Alpha-Bungarotoxin as a tool for exploring naturally insensitive ion channels Liu, Jing creator 2009 xx, 195 p. born digital Thesis (Ph.D.) -- Brown University (2010) Hawrot, Edward director Zimmerman, Anita reader Kauer, Julie reader Bowen, Wayne reader Jacob, Michele reader Brown University. Division of Biology and Medicine. Molecular Pharmacology, Physiology, and Biotechnology sponsor theses In this thesis,alpha-bungarotoxin (Bgtx) was validated as a tool for exploring the function and regulation of three otherwise Bgtx-insensitive ion channels: the first is a transgenic mouse model featuring a genetic modified neuronal nicotinic alpha3 subunit; the second is voltage-gated potassium Kv4.2 channel; the third is a subtype of gamma-aminobutyric acid type A receptors (GABAARs) containing alpha1beta2 subunits. The findings of this thesis are summarized below. a) We devised a novel genetic knock-in mouse model with a modified neuronal nicotinic alpha3 subunit. The alpha3 subunit bears 5 amino acid substitutions (alpha3/alpha1[5]), which confer a functional sensitivity to Bgtx. Using whole-cell patch clamp technique, the initial electrophysiological characterizations show that alpha3/alpha1[5]-containing receptors were functionally expressed and readily identifiable in cultured dissociated mouse superior cervical ganglion (SCG) neurons. Moreover, its pharmacological and biophysical properties are also largely unaltered at the macroscopic level. b) To better understand the trafficking mechanisms that regulate voltage-gated Kv4.2 channel and to facilitate its fluorescence imaging of the channels in neurons, a short Bgtx binding sequence HAP, was introduced into the extracellular loop of the channel. The results show that Bgtx binds specifically to HAP-tagged Kv4.2 expressed in mammalian cells and primary neuron cultures. The data also show that the introduced Bgtx binding sequence is functionally well-tolerated. c) Using two-electrode voltage clamp measurements, we show that Bgtx also binds to and blocks a subtype of un-modified GABAARs expressed in oocytes, containing solely alpha1 and beta2 subunits. These results may also introduce a previously unrecognized tool for analysis of GABAARs. alpha-bungarotoxin nicotinic receptor voltage gated potassium channel GABA type A receptor Ion channels RPB 20091218 engEnglish10.7301/Z0FF3QMGIn CopyrightCollection is open for research.dissertations