Large conductance voltage and Ca2+ activated K+ (BK) channels control electrical excitability in many cell types. BK channels have the largest conductance (~250 pS) of all K+ selective channels. To explore whether a large entrance to the inner cavity of BK channels is required for their large conductance, I examined if changing the size of the entrance alters the single-channel current amplitude. Previous studies suggest that residues E321/E324 in BK channels are located at the entrance to the inner cavity. To test if positions 321/324 are accessible to intracellular ions, I compared single-channel outward current before and after attaching thiol reagents at E321C/E324C. Attachment of MBB and MTSET altered single-channel currents, indicating that positions 321/324 are accessible to the conduction pathway. Decreasing the size of the entrance to the inner cavity by substituting residues with larger side chains, such as tyrosine and tryptophan, at positions 321/324 decreased the conductance, whereas increasing the size of the entrance had little effect on conductance. Increasing [K+]i from 0.15 to 2.5 M negated differences in single-channel outward current associated with side chain volume. Substitutions had less effect on inward currents. Plots of conductance vs. substituted side chain volume could be approximated with a simple model for the conduction pathway described by two resistors in series, R1 and R2. R2 is a variable resistor, with the resistance proportional to the inverse of the volume of the entrance to the inner cavity not occupied by the side chains. R1 is a fixed resistor arising from the other parts of the conduction pathway including the selectivity filter. Fitting the experimental observations indicated that R1+R2 ~5.4 GΩ for glycine substitution, with an R1/R2 ratio of ~17, and an effective radius and length of the entrance to the inner cavity of ~9.0 and 5.4 Å, respectively. The volume of K+ and water were not taken into account. Taken together, the above observations suggest that a large entrance to the inner cavity is needed for the large conductance of BK channels, as my study shows that the entrance is large and that decreasing the entrance size decreases the currents.

Speech intelligibility represents how comprehensible a speech is. It is more important than speech quality in some applications. Single channel speech intelligibility enhancement is much more difficult than multi-channel intelligibility enhancement. It has recently been reported that training-based single channel speech intelligibility enhancement algorithms perform better than Signal to Noise Ratio (SNR) based algorithm. In this thesis, a training-based Deep Neural Network (DNN) is used to improve single channel speech intelligibility. To increase the performance of the DNN, the Multi-Resolution Cochlea Gram (MRCG) feature set is used as the input of the DNN. MATLAB objective test results show that the MRCG-DNN approach is more robust than a Gaussian Mixture Model (GMM) approach. The MRCG-DNN also works better than other DNN training algorithms. Various conditions such as different speakers, different noise conditions and reverberation were tested in the thesis.

Transient receptor potential vanilloid 2 channel (TRPV2) is a Ca2+-permeable ion channel that is highly expressed in leukocytes but is also present in skeletal and cardiac muscle and endocrine cells. The TRPV2 function is implicated in a number of physiological processes, including bacterial phagocytosis, pro-inflammatory cytokine production, cardiac hypertrophy, and cancer development. TRPV2 knockout mice exhibit a high incidence of perinatal mortality, arguing that the channel plays essential roles in physiology. Despite the importance of TRPV2 for normal homeostasis, the mechanisms that control TRPV2 gating in response to pharmacological agonists, heating, membrane stretch, bioactive lipids and reactive oxygen species (ROS) remain poorly understood. Here we demonstrate that TRPV2 is functionally expressed in microglia (i.e., ‘brain macrophages’) and the microglia-like BV-2 cell line, and demonstrate that the gating of an endogenous TRPV2-like conductance is positively modulated by the bacterial toxin lipopolysaccharide (LPS), which is known to cause pro-inflammatory (M1) activation and increase ROS production by NADPH oxidase. To determine how TRPV2 gating is modulated by ROS, we recorded single channel activity in inside-out patches excised from HEK-293 cells expressing GFP-rTRPV2. Unitary currents elicited by the TRPV2 agonist 2-aminophenyl borinate (2-APB) or cannabidiol (CBD) are linear in monovalent recording solutions and give rise to an estimated unitary conductance of ~100pS, which is similar to TRPV1 but significantly smaller than TRPV3. Intriguingly, we find that although TRPV2 is insensitive to ROS (in the form of exogenously applied H2O2) alone, apparent open probability is synergistically enhanced when H2O2 is applied together with CBD. We identify two intracellular Cys residues that are necessary for TRPV2 responses to H2O2 sensitivity and find that these residues are located close to one another, albeit in different subunits, in the TRPV2 structure, suggesting that ROS promote the formation of an inter-subunit disulfide bond that alters sensitivity to pharmacological agonists. We hypothesize that ROS-dependent modulation of TRPV2 activity may be an important contributor to pro-inflammatory activation of microglia underline central nervous system diseases and that TRPV2 antagonism could be a useful therapeutic strategy in the treatment of neuroinflammation.

