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Electrogenic transport of ammonium across the cell membrane

Nitrogen is an essential building block of proteins. Bacteria and plants have developed special membrane proteins that are able to transport nitrogen, which comes in the form of ammonium ions, into cells. Such ammonium transporters have also been discovered in humans. Crystallographic investigations have led to the elucidation of the complex structure of numerous ammonium transporters. Dr. Susana Andrade is the head of a group of junior researchers at the Institute of Organic Chemisty and Biochemistry at the University of Freiburg, funded through the German Research Foundation’s (DFG) Emmy Noether programme. Dr. Andrade and her team are taking a very close look at these transport molecules.

Import and export are the “business” of all cells. Important low molecular compounds such as phosphate or ammonium are essential building blocks of proteins and lipids. Many of these compounds need to be taken up from the environment. Membrane proteins that are able to actively or passively transport such compounds into cells have evolved and are present in all organisms, even those as simple as bacteria. The root cells of plants can contain millions of ammonium transport proteins. In order for plants and animals to be able to use nitrogen, it must first be converted to a chemically available form such as ammonium. Complex biochemical pathways lead to the integration of nitrogen into amino acids and proteins. Ammonium transport proteins have also been found in humans, including, for example, representatives of the rhesus (Rh) protein family which is one of the many human blood group systems. The Rh proteins are found in the membranes of blood and kidney cells where they regulate the cellular pH and ion concentration. “The crystal structure of some ammonium transport proteins has been clarified over the last few years,” said Dr. Andrade. “However, hardly anything is known about the function of these molecules.”

The transport mechanism is still unknown

Over the last few years, Andrade has worked on the elucidation of the crystal structure of an Archaeoglobus fulgidus ammonium transporter. Andrade and her team succeeded in determining the high-resolution (1.4 Angström; 1 Angström corresponds to 0.1 nanometre) crystal structure of the molecule, which enables them to look at single amino acids. The Archaeoglobus fulgidus ammonium transporter consists of three identical monomers. Each monomer has eleven transmembrane helices and a channel that can be closed upon the specific arrangement of two amino acids. The researchers know from other investigations that the protein mediates the transport of nitrogen into bacterial cells, but this is currently all they know. Nothing is known about the transport mechanism: is the transport protein a passive channel that enables positively charged ammonium ions (NH4+) to pass through as long as the ion gradient allows it? Or is the protein an active, energy-dependent sluice that transports its substrate by way of conformational changes?

These questions are extremely difficult to answer. It is still unknown what type of substrate the protein depends on. Is it positively charged ammonium ions? Or is it electrochemically neutral ammonia gas? “Crystallography enables the researchers to look at the molecular structure of the protein and hence is one of the methods that provides the researchers with the greatest insights,” said Andrade. “However, in our case, crystallography has proven unsuitable for providing information regarding the function of the transport protein.”

When researchers succeed in crystallizing transport proteins along with the molecule that they are transporting, they have information about the type of substrate specific transporters depend on. Crystallography visualizes molecular structures as it measures the electron distribution in a crystal, which can differ from atom to atom and from molecular bond to molecular bond. The crystallized Archaeoglobus fulgidus ammonium transport protein revealed the presence of water molecules that have a similar electron distribution to that of ammonium. Andrade’s researchers are therefore unable to distinguish between water and ammonium molecules. “We need to find a different method in order to distinguish these two molecules in the channels,” said Andrade.

Is the freight charged?

The researchers led by Dr. Susane Andrade use the electrodes of the apparatus shown in the photo to investigate small membrane pieces. © Dr. Susana Andrade

At present, the biochemists from Freiburg are trying to solve the question using electrophysiological methods. They clamp small artificial membrane pieces between two chambers that contain a physiological solution and integrate the transport protein under investigation into the membrane fragments. The application of a charge triggers ions to move across the membrane. The researchers are able to measure the flow of electrical current when the transport proteins use ammonium ions as substrate; however, no electrical current can be measured when electrochemically neutral ammonia gas is used as substrate. The method also enables the researchers to glean information about the function of the channel, which is closely related to the flow of electrical current. "We hope that this method will provide us with the information we need. If we are unable to measure electrical current, this means that the transport protein under investigation does not use ions as substrate," said Andrade highlighting that her group of researchers is currently analyzing their initial results.

Crystal structure of the Archaeoglobus fulgidus ammonium transport molecule (top) and of the intracellular GlnK protein that seems to affect the opening of the channel. © Dr. Susana Andrade

Initial results suggest that the transport mechanism is rather complex: the researchers assume that an amino acid barrier inside the channel cannot be the only site where the transport of substrate can be controlled. Experiments in which the researchers manipulated the two amino acids of the barrier suggest that the barrier needs to be open for the cell to be able to take up ammonium; the findings also suggest that the absence of the barrier does not mean that the channel is automatically open. The issue becomes even more complicated as a result of the identification of a molecule inside an Archaeoglobus fulgidus cell that interacts with the ammonium transport protein. This protein, which is known as GlnK protein, is able to monitor and respond to cellular ammonium and energy levels. “Our findings suggest that this protein regulates the opening of the ammonium transport protein,” said Andrade highlighting that the protein has a kind of loop at one of its ends. Crystallographic investigations have shown that this loop fits into the channel, thereby closing it. In biological terms, the ability of GlnK proteins to close and open the channel in relation to the intracellular ammonium and energy level makes sense; the cells do not take up ammonium when the intracellular ammonium level is high enough and when too little energy (ATP) is available.

In addition to electrophysiological experiments, Andrade and her team will in future also focus on the molecular function of GlnK. They hope that they will eventually be able to clarify the mechanism and regulation of ammonium transport in all its complexity. “This is a very competitive field of research,” said Andrade, adding “but the insights we are gaining are of great importance, for example with regard to the function of the human ammonium transporter counterparts, i.e. rhesus proteins.”

Further information:

Dr. Susana Andrade
Head of Emmy-Noether Junior Research Group
Institute of Organic Chemistry and Biochemistry
Department of Biochemistry
Albertstrasse 21
79104 Freiburg
Tel.: +49 (0)761/ 203 8719
Fax: +49 (0)761/ 203 6161

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/electrogenic-transport-of-ammonium-across-the-cell-membrane