A Senior Honors Thesis Presented in partial fulfillment of the requirements for graduation with distinction in Molecular Genetics in the undergraduate colleges of the Ohio State UniversityStroke is the third leading cause of death in the United States and survivors of stroke suffer permanent neural damage. During such conditions, extracellular pH falls and becomes acidic and consequently activates the Acid Sensing Ion Channels (ASICs). ASICs are expressed in neurons throughout the mammalian central and peripheral nervous systems. These channels are known to impact synaptic plasticity of the brain and affect learning and memory. Activation of ASIC is the major cause of neuronal death. Thus, ASICs represent novel targets for design of drugs to treat disorders of learning and memory as well as for the prevention and restriction of stroke-induced brain damage. However the specific protein domains responsible for desensitization, the closing of a channel in the presence of an agonist, are yet to be determined.
The first project was done to identify the protein regions responsible for determining desensitization rate. In vitro over-expression studies of various individual ASIC subunits show that each subunit has a unique desensitization rate. Therefore, chimeras of an ASIC protein with a fast desensitization rate and an ASIC protein with a slow desensitization rate were made. Since the ASIC 1a protein has a very fast desensitization rate when compared to the ASIC 2a protein, these ASIC subunits were selected for the study. Chimeras, or fusions of different proteins, were made with a portion of ASIC1a replacing a similar conserved locus of an ASIC2a subunit. These chimeras were injected into Xenopus eggs and the desensitization rate was measured using the two electrode clamp technique. We hypothesized that if the domain responsible for a fast desensitization rate were present in the chimera, the desensitization rate of the chimera will be much faster than ASIC2a. As a result of this project, we narrowed down two domains which are essential for a fast desensitization rate.
The second project was done to isolate specific amino acids that may dictate the fast desensitization rate. So, the general domains essential for this, determined in the previous project, were selected for site directed mutagenesis. One set of data shows the desensitization rates resulting from mutations of codons for charged amino acids such as glutamate and aspartate to code for uncharged amnio acids such as glycine and alanine. The second set of data shows the desensitization rate resulting from ASIC1a mutants that have codons changed to their corresponding codons from ASIC2a.
The third project will focus on the pH dependence of human ASIC 1a desensitization rates. There have been no findings that report a pH dependence on the rate of densensitization. Using the data from wild type human ASIC1a and 2a, the pH and the desensitization were compared. We saw a decreased desensitization rate correlated with increased pH in ASIC 1a. However, no such definite pattern was determined for the data from wildtype ASIC 2a.
The discovery of domains responsible ASIC desensitization can potentially lead to development of drugs that can target them to prevent ASICs from opening and consequently curtail neuronal damage in patients who suffer stroke.Department of Neuroscience, Dr. Candice Askwit
