The success of any endeavor often depends on transportation. Whether it’s passengers moving through airports or little metal balls in a pinball machine, a minor glitch can throw the entire system out of whack. Mayo Clinic physiologist Michael Romero, Ph.D., studies glitches in the world’s smallest transportation system — ion transporters. These specialized proteins move charged atoms in and out of cells. The Romero lab focuses on bicarbonate transporters because they are crucial to maintaining pH balance in cells.

The pH is a measurement of acidity or alkalinity based on the percentage of hydrogen ions in solution. On a scale of 14, where a pH of 7.0 is neutral, anything below 7.0 is acid and above 7.0 is alkaline. Anyone who has ever taken care of a swimming pool knows that if the pH of the water is too low your eyes will sting and if it’s too high algae will start to grow. In the human body the stakes are much higher.

“In a biologic system, if a pH is too high — that’s alkalosis, or too low — that’s acidosis. Either way you die,” Dr. Romero says. “We want to know the nuances and details of the transport system to figure out what’s really going on.”

A nano transportation system

To understand this vital process, Dr. Romero measures ion movements across cell membranes using microelectrodes — cylinders of glass with tips measuring half a micron, or millionth of a meter. The nano scale is necessary because pH is very finely tuned. A level below 7.0 is too acidic to support life; above 7.9 it’s too alkaline. A pH of 7.0 has 100 nanomole (or billionths of a mole, the molecular weight unit) of protons; a pH of 7.9 has 12 nanomole.

“So all of life exists between 12 and 100 nanomole of protons,” Dr. Romero says. “That means things are very tightly controlled. We want to understand that control on the molecular and atomic level.”

Acid is a natural by–product of the breakdown of fats and other processes in the body. The body controls pH by transporting bicarbonate, which is an alkaline, in and out of cells to buffer the protons that are a normal byproduct of metabolism. This buffering occurs in the kidneys, which absorb the equivalent of a one pound box of sodium bicarbonate — baking soda — a day. The lab members looked to the kidneys, the body’s absorption experts, as a logical focus for research on ion transport across cell membranes. But first, they needed cells they could manipulate in a lab.

Cloning a gene to get the right protein

About a decade ago, Dr. Romero discovered how to clone the gene for a protein called NBCe1, a sodium bicarbonate transporter, by following function.

“Once we had one gene, we were able to clone the NBCe1 gene from a variety of different species,” he says. “Species variation allows us to determine how evolution has changed the protein and its physiology.”

That accomplished, the team was ready to begin sorting out how bicarbonate transporters work. They manufactured proteins by injecting the immature egg cells (oocytes) of lab animals with the cloned NBCe1 RNA. Then, using the microelectrodes, they studied ion transport across cell membranes under a variety of conditions.

“The beauty of this system,” he says, “is that it allows us not only to measure what’s going on inside the oocyte, but also to control the outside environment while we’re making measurements.”

The research team discovered mutations in the NBCe1 transporter. Subsequently, Dr. Romero found that both lab animals bred to lack the transporter, and people who have the genetic mutation in the transporter have “…significant metabolic acidosis.” He explains:

“The protein either doesn’t function correctly or doesn’t make it to the cell membrane where it’s supposed to go. That means the blood has very low bicarbonate, and the kidney just can’t move bicarbonate out of the cell back to the blood.”

Dr. Romero anticipated that all of the patients with the mutated bicarbonate transporter gene would have severe acidosis and significant kidney disease. He was right.

The NBCe1 protein “is the only bicarbonate transporter in an area of the kidney where most of absorption is done,” Dr. Romero says. “And if you don’t absorb that pound of sodium bicarbonate a day, you have problems.”

The discovery made Dr. Romero wonder how the mutant proteins in his lab compare to those in human patients. The problem was the mutation has been detected in only eight patients worldwide. Dr. Romero was, therefore, extremely fortunate to obtain human DNA samples from collaborators in California and Israel. After the mutation in the NBCe1 gene was found, Dr. Romero and Min–Hwang Chang, Ph.D., another physiologist in his lab, were able to fabricate the same mutant protein.

Collaborating with eye researchers

What the researchers did not anticipate was a link between kidney and eye problems in patients with the NBCe1 bicarbonate transporter mutation. When they learned that these patients develop glaucoma, they were baffled. The lab animals they had bred with a mutated bicarbonate transporter developed acidosis, yet their eyes were not affected. Why the difference in humans?

At Mayo, it is not unusual for two researchers from entirely different fields to put their heads together to solve a problem. Mayo’s hallmark is collaboration. In fact, much of its architecture is specifically designed to enhance cooperation among physicians, investigators and educators. It was a casual hallway conversation with ophthalmologist researcher Michael Fautsch, Ph.D. that led Dr. Romano to a solution. When Dr. Fautsch explained that human eyes are virtually unique among mammals, moving salt and water around the organ very differently from rodents, it suddenly all made sense because sodium bicarbonate and water move together through the human body.

“So if the sodium bicarbonate transport system malfunctions,” explains Dr. Romero, “fluid builds up in the eye, increasing pressure and causing glaucoma.”

Dr. Romero’s discoveries offer hope of improved glaucoma treatment.

“If we can control this sodium bicarbonate transporter protein and either tune its activities so it moves salt faster or slower, the water, of course, will go faster or slower,” Dr. Romero says. “With a molecular switch, you could potentially tune up or tune down the amount of water that’s moved and control the pressure in the eye.”

It is a significant discovery for people with glaucoma because it involves one of only eight gene mutations that cause the disease.

“Dr. Romero’s work is very important to understanding the basic physiology of the eye,” says ophthalmologist researcher Arthur Sit, M.D. “Understanding the flow of fluid in the eye is critical to understanding the mechanisms of current and future treatment for glaucoma.”

Dr. Sit believes this research “could lead to a significant advance” in glaucoma therapy. “There is no cure for glaucoma, so clinicians are always looking for new treatment options,” he says. “If the bicarbonate transport system can be manipulated without affecting other systems, it has the potential to provide therapy that is free of the many potential side effects in current treatments. Since it would use a fundamentally different mechanism than existing therapies, it could be added to current medications.”

Besides studying the bicarbonate transporter’s function, Dr. Romero also is studying its structure. He and proteomics researcher James Thompson, Ph.D., recently discovered that one of the human NBCe1 mutations loses part of its function because a portion of the protein structure isn’t held together correctly.

“That means that even the atomic shape and form of this bicarbonate transporter are important for understanding human disease,” Dr. Romero says.

His lab is now trying to use atomic pictures of this protein as another way of finding the molecular on/off switch. Dr. Romero hopes his discoveries will eventually lead to new treatments. He appreciates what he calls an ideal environment for basic research to ultimately benefit patients.

“The sort of conversation that I had with Dr. Fautsch is much more possible here at Mayo than at other places that I’ve worked. There, physicians that I talked to ran research labs, but did not see many patients. It’s not quite the same as talking to folks who see patients routinely and have an intimate knowledge of what’s going on with them.”

Dr. Sit agrees.

“Finding the link between basic research and clinical application is not always a simple process. But having us all under the same roof greatly accelerates the translation of basic science to clinical application. The free exchange of information is something that is truly exceptional at Mayo.”

— Barbara Toman, February 2009