Closing the gaps

04.15.09 -

Biophysicists at the University of Pennsylvania used NCSA's Abe to clarify a mysterious interaction between cholesterol and neurotransmitter receptors.

Research into how anesthesia works may eventually unlock not only that mystery but dozens of others as well.

"Anesthetics have improved significantly over the last hundred years, but the mechanism of anesthesia is not understood at all," says Grace Brannigan, a researcher at the University of Pennsylvania's Center for Molecular Modeling (CMM).

To gain insight into how anesthetics work, a team consisting of Brannigan and fellow researcher Jérôme Hénin, University of Pennsylvania professors Michael Klein and Roderic Eckenhoff, and Richard Law of the Lawrence Livermore National Laboratory, is focusing on the nicotinic acetylcholine receptor (nAChR).

This receptor, found in both brain and muscle cells, is a ligand-gated ion channel. The channel opens or closes in response to binding with a chemical messenger (ligand) such as a neurotransmitter, like acetylcholine. When the channel is open, ions can cross the membrane. Anesthetics are believed to close the channel, thus reducing sensations and possibly causing the memory loss associated with being under general anesthetic.

Structural gaps

The basic nAChR structure consists of five protein subunits symmetrically arranged. Cryo-electron microscopy examination of the structure by British and Japanese researchers revealed more particulars about the receptor's structure than previously known, including holes (gaps) in the protein's density that were thought to hold water.

The CMM team looked at those holes, however, and realized they were the perfect size and shape for cholesterol. Knowing that cholesterol is essential for brain function, and that nAChR can only do its job properly if cholesterol is nearby, the team decided to explore the relationship between the gaps and cholesterol.

As nAChR is also involved in inflammation, Alzheimer's disease, Parkinson's disease, schizophrenia, and epilepsy, the team's findings could lead to increased understanding of these health issues as well. In addition, the team's results likely apply to closely related receptors, such as the GABA receptor. These related neurotransmitter receptors are involved in regulating mood and sleep.

Modeling the hypothesis

Since modeling and simulation is the domain of the Center for Molecular Modeling, the team directed their expertise to their hypothesis that the nAChR protein density gaps were occupied by cholesterol rather than by water.

The CMM team used the basic shape of the nAChR transmembrane domain as revealed by electron microscopy (EM) structure 2BG9. Improvements in EM allow researchers to observe specimens in their native environment, affording more accurate observations and better data to use in computer modeling and simulations. The team then calculated potential binding sites (colored yellow, orange, and red in B) within the protein's transmembrane domain. This was done by carefully examining the receptor using advanced visualization software, identifying holes, and then relying on their chemical intuition as to where to place cholesterol, as well as using an automated docking program that found holes and inserted cholesterol.

The researchers say their proposed sites for cholesterol that are colored orange and red contradict the 1993 publication of hypothetical binding sites for cholesterol for nAChR. The earlier research primarily considered cholesterol in the interface with the surrounding membrane only (A). This new research shows that cholesterol can be deep within nAChR.

Using the prior data, the researchers used the Nanoscale Molecular Dynamics (NAMD) code on NCSA's Abe cluster to test their theory. Running four simultaneous jobs requiring 800 processors each, the team simulated more than 230,000 atoms for 100 nanoseconds (C and D).

They observed that after 25 nanoseconds in the control simulation, the protein significantly redistributed its density, with gaps closing because of the collapse of individual subunits (D). Placing cholesterol in the gaps reduced the number of subunits collapsing.

Next steps

The molecular dynamics simulations supported the team's hypothesis that the published experimental structure is consistent with cholesterol molecules buried within the receptor. Their findings were published in September 2008 in the Proceedings of the National Academy of Sciences. Although the findings need to be confirmed by experimental researchers to become accepted scientific fact, the simulations will aid the team in the next phase of their anesthetics research.

"There are several next steps, but the most important in the framework of this project is to now actually start looking at how small anesthetic molecules are going to interact with the protein," says Hénin. "And now we know that when we do these simulations, we'll need to include cholesterol molecules at the places where we think they are interacting (E). We think that is going to be more of a multiple interaction. Not just a protein-anesthetic interaction, but also a protein-cholesterol-anesthetic interaction."

The researchers hope their work leads to improved drug design. "You could fine-tune the properties of the drug if you could understand how the mechanism works," says Brannigan. "For instance, by understanding how anesthetics work, you could design new anesthetics that could be more powerful yet maybe wouldn't have some of the side effects that current ones do."

This project was funded by the National Institutes of Health and the National Science Foundation.

Team members
Grace Brannigan
Jérôme Hénin
Michael Klein
Roderic Eckenhoff
Richard Law