Browsing by Subject "ion channels"
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Item DIETARY SUPPLEMENTS INFLUENCE ACTIVITY OF ACID-SENSING ION CHANNELS(2014-03) Agharkar, Amruta S.; Gonzales, Eric B.Dietary supplements or nutraceutical industry contributes billions of dollars to Unites States economy every year. The dietary supplement we intend to study is one of the most commonly used nutraceutical available in the market over the counter. It is used primarily by athletes and body builders to build lean body mass. Studies have shown that DS is effective in neuroprotection after stroke and also helps in improving muscle strength in patients suffering from muscle weakness or osteoarthritis. We are studying the effect of DS on acid-sensing ion channels (ASICs) which are the major contributors to neuronal damage after ischemia and pain. Determining the activity of DS on ASICs will give the new preventive measure for stroke and pain. Purpose (a): Dietary supplements and nutraceuticals have been the focus of research to identify novel therapeutics for a variety of pathologies, including the prevention of long-term consequences of stroke and reducing pain. Ion channels offer a growing group of molecular targets for treatment, which include the acid-sensing ion channels (ASICs). Acid-sensing ion channels (ASICs) are sodium channels that are sensitive to changes in extracellular pH, specifically those changes following injury and ischemia. These channels are expressed most prominently in peripheral and central nervous system. Their role in physiology is yet to be fully understood, but these channels have been implicated in pain sensation and centrally in the neurodegeneration following ischemic stroke. We identified an over-the-counter dietary supplement (DS) that shares similarity to guanidine compounds that selectively modulate acid-sensing ion channels. Thus, we hypothesize that this dietary supplement inhibits channel activity in acid-sensing ion channels. Methods (b): We will utilize whole cell patch-clamp electrophysiology technique to determine the intrinsic activity of DS on ASICs. The current elicited in absence and presence of DS at various pH will be normalized to maximum peak current obtained with control. Results (c): Our preliminary data show that DS decreased the ASIC1a pH sensitivity by shifting the observed proton activation profile to the right. Furthermore, we observed a change in the Hill coefficient of the DS influenced ASIC1a steady-state desensitization profile. Conclusions (d): Based on our preliminary data, we can conclude that DS influences ASIC current amplitude and steady state desensitization profile. Future experiments will focus on determining the influence of DS on other acid-sensing ion channel subtypes and identifying the DS binding site with the protein structure.Item Psalmotoxin-1 and nonproton ligand interactions with acid-sensing ion channels(2015-05-01) Smith, Rachel N.; Gonzales, Eric B.; Dillon, Glenn H.; Sumien, NathalieAcid-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.