Scott Hollingshaus and Dr. James P. Lewis, Physics and Astronomy

Trans-membrane channels are the highway mechanisms of biological cells. Through these channels, cells transfer information in the form of ions and other molecules. These transferred materials are called permeating ions. It is well known that most channels work on the principles of size, shape, and charge. Charged channels allow permeating ions of the opposite charge and appropriate shape to pass, while similarly charged ions cannot pass. However, the fluid inside and outside a cell contains many atomic ions, such as sodium (Na+) ions. Because these atomic ions are both small and charged they are able to fit through many different types of channels. At times, they may even pass into a channel that is not designed to transport atomic ions. It seems that atomic ions compete with other permeating ions for access to these channels. This theory is called “Charge-Space Competition Theory,” and was developed at Brigham Young University. My research studied the effects of an increase in solvent ion concentration on the ability of a channel to bind with its intended permeating ion.

Being the first experiment in a series of investigations, my project was simplified in two important ways. First, instead of running experiments with actual ion channels, I performed computational experiments on the Marylou10 supercomputer at the Ira and Marylou Fulton Supercomputing Center at BYU. Computational molecular dynamics simulations use computers and the basic mathematics of Newtonian physics to calculate the motions of atomic models through time. Compared to experiments with actual matter they are very inexpensive to perform and provide valuable theoretical data.

Second, I used an ion channel analog for my computer model, rather than an ion channel model. An analog model is one that posses many of the same characteristics of a true model but is simplified in one way to make it easier to work with. In this project I used the Acetylcholinesterase (AChE) enzyme along with its substrate, the Acetylcholine (ACh) neurotransmitter. AChE is like an ion channel in that its substrate must pass through a 20 Å long channel in order to bind at the active site. However, because AChE has only one entrance to the active site, it acts like a simplified trans-membrane ion channel with only one opening (see fig. 1). I studied the validity of the Charge-Space Competition Theory by comparing the binding affinity of AChE for ACh in different ion concentrations. Results from this simplified system provided insight into more complex ion channel systems.

To calculate the relative binding affinity I ran a Free Energy Perturbation (FEP) simulation on an AChE-ACh system resting in a bath of water molecules with a physiologic Na+ concentration of 140 mmol. This simulation involved changing a single parameter of the system slowly over time and calculating the change in the total free energy (G) of the system. Since we wanted to see how increasing the ionic concentration of the bath would influence the binding affinity, I varied the charge of a Na+ placed in the active site of AChE from 0 to +1, thus simulating the entry of an ion into the enzyme from a highly concentrated bath. An increase in the free energy of the system would represent a decrease in binding affinity. In order to make the FEP simulation work correctly, I performed two simulations: one on the AChE with an ACh in the active site and one on the AChE without an ACh in the active site.

We acquired the AChE-ACh model files from the Andrew McCammon group at UCSD, and I ran all the simulations with NWChem, Version 4.5, a computational chemistry package from Pacific Northwest National Laboratory. Interestingly, the NWChem program presented me with a problem in that it would not output reasonable energies when charging the Na+. Therefore, instead of charging the ion to +1 charge I discharged it from +1 to 0 charge and took the negative of the energy differences produced. I was able to do this because FEP does not distinguish between charging and discharging; mathematically the difference is a minus sign.

The results of the experiment were very pleasing. The FEP simulation of the AChE-ACh complex produced a G of -115 ± 2 kcal/mol and the simulation of the AChE in the absence of ACh produced a G of -196 ± 2 kcal/mol. Put together, these results provide a change in binding affinity of 81 ± 4 kcal/mol with the presence of a Na+ in the active site (see fig. 2). Examination of these data with respect to the Charge-Space Competition Theory suggests that solvent ion concentration could be an important factor in the affinities of trans-membrane channels as well. As observed, the competition between the ions in the bath and the ACh for access to the AChE active site is considerable.

The results of this experiment immediately inspire various other experimental questions. For example, further research on the AChE-ACh system could include FEP simulations with two Na+ ions in the active site, or perhaps a Na+ being charged from 0 to +2 charge. Other simulations could be run with negative ions in the active site to compare the energy differences. We would also like to run longer simulations with both ACh and Na+ in the active site to see if the sodium will be displaced completely out of the enzyme. Aside from these computational simulations, we would like to run a physical experiment to verify the results of this recent project. We would like to experimentally measure the effect of the solvent ion concentration on the binding affinity of the AChE enzyme by running kinematics tests on the enzyme in solution.