Date of Award

5-1-2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Eric B. Gonzales

Second Advisor

Glenn H. Dillon

Third Advisor

Nathalie Sumien

Abstract

Acid-sensing ion channels (ASICs) are trimeric, sodium-selective channels activated by extracellular protons. Although ASICs are intriguing molecular targets for pharmacological agents, there remains a lack of selective compounds that differentiate ASIC subtypes. The peripherally located ASIC3 activates with the simple removal of calcium. Additionally, nonproton ligands, like 2-guanidine-4-methylquinazoline (GMQ), have been identified to selectively activate ASIC3 via the nonproton ligand sensor domain (NPLSD). A pair of glutamates in rat ASIC3 (E79 and E423) responsible for GMQ activation is present in ASIC1, despite having no direct modulation effect on the channel. We proposed that nonproton ligand activation of ASIC1 may be state dependent, and relies on expansion of the NPLSD in order for GMQ to reach the binding site and exert its effects. We utilized two features of ASICs in order to test our hypothesis with whole cell and outside out patch-clamp electrophysiology. First, we induced a persistent current in chicken ASIC1 (cASIC1) via a natural venom toxin, Psalmotoxin-1 (PcTx1). We determined that GMQ acts as a molecular wedge by prying apart the transmembrane domains of the cASIC1-PcTx1 protein complex and potentiating ASIC-current. This led us to better understand that the NPLSD is intact in cASIC1 and is sensitive to GMQ additions, albeit in a state-dependent manner. We then theorized that direct activation of rASIC3 by GMQ is possible due the channel’s interaction with extracellular calcium, and were interested in introducing feature into the cASIC1 channel. We generated a chimeric ASIC combining the extracellular, transmembrane, and intracellular domains of rASIC3 and cASIC1 in order to individually isolate the calcium and nonproton ligand effects on channel activation. This chimera, termed cASIC1 (rASIC3-TM/ITC), is comprised of the extracellular domain of cASIC1 and the transmembrane/intracellular domains of rASIC3, and can be activated by GMQ in the absence of calcium similarly to wild-type rASIC3. Thus, GMQ activation was introduced in cASIC1 by replacing the transmembrane domains with those of ASIC3 suggesting that the ASIC3 TM domains dictate NPLSD influence on channel activity. Taken together, we identified that channel state influences nonproton ligand interaction with ASICs, and the transmembrane domains are critical for this interaction.

Comments

Smith, Rachel N., Ionotropic purinoceptors and acid-sensing ion channels in epithelial cancers. Master of Science (Biomedical Sciences), August 2011, 206 pp., 41 figures, 12 tables, references.