Suspended lipid bilayers, or black lipid membranes (BLMs), have been used to study the electrophysiological properties of ion channels (ICs); however, BLMs assembled from natural, non-polymerizable lipids are inherently unstable due to the non-covalent associations on which they are based. Lifetimes of several hours are commonly observed in BLMs until rupture due to mechanical, thermal, or chemical insults. One potential improvement is the use of polymerizable phospholipids (poly(lipids)). BLMs prepared using dienoyl functionalized poly(lipids) and binary mixtures of fluid, non-polymerizable lipids with poly(lipids) were investigated for IC recordings.poly(BLMs) exhibited enhanced lifetimes from several hours to upwards of 4 weeks while maintaining IC functionality for one week. Activity of ICs that require membrane fluidity was retained using binary phospholipid mixtures of fluid and polymeric phospholipids. IC activity was retained by inducing domain formation, wherein ICs incorporated into the fluid domains. The binary membranes exhibited marked enhancement in stability resulting from fractional poly(lipids) polymerization. Additionally, ICs can be reconstituted into the fluid domains following photopolymerization and subsequent domain formation, a key requirement when UV-sensitive ICs are utilized. Here, the electrical properties, stability, and incorporation of pore-forming ICs, including hemolysin, alamethicin, and gramicidin, into poly(lipid) membranes are reported. Potential applications developing ligand-gated IC based sensors for high throughput screening are being investigated.In parallel to the characterization of poly(lipids) for potential long-term IC membranes, a model ligand-gated IC was expressed, characterized, and reconstituted into non-polymerizable lipids. Mutant K_ATP channels were expressed in mammalian and yeast systems. The orientations of mutant K_ATP channels were studied using electrophysiological and immunohistochemical techniques. Large quantities were expressed and purified from <italic>Pichia pastoris</italic> and functionally reconstituted into BLMs. ATP and long-chaing coenzyme A ester sensitivity was maintained in reconstituted in BLMs. K_ATP channels will serve as a model system for testing the effect of poly(lipid) BLMs on IC function. Future utilization of poly(lipid) BLMs in combination with ligand-gated ICs offer major advancements to potential increased throughput for IC screening.

The voltage-gated potassium channel Kv1.2 is a critical modulator of neuronal physiology, including dendritic excitability, action potential propagation, and neurotransmitter release. However, mechanisms by which Kv1.2 may be regulated in the brain are poorly understood. In heterologous expression systems Kv1.2 is regulated by endocytosis of the channel from the plasma membrane, and this trafficking can be modulated by adenylate cyclase (AC). The goal of this dissertation was to determine whether AC modulated endocytic trafficking of endogenous Kv1.2 occurred in the mammalian nervous system. Within the brain, Kv1.2 is expressed at its highest levels in the cerebellar cortex. Specifically, Kv1.2 is expressed in dendrites of Purkinje cells (PC), the sole efferent neurons of the cerebellar cortex; Kv1.2 is also expressed in axon terminals of Basket cells (BC), which make inhibitory synapses to Purkinje cells. The loss of functional Kv1.2 in PC dendrites or BC axon terminals causes profound changes in the neurophysiology of Purkinje cells, and aberrant loss of Kv1.2 produces cerebellar ataxia. Therefore, the cerebellum offers a brain structure where Kv1.2 is abundant and has known and important roles in synaptic physiology. A candidate regulator of Kv1.2 trafficking in cerebellar synapses is the secretin peptide receptor: the receptor is also located in both PC dendrites and BC axon terminals, and ligand binding to the secretin receptor stimulates AC. Although secretin affects cerebellar neurophysiology and cerebellar dependent behavior, the mechanisms are not well resolved. By cell-surface protein biotinylation and subsequent immunoblot quantitation of secretin treated rat cerebellar slice lysates, secretin was found to decrease cell-surface Kv1.2. This effect could be mimicked by stimulating AC with forskolin, and could be occluded by inhibition of the secretin receptor, AC, or protein kinase A. The secretin receptor stimulated loss of surface Kv1.2 was not accompanied by decreased total Kv1.2 protein levels, but did involve enhanced channel endocytosis. Microscopy studies using two novel independent techniques provided evidence that both BC axon terminals and PC dendrites are sites of AC-stimulated Kv1.2 endocytosis. The physiological significance of secretin mediated suppression of Kv1.2 was supported by collaborative studies which found infusions into the cerebellar cortex of either a toxin that inhibits Kv1.2, or of secretin, enhanced eyeblink conditioning, a form of cerebellar dependent learning, in rats. These studies provided the first evidence that Kv1.2 is regulated by endocytic trafficking in the brain. However, to address the role of that trafficking in synaptic physiology requires knowledge about the determinants of Kv1.2’s endocytic potential, and non-destructive assays to measure Kv1.2 endocytosis in neural circuits. This dissertation therefore concludes with preliminary studies that explore an ancient motif regulating Kv1.2 trafficking, and that discuss a novel dual fluorescent fusion protein reporter of Kv1.2’s subcellular localization.

The green algae Chlamydomonas reinhardtii senses light through two photosensory proteins, channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2). The initial discovery of these two photoreceptors introduced a new class of light-gated ion channels. ChR2 is an inwardly-rectified ion channel that is selective for cations of multiple valencies. Similar to microbial-rhodopsin ion pumps, ChR2 has a seven transmembrane domain motif that binds the chromophore all-trans retinal through a protonated Schiff base linkage. Physiologically, ChR2 functions to depolarize the membrane which initiates a signaling cascade triggering phototactic response. This fundamental property has been pivotal in pioneering the field of optogenetics, where excitable cells can be manipulated by light. ChR2 reliably causes neuronal spiking with high spatial and temporal control. Moreover, the recent discovery of new chloride-conducting channelrhodopsins (ChloCs) has further expanded the optogenetic toolbox. Although structurally similar to microbial-rhodopsin ion pumps, ChR2 undergoes more complex conformational rearrangements that lead to ion conductance. Currently, the molecular basis for ChR2 gating remains unresolved. Revealing the specific structural interactions that modulate ChR2 function have important implications in understanding the intricacies of ion transport and molecular differences between ion pumps, channels, and transporters. Here we describe a combined computational and experimental approach to elucidate the mechanism of ion conductance, channel gating, and structure-function relationship of ChR2. Our results have contributed to expanding our understanding of the fundamental properties of ion channels.

