Browsing by Subject "Ion channels"
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Item Approaches and evaluation of architectures for chemical and biological sensing based on organic thin-film field-effect transistors and immobilized ion channels integrated with silicon solid-state devices(2007) Fine, Daniel Hayes, 1978-; Dodabalapur, Ananth, 1963-There is significant need to improve the sensitivity and selectivity for detecting chemical and biological agents. This need exists in a myriad of human endeavors, from the monitoring of production of consumer products to the detection of infectious agents and cancers. Although many well established methodologies for chemical and biological sensing exist, such as mass spectrometry, gas or liquid phase chromatography, enzymelinked immunosorbent (ELISA) assays, etc., it is the goal of the work described herein to outline aspects of two specific platforms which can add two very important features, low cost and portability. The platforms discussed in this dissertation are organic semiconductor field-effect transistors (OFETS), in various architectural forms and chemical modifications, and ion channels immobilized in tethered lipid bilayers integrated with solid state devices. They take advantage of several factors to make these added features possible, low cost manufacturing techniques for producing silicon and organic circuits, low physical size requirements for the sensing elements, the capability to run such circuits on low power, and the ability of these systems to directly transduce a sensing event into an electrical signal, thus making it easier to process, interpret and record a signal. In the most basic OFET functionality, many types of organic semiconductors can be used to produce transistors, each with a slightly different range of sensitivities. When used in concert, they can produce a reversible chemical "fingerprint". These OFETS can also be integrated with silicon transistors - in a hybrid device architecture - to enhance their sensitivity while maintaining their reversibility. The organic semiconductors themselves can be chemically altered with the use of small molecule receptors designed for specific chemicals or chemical functional groups to greatly enhance the interaction of these molecules with the transistor. This increases both sensitivity and selectivity for discrete devices. Specially designed nanoscale OFET configurations with individually addressable gates can enhance the sensitivity of OFETS as well. Finally, ion channels can be selected for immobilization in tethered lipid bilayer sensors which are already inherently sensitive to the analyte of choice or can be genetically modified to include receptors for many kinds of chemical or biological agents.Item Bayesian approaches for modeling protein biophysics(2014-08) Hines, Keegan; Aldrich, R. W. (Richard W.)Proteins are the fundamental unit of computation and signal processing in biological systems. A quantitative understanding of protein biophysics is of paramount importance, since even slight malfunction of proteins can lead to diverse and severe disease states. However, developing accurate and useful mechanistic models of protein function can be strikingly elusive. I demonstrate that the adoption of Bayesian statistical methods can greatly aid in modeling protein systems. I first discuss the pitfall of parameter non-identifiability and how a Bayesian approach to modeling can yield reliable and meaningful models of molecular systems. I then delve into a particular case of non-identifiability within the context of an emerging experimental technique called single molecule photobleaching. I show that the interpretation of this data is non-trivial and provide a rigorous inference model for the analysis of this pervasive experimental tool. Finally, I introduce the use of nonparametric Bayesian inference for the analysis of single molecule time series. These methods aim to circumvent problems of model selection and parameter identifiability and are demonstrated with diverse applications in single molecule biophysics. The adoption of sophisticated inference methods will lead to a more detailed understanding of biophysical systems.Item Circadian and Social Cues Regulate Ion Channel Trafficking(Public Library of Science, 2009-09-29) Markham, Michael R.; McAnelly, M. Lynne; Stoddard, Philip K.; Zakon, HaroldElectric fish generate and sense electric fields for navigation and communication. These signals can be energetically costly to produce and can attract electroreceptive predators. To minimize costs, some nocturnally active electric fish rapidly boost the power of their signals only at times of high social activity, either as night approaches or in response to social encounters. Here we show that the gymnotiform electric fish Sternopygus macrurus rapidly boosts signal amplitude by 40% at night and during social encounters. S. macrurus increases signal magnitude through the rapid and selective trafficking of voltage-gated sodium channels into the excitable membranes of its electrogenic cells, a process under the control of pituitary peptide hormones and intracellular second-messenger pathways. S. macrurus thus maintains a circadian rhythm in signal amplitude and adapts within minutes to environmental events by increasing signal amplitude