Research into ion transport through cell membranes aided by fluorescence spectroscopy

Some diseases are more unkind than others.

Cystic fibrosis, an inherited disorder often inflicting the young, causes severe damage to the lungs, digestive system and other organs in the body (Clinic, 2021). Worldwide, more than 700,000 people suffer from this disease (Foundation, 2021).

If affects mucus, sweat and digestive juices production. Normally, the secretions are thin and slippery. But because of a defective gene, the secretions become sticky and thick. Instead of acting as lubricants, the secretions plug up tubes, ducts and passageways, especially in the lungs and pancreas.

It produces a constellation of symptoms. Persistent coughing, frequent lung infections including pneumonia or bronchitis, wheezing or shortness of breath, poor growth or weight gain, and male infertility are among its debilitating effects.

For those suffering from the disease, “mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the CFTR protein to become dysfunctional. When the protein is not working correctly, it’s unable to help move chloride ― a component of salt ― to the cell surface. Without the chloride to attract water to the cell surface, the mucus in various organs becomes thick and sticky” (Foundation, 2021).

Ion transport is at the heart of most biological processes, in everything from vision to muscle movement to energy production. When these things go wrong, when these membrane proteins that transport ions and regulate the ion concentration in the cell go awry, that becomes a big issue. Like in cystic fibrosis.

Humans have these chloride channel proteins in the cell membrane that regulate chloride transport. With cystic fibrosis, it essentially shuts down or become misregulated. But if you can have a small molecule that essentially bypasses that faulty protein and transports chloride in a controlled way, in the correct type of cell and in the correct location in the body, then we have something that's potentially a very useful therapeutic. It’s essentially making a small molecule do what the protein should but doesn't do.

Hope for this and many other diseases is fueled a United Kingdom researcher who is trying to develop delivery systems that bypass these faulty cell membrane proteins to properly orchestrate the cell transport system.

Matthew Langton is an Associate Professor and Royal Society University Research Fellow in the Department of Chemistry at the University of Oxford. His group’s research interests are at the interface of synthetic supramolecular chemistry, biological chemistry and nanotechnology.  (Oxford, 2021)

Supramolecular chemistry is the field of chemistry that concerns intermolecular interactions. The study of these non-covalent interactions is crucial to understanding many biological processes. Controlling these in artificial systems enables new self-assembled systems, responsive molecular devices and nano-scale architectures to be engineered for a wide range of applications, like a cure for cystic fibrosis

“In our group we seek in particular to design, synthesize and study functional supramolecular devices which can interface with biological systems,” he said.

At the heart of his research is a desire to design, make and study responsive supramolecular systems that operate in lipid bilayer (cell) membranes and can be “remote-controlled” by an external stimulus like chemicals, light, pH and so forth (Oxford, 2021).

Langton is interested in in putting functional molecules into cell membranes. And there are two reasons for doing that. One is to manipulate living cells.

“For example, we're interested in ion transporters, which are small molecules that can facilitate the transport of ions such as chloride or potassium across cell membranes,” he said. “In biology this is done by membrane proteins. And when these go wrong, then that leads to diseases. So instead we’re designing synthetic molecules that can transport ions and other molecules, which may ultimately lead to new types of therapeutics.”

The other aspect he’s studying is developing completely artificial systems where you can use artificial cells that do useful things, like nanoscale chemical reactors or devices to communicate with living cells.

“And so it's important in these applications to be able to control how things cross the membrane,” he said. “We need to worry about how to get molecules in and out of the compartment. What we try and do is use small molecule systems that we can synthesize in the lab, that we can design to do a specific task and respond to a specific stimulus that allows us to control it. This is rather than trying to use an existing protein and modify that in some way ― we're coming at it from a bottom up approach, really.”

He designs molecules that he can make and put into the membranes of artificial cells that can be made quite simply in the lab. These are similar to a cell, except that he’s stripped out most of the biology and is left with the cell membrane.

One of the applications of artificial cells is trying to make a cell-like system that can do something useful. For example, you might want a system that can generate drug molecules from inactive precursors, and then release them on demand. Many researchers across the globe are interested in trying to do that.

“So you can imagine having a sort of cell or a miniature cell that has all the components necessary in it, but in an inactive state,” he said. “And then you program them in some way to carry out the synthesis of the drug molecule, and release it in the correct location in the body. That's a long way down the line, but it’s a very exciting goal.”

