A fluorescent biosensor is the first to quantify the ionic strength inside living cells (ACS Chem. Biol. 2017, DOI: 10.1021/acschembio.7b00348). The sensor could enable cell biologists to follow changes in ionic strength over time or at different locations in a cell, changes that are key for controlling protein aggregation.
Ionic strength measures the concentration of unbound ions floating in a cell. It affects many biological processes including protein folding and assembly and the catalytic activity of enzymes and ribozymes. Measuring ionic strength could also help researchers better understand protein misfolding and aggregation, two key processes in the formation of amyloid fibers, which are linked to neurodegenerative disease and type 2 diabetes.
To measure the total concentration of sodium and potassium ions in cells, researchers have burned dry, dead cells and used the resulting flame color changes to quantify the levels of ions per cell. But these flame measurements do not provide information about the ionic strength, which can change when ions bind to biomolecules or the internal volume of the cell changes.
To build a cellular ionic strength sensor, Boqun Lu, Bert Poolman, and Arnold J. Boersma at the University of Groningen, made use of Förster-type resonance energy transfer (FRET), which relies on the transfer of energy from one molecule to another, producing light with an intensity that depends on the molecules’ distance from one another. It is commonly used as a signal for biosensing and molecular imaging.
The researchers built molecules carrying a cyan and a yellow fluorescent protein, separated by two charged peptide helices. In a solution of low ionic strength, positive charges on one helix attract the negative charges on the other. The helices fold together like the arms of tweezers, drawing the fluorescent proteins at the tips close together. In a solution of higher ionic strength, ions surround the helices and neutralize their charges, keeping the helices, and thus the fluorescent proteins, separated.
When the proteins come close together at low ionic strength, the cyan fluorescent protein, excited by blue light, efficiently transfers energy to the yellow fluorescent protein, which emits yellow light. But in high ionic strength the proteins remain separated and the glowing cyan protein dominates the fluorescent signal since it is unable to transfer its energy to the other protein.
The researchers first confirmed that the sensors could distinguish different concentrations of sodium and potassium salts. Then they introduced a sensor to human embryonic kidney cells and correlated the sensor’s response to the concentration of potassium chloride inside the cells. They controlled the intracellular potassium chloride concentration by adding the salt to the culture medium along with two potassium transporter molecules that shuttle potassium across the cell membrane. The transporters shuttled ions into the cells until the concentrations were equal across the membrane.
The sensor with the best-performing peptide sequence measured potassium at 130 mM within cells. That was, as expected, a little lower than the 140 mM total potassium ions measured using the flame method. In the future, it may be possible to modify the sensor so it travels to specific locations in the cell and provides localized measurements, Boersma says.
“This is a beautiful example of using fundamental chemistry to design a sensor that is simple but intelligent,” says Xin Zhang, who studies cellular protein folding at Pennsylvania State University.