzing these experiments, we found that about 25 calmodulin molecules associate with each calcium channel,” says Yue. “That is a remarkable concentration compared to the rest of the cell.”
In fact, the concentration of calmodulin around the channel is 100,000 times higher than elsewhere in the cell. Why and how calmodulin stays by the channels are still unknown.
To determine how many calmodulins were needed to close the channel door, the researchers borrowed a concept from plants. Plant calmodulin kinase is naturally tethered to a calmodulin-like module, thus ensuring quick binding and signaling.
Mimicking this, the scientists fused their calmodulin protein onto a calcium channel using “bridges” of different lengths and, in a series of experiments, observed whether the channels were closing. They discovered that proteins with a short bridge signaled properly, whereas those with a long bridge did not, since it let other free-floating proteins get in the way.
In complex calculations, the researchers took the bridges’ lengths and the channel’s activity to determine the optimal bridge length. They also figured out that one calmodulin was enough to close the channel.
“We don’t know why only one of many nearby calmodulin molecules is needed to close the channel,” says Yue. “Many nearby calmodulin molecules are available, ready to bind the cloud of calcium ions that enter after the gates are “zapped” open, so at this point it’s still a mystery.”
That it took Mori and Erickson two years to complete the study shows the problem’s difficulty. “It was actually quite crazy to spend time and resources on this complicated problem,” says Yue, “but in science, you need enough creativity and energy to try high-risk, but important, projects.”
Now the scientists will try to figure out why calmodulin hangs out at the channel, how the proteins stay there in such high concentrations, and whether any other proteins help keep calmodulin near the calcium channel.
This study was supported by grants from the National Institutes of Health. Authors on the study are Mori, Erickson and Yue of the Department of Biomedical Engineering and Neuroscience, Johns Hopkins School of Medicine.
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