With more states revealed, and more reliable rates inferred, mechanistic schemes for ion channels have increased in complexity over the history of single-channel studies. At the forefront of single-channel studies we are faced with a temporal barrier delimiting the briefest event which can be detected in single-channel data. Despite improvements in single-channel data analysis, the use of existing methods remains sub-optimal. As existing methods in single-channel data analysis are unquantified, optimal conditions for data analysis are unknown. Here we present a modular single-channel data simulator with two engines; a Hidden Markov Model (HMM) engine, and a sampling engine. The simulator is a tool which provides the necessary a priori information to be able to quantify and compare existing methods in order to optimize analytic conditions. We demonstrate the utility of our simulator by providing a preliminary comparison of two event detection methods in single-channel data analysis; Threshold Crossing and Segmental k-means with Hidden Markov Modelling (SKM-HMM).

One of the important measures for sleep quailty is sleep structure. Normal sleep consists of awake, rapid eye movement (REM) sleep and nonrapid eye movement (NREM) sleep states. NREM sleep can be further classified into stage 1, stage 2 and slow wave sleep (SWS). These stages can be analyzed quantitatively from various electrical signals such as the electroencephalogram (EEG), electro-oculogram (EOG), and electromyogram (EMG). The goal of this research is to develop a simple four-stage process to classify sleep into wake, REM, stage 1, stage 2 and SWS by using a single EEG channel. By applying the proposed approach to 48727 distinct epochs which are acquired from 62 persons, the experimental results show that the proposed method is achieves 76.98% of accuracy. The sensitivity and PPV for wake are 85.96% and 68.35%. Furthermore, the sensitivity and PPV for REM are 82.13% and 74.11%, respectively. The sensitivity and PPV for the stage 1 are 9.02% and 39.00%. The sensitivity and PPV for the stage 2 are 84.19% and 79.36%. The sensitivity and PPV for SWS are 81.53% and 85.40%.

The glycine receptor (GlyR) is a ligand-gated ion channel and member of the nicotinic acetylcholine receptor superfamily. Glycine and the partial agonist taurine are both believed to be the endogenous ligands of the receptor. Partial agonists have lower efficacies than full agonists, eliciting submaximal responses even at saturating concentrations. Recent evidence suggests that efficacy at these receptors is determined by conformational changes that occur early in the process of receptor activation. We previously identified a mutation of the aspartate-97 residue to arginine (D97R), which produces a spontaneously active mutant with behavior that mimics the effects of saturating glycine concentrations on wildtype (WT) GlyR. This D97 residue is hypothesized to form an electrostatic interaction with arginine-119 on an adjacent subunit to stabilize a closed channel closed state. We found that the disruption of this bond converts taurine into a full agonist and greatly increases the efficacies of other [beta]-amino acid partial agonists. Our findings suggest that the determination of efficacy in the GlyR involves the disruption of an inter-subunit electrostatic interaction soon after binding. We next investigated whether the taurine efficacy could be enhanced by ethanol, a well-studied positive allosteric modulator of receptor function. Whole-cell recordings of WT GlyRs demonstrated that alcohol could potentiate the effect of low concentrations of taurine, but did not increase the efficacy of a saturating concentration. Therefore we sought to understand the mechanism by which alcohol enhances the GlyR, because ethanol's actions at inhibitory receptors in the brain are thought to produce many of the physiological effects associated with its use. We examined the effects of 3 [mu]M glycine ± 50 or 200 mM ethanol on outside-out patches expressing WT [alpha]1 GlyR, to determine the effects of alcohol at the single-channel level. Alcohol enhanced GlyR function in a very specific manner. It had minimal effects on open and closed dwell times. Instead, ethanol potentiated GlyR function almost exclusively by increasing burst durations and increasing the number of channel openings per burst, without affecting the percentage of open time within bursts. Kinetic modeling suggests that ethanol increases burst durations by decreasing the rate of glycine unbinding. / text

Calcium ions (Ca2+) are admitted into presynaptic nerve terminals through voltage gated calcium channels and diffuse to bind and activate the secretory vesicle discharge mechanism. Current research favors a highly ‘modal’ organization where the release sites are activated by one or a few closely apposed channels (Stanley, 1997). To fully understand the nanophysiology of transmitter release site activation, it is necessary to determine the rate of Ca2+ flux through individual channels at normal physiological external concentrations. OBJECTIVE: To explore the relationship between CaV2.2 channel conductance and external Ca2+ across the physiological range. CONCLUSION: The conductance of the CaV2.2 channel was determined across the range of 1-100 mM [Ca2+]EXT . With 2 mM [Ca2+]EXT, the conductance was determined to be 2.76 ± 0.24 pS.

Calcium ions (Ca2+) are admitted into presynaptic nerve terminals through voltage gated calcium channels and diffuse to bind and activate the secretory vesicle discharge mechanism. Current research favors a highly ‘modal’ organization where the release sites are activated by one or a few closely apposed channels (Stanley, 1997). To fully understand the nanophysiology of transmitter release site activation, it is necessary to determine the rate of Ca2+ flux through individual channels at normal physiological external concentrations. OBJECTIVE: To explore the relationship between CaV2.2 channel conductance and external Ca2+