Principal neurons of the medial superior olive (MSO) encode low-frequency sound localization cues by comparing the relative arrival time of sound to the two ears. In mammals, MSO neurons display biophysical specializations, such as voltage-gated sodium (Na[subscript v]) and potassium (K[subscript v]) channels that enable them to detect these cues with microsecond precision. In this dissertation electrophysiological techniques were used to examine the specific channel properties and functional role these channels play in MSO neurons following hearing onset. In addition, computational models that incorporated these physiological data were used to further study how the specific properties of these channels facilitate MSO function. Experiments in this dissertation showed that Na[subscript v] channels are heavily expressed in the persisomatic region of MSO neurons, but unlike those expressed in other neurons they minimally contribute to action potential generation. This is likely due to the low percentage of channels available for activation at the resting membrane potential. Current clamp recordings determined that Na[subscript v] channels counterbalance K[subscript v] channels voltage rectification by boosting near action potential threshold excitatory post-synaptic potentials (EPSPs). Further, computational modeling revealed that synaptic inputs are larger at the soma with Na[subscript v] channels restricted to the soma than when they are evenly distributed throughout the soma and dendrites. During the first few weeks after hearing onset current clamp experiments showed EPSP duration decreased while the temporal resolution for detecting the arrival time of synaptic inputs increased. These changes in EPSP duration are due in part to both the development of faster membrane response properties and increases in the expression of low voltage-activated K[subscript v] channels (K[subscript LVA]). Further investigation determined these channels display a somatically enriched distribution and act to counterbalance the distortions that result from dendritic cable filtering. This is accomplished by K[subscript LVA] actively decreasing the duration of EPSPs in a voltage dependent manner. Computational modeling confirmed these results as well as illustrating their effects on the integration of mono- versus bilateral excitation. Together these findings indicate that the expression of specialized Na[subscript v] and K[subscript v] channels facilitate the neuron’s computational task, detecting and comparing the relative timing of synaptic inputs used in low frequency sound localization. / text

The glycine receptor (GlyR) is the major inhibitory ligand-gated ion channel in the brainstem and spinal cord. It is a member of the Cys-loop superfamily of ligand-gated ion channels that includes serotonin-3, GABA[subscript A] and nicotinic acetylcholine (nAChR) receptors. Individual subunits are comprised of a large extracellular N-terminal agonist binding domain, four transmembrane (TM) segments and a large cytoplasmic loop between TM3 and TM4, containing phosphorylation sites (Brejc et al. 2001, Unwin, 2005). These receptors are pentameric in structure, with the TM2 region of each subunit contributing to the formation of a central ion pore (Lynch 2004). While the TM2-3 linker region has been hypothesized to be important for signal transduction thoughout the Cys-loop family, the precise structure and function of this region is unclear. We hypothesized that the TM2-3 linker region is a point of connection between subunits. We used disulfide bond trapping to show that the TM2-3 is able to interact with adjacent subunits and plays a critical role in signal transduction. In addition, we provide experimental evidence that the structure of the TM2-3 linker region in the [alpha]1 GlyR is a [beta]-sheet. We next sought to determine the role of the TM2-3 linker region in allosteric modulation. Using two-electrode voltage clamp electrophysiology we found that the TM2-3 linker can determine the direction of modulation without affecting modulator binding. Finally, we wanted to determine if a single alcohol and anesthetic binding site could be occupied to prevent EtOH molecules from binding. Using a combination of thiol reagents and disulfide bond trapping we show that a residue previously identified as important for the binding of alcohols and anesthetics interacts within the pore. We were unable to increase the volume at residue-267 such that EtOH was unable to bind, suggesting that EtOH may have more than one binding pocket. Together, these findings suggest that the TM2-3 linker plays a critical role in signal transduction and receptor modulation providing a foundation for future work on this region in the GlyR. / text

published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

abstract / Medicine / Doctoral / Doctor of Philosophy

No description available.

No description available.

Three distinct K$ sp+$ currents were identified in corn (Zea mays) protoplasts using the whole-cell patch-clamp technique. Inward-rectifying K$ sp+$ currents were evoked at membrane potentials more negative than $-$100 mV. The activation range was sensitive to external K$ sp+$ and shifted in the positive direction as the K$ sp+$ concentration was elevated. The second K$ sp+$ current was voltage-independent and contributed to the resting membrane conductance of the protoplast. Finally, a voltage- and Ca$ sp{2+}$-dependent K$ sp+$ current was observed at potentials positive to $-$60 mV. This current was inhibited by reagents which antagonize plasmalemma Ca$ sp{2+}$ influx (e.g. nitrendipine, verapamil). In contrast, currents were enhanced by increasing the cytosolic free Ca$ sp{2+}$ concentration from 40 to 400 nM. The Ca$ sp{2+}$-dependent K$ sp+$ current was inhibited by tetraethylammonium ions, Cs$ sp+$, Ba$ sp{2+}$, and charybdotoxin which suggested that the channel protein has structural similarities to the high conductance Ca$ sp{2+}$-dependent K$ sp+$ channel observed in animal systems.