through the rapid trafficking of ion channels, a process that directly modifies an ongoing behavior in real time.Item The structure of the TM2-3 linker in the [alpha]1 GlyR and its role in gating and modulation(2008-12) Dupré, Michelle Louise, 1979-; Mihic, S. JohnThe 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.Item The study of multiple ion channel gating models and mechanisms(2023-08) Yeh, Frank R.; Senning, Eric Nicolas, 1978- ; Aldrich, R. W. (Richard W.); Zakon, Harold; Pierce, Jon; Horrigan, Frank; Goldschen-Ohm, MarcelRecent advancements in protein structural determination, structural predictions, structural modelling, and bioinformatics have significantly improved our understanding of ion channel gating models and mechanisms. Despite these improvements in technology, the transient and sporadic nature of multiple open and closed states in ion channels remains a challenge to study. These states are observable in electrophysiology studies but might be difficult to capture through structural determination techniques such as cryo-electron microscopy (cryo-EM) or may not appear as stable structures in prediction and modeling approaches. Bioinformatics are instrumental in generating hypotheses for further investigation. By examining functional and sequence differences across species or isoforms, these methods yield profound insights into ion channel gating mechanisms. Nevertheless, these hypotheses must be rigorously tested in functional studies. Hence, although ion channel research has seen huge advancements from these recent technological improvements, there is still no technique that can substitute electrophysiology experiments in the degree of functional information provided. To gain a strong understanding of ion channel gating models and mechanisms and the integration of knowledge provided by recent improved technological advances of, I chose to study thermoTRP channels, six-transmembrane voltage-gated or ligand-gated ion channels, as well as BK channels, the voltage- and calcium-gated potassium channels. Particularly, I have developed a theory for temperature-dependent gating in thermoTRP channels, generated hypotheses for voltage-gating mechanisms in six-transmembrane voltage-gated channels from sequence-based bioinformatics integrated with structural knowledge, and investigated BK’s voltage-gating mechanism by performing single-channel electrophysiology studies of BK based on hypotheses generated from the bioinformatics approach.Item Voltage gated ion channel control of CA1 pyramidal neuron function in wild type and fmr1 KO mice(2021-05-07) Ordemann, Gregory James; Brager, Darrin H.; Golding, Nace L.; Johnston, Daniel; Colgin, Laura; Pierce, JonathanChanges in the complement or function of ion channels can drastically affect individual neurons and their constituent circuits. Neuron function is a highly tunable system. The difference between function and dysfunction can exist on a razor’s edge. Studying neurons in disease can provide insight into the operation of brain structures. This dissertation focuses on ion channel control over individual neuron and neuronal circuit function of CA1 pyramidal neurons in wild type mice and a model of Fragile X syndrome. Using somatic recordings, we investigated differences in CA1 pyramidal neurons across the dorsoventral axis of mouse hippocampus. Ventral neurons show depolarized resting V [subscript m], have greater R [subscript N], and have reduced dendritic branching compared with dorsal neurons. Action potential firing was not different across the dorsoventral axis of mouse hippocampus. However, ventral neurons have a more depolarized action potential threshold compared to dorsal neurons. Action potential threshold in ventral neurons was more sensitive to block of KV1 channels compared to dorsal neurons. Outside-out voltage clamp recordings showed larger slowly inactivating K⁺ currents in ventral neurons. Despite differences in subthreshold properties between dorsal and ventral CA1 neurons, action potential output is normalized by the differential functional expression of D-type K⁺ channels. In investigating the effects of fmr1 KO on CA1 pyramidal neurons no difference was identified in intrinsic function across the dorsoventral axis of mouse hippocampus between wild type and fmr1 KO neurons. We further investigated differences in wild type and fmr1 KO CA1 neurons using somatic and dendritic recordings to investigate synaptic transmission at distal inputs from entorhinal cortex. We found that TA-LTP was impaired in male fmr1 KO mice. Synaptically evoked dendritic Ca²⁺ signals were smaller in fmr1 KO neurons. Threshold for Na⁺ dependent dspikes was depolarized in fmr1 KO mice. Dspike threshold and TA-LTP were restored by block of A-type K⁺ channels. TA-LTP impairment, coupled with previously described enhanced Schaffer collateral LTP, may contribute to spatial memory alterations in FXS. Furthermore, as both of these LTP phenotypes are attributed to changes in A-type K⁺ channels in FXS, our findings provide a potential therapeutic target to treat cognitive impairments in FXS.Item Voltage gated ion channels shape subthreshold synaptic integration in principal neurons of the medial superior olive(2008-12) Mathews, Paul James, 1978-; Golding, Nace L.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.