It’s one of the potential applications, but in all of these applications, researchers need some way of controlling what crosses the membrane, also referred to as lipid bi-layer membranes, or the cell shell.

“If you're trying to make a molecule in the lab, we usually do it in a flask. You can add things into the flask, through the neck, and you can take things out. If you use a cell as a reaction vessel, you can't do that because it's a cyclical object that's completely encapsulated. So we try and control what goes in and out using chemistry.”

Langton and his team often work with suspensions of vesicles, which are small fluid-filled bladders surrounded by a cell membrane, to study nanomolar concentrations of material. Fluorescence spectroscopy is the only technique that’s sensitive enough to study these suspensions. The researchers use fluorescence to detect the transport through the membrane.

“For example, we've recently reported a system that can transport ions across a membrane when we irradiated it with certain wavelengths of light,” he said. “So we can shine a red light on it and turn on ion transport, and then if you shine blue light on it, you turn it off again. We started studying these systems with these artificial cells in the spectrometer, and these cells encapsulate fluorescent molecules which responds to ion concentration. So we can measure the rate of change of the fluorescence, which tells us about the rate of ion transport across the membrane. The sensitivity of the florescence experiment is vital really to look at these very low concentrations of molecules in nanoscale compartments.”

Langton’s group uses a HORIBA Duetta™ benchtop spectrofluorometer, which combines, simultaneously, the functions of fluorescence and absorbance spectrometers. With its high-speed built-in CCD detector, the Duetta can acquire a full spectrum from 250 nm to 1,100 nm in less than one second, making it the fastest fluorescence spectrometer on the market.

“All of my group, which is around eight to 10 researchers, spend, say, half their time doing synthesis and the other half of their time measuring the properties of these molecules, typically by fluorescence spectroscopy,” he said. “So (the Duetta) runs day in, day out. The poor thing. It's used a lot.”

Langton’s group is trying to develop different types of vehicles for drug delivery. In one study, they showed that a light-activated molecular switch essentially grabs hold of an ion, carries it across the membrane and then release it on the other side. It's an artificial transport vehicle that he can control (Langton, 2020).

“In that particular case, when we shine red light on it, it grabs hold of the ion. And when you shine blue light on it, it releases it,” he said.

Medical applications of the discoveries Langton’s group is making are some way down the road. He leads a primarily fundamental science group interested in all of the aspects of how molecules behave inside membrane and how you can control the functions of synthetic molecules, like pharmaceuticals, using external stimuli. But the longer term application of all of this is biotechnology and medicine.

How long will it take before this basic research becomes commercialized?

“I would say we're looking at a decade or so, maybe optimistically,” Langton said. “Many research groups are looking in the area of responsive medical therapeutics and nano-medicine, and many of these applications are making real progress. And maybe, it's realistic to say within the decade we may see some synthetic ion transporters as therapeutics in the clinic. Perhaps using artificial cells to synthesize and deliver drugs may take longer, but it’s a very active field so you never know”

Langton’s biggest practical hurdle has been operating during COVID-19, limiting the number of researchers available to 40 percent.

But the major scientific problem is control, he said.

“Membrane proteins, like the visual receptors in your eye, are extremely sensitive, and it's a very well controlled process. Being able to achieve such a high level of control over ion transport with a synthetic system is very difficult. But often you want to design a drug molecule that only targets are particular type of cell in a particular location in the body, or control a chemical reaction in a particular artificial cell. And so you need a system that can be activated when and where you want it to. Control is key in all of this ― that's the main scientific hurdle.”

Clinic, M. (2021). Cystic fibrosis. Retrieved from Mayo Clinic Diseases and Symptoms:
www.mayoclinic.org/diseases-conditions/cystic-fibrosis/symptoms-causes/syc-20353700

Foundation, C. F. (2021). About Cystic Fibrosis. Retrieved from What is CF:
www.cff.org/What-is-CF/About-Cystic-Fibrosis/

Langton, A. K. (2020). Reversible photo-control over transmembrane anion transport using visible-light responsive supramolecular carriers. Chemical Science(Issue 24). Retrieved from
pubs.rsc.org/en/content/articlelanding/2020/sc/d0sc02745f

Oxford, U. o. (2021). Professor Matthew Langton. Retrieved from University of Oxford Department of Chemistry:https://www.chem.ox.ac.uk/people/matthew-langton