This interdisciplinary project brings together five complementary computational and experimental researchers to build an integrated and experimental analysis that can decipher how a change in the external environment in the form of a model insult (loss of sialic acid on a glycosylation site) is manifested in changes in the electrical properties of potassium channels that ultimately leads to paralytic behaviors in Drosophila organisms carrying this defect. Sialic acid was chosen to represent a model external biochemical stimulus, but the exact physiological role of this biochemical modification remains a relative poorly understood area especially in neuronal and in vivo animal models. To understand the effect of sialic aicd, the Principal Investigators (PIs) will utilize molecular scale dynamics simulations; create a Markov model of the kinetic and electrophysiological properties which connects to the molecular scale dynamics; and compare these predictions to experimental cell culture systems for different potassium Shaker family ion channels. This project will compare the neural behaviors predicted from molecular simulation, Markov models, and cell culture system to electrophysiology and behavior phenotypes of a Drosophila mutant in which the sialic acid gene is deleted. Understanding the role of the biochemical environment and particular sialic acid on electrical properties and cell dynamics will help one to identify possible methodologies for alleviating the symptoms, and potential pharmacological lipid agents to rescue or minimize the defect will be tested.
This project will create a new interdisciplinary network of researchers with expertise in molecular modeling; ion channel modeling; electrophysiology, biochemical engineering, and Drosophila genetics to investigate the role that the biochemical environment, and in particular glycosylation and sialic acid, plays on electrical properties and function of ion channels, neuronal processes, and neurological disease. The group is drawn from a variety of different geographic institutions and diverse ethnic backgrounds, and two of the co-PI''s are female. The proposal will broaden the participation of high school and middle school students from underrepresented groups through an educational partnership with city school teachers in modeling the brain and brain disease. This program will excite students all the way from middle school and high school to graduate and post-doctoral levels about the integration of engineering and science by showing how behaviors and disease of the most complex organ, the brain, can be modeled and examined using experimental systems such as flies.
The overall goal of the project was to determine the effects of sialic acid on the function of ion channels through an integrated approach ranging from mathematical modeling to an animal model. Sialic acid is added to proteins in cells during glycosylation. Glycosylation is the addition or removal of carbohydrates to proteins, which can affect the protein’s function and targeting and hence overall organism behavior. Sialic acid is particularly relevant because this is the only charged sugar added during glycosylation and charge may alter the behavior of membrane proteins, especially voltage-dependent ion channels. Ion channels controls a number of important functions in humans including neuronal and chemical signaling. Broader Impacts: If sialic acid affects ion channel function, then it may alter critical neuronal and metabolic outputs. A change in the sialic acid content could lead to ion channel malfunction and neurobiological diseases. A number of people suffer from ion channel dysfunction, called channelopathies, and this project demonstrated a link between sialic acid and ion channel function. By understanding what causes ion channel malfunction using animal and mathematical models, the development of more effective treatments for the diseases will be possible. In addition, the project has contributed to the education of four PhD graduate students in chemical and biomolecular engineering and biology who are now in post-doctoral studies or are working in the biotechnology industry. Numerous undergraduates and several masters’ students were educated in the project as well, and these graduates have gone on to graduate study or work in industry. Intellectual merit: An initial goal was to determine the glycosylation of proteins in different model organisms and cell lines. Solid phase extraction of N-linked glycopeptides (SPEG), was used in combination with mass spectrometry to determine the glycosylation of proteins from multiple cell types including the fruit fly (Drosophila), zebrafish (Danio rerio), and Chinese hamster ovary (CHO) cells. These glycosylated proteins were published and are available on the web. In order to examine the role of sialic acid in a model organism, a Drosophila mutant was generated in which the sialic acid synthase gene has been knocked out. The mutant flies exhibited differences in their brain pathology when compared to wild type Drosophila and also the mutant flies were less able to perform basic physiological functions, resulting in significantly shorter life times. Changes in cellular metabolism were also detected in the mutant flies as well. Current efforts are under way to determine what specific sections of the brain in flies are affected when sialic acid is eliminated. In order to examine further the effects of knocking out the sialic acid step, our group established central nervous system neuronal cultures from mutant and wild type fly larvae. The incorporation of sialic acids into these neuronal cultures was then examined by incubating the cultures with chemically (azide)-modified sialic acid precursors that could then be detected using fluorescent microscopy. Only the wild type fly neurons were observed to generate sialic acid while the mutant flies were unable to produce any, verifying the deletion of the sialic acid synthase in the mutant fly cultures. Chemical labeling also revealed that sialic acid is attached to proteins located at the plasma membrane, consistent with sialic acid attachment to ion channels found principally at the plasma membrane. In the area of molecular modeling, new techniques have been developed to model and describe ion channels, which are an important system to study for channelopathy diseases and other biomedical applications. In addition, novel techniques have been developed to model glycosylation of ion channels. In the area of kinetic models, the project resulted in one of the first systems to model all the different components of specific ion channels in flies. The role of sialic acid on channel opening and closing kinetics has been incorporated as well. These model predictions have then been compared with ion channel function obtained using experimental electrophysiology measurements, which validated the ability of the models to predict the effects of sialic acid on ion channel opening and closing. These models will now be used to evaluate approaches for overcoming ion channel dysfunction due to sialic acid limitations that lead to channelopathies. From these modeling studies, new experimental methods will be designed that can be used to treat channelopathies. These treatments can be evaluated using the cell culture and animal models created as part of the current study. As a result, this study has provided a fundamental understanding of the role of sialic acid in ion channel function. Most importantly, modeling and experimental systems have been put into place to develop and test new biomedical treatments for the wide range of diseases ranging from ataxia to heart disease to metabolic disorders that are caused by sialic acid deficiencies in ion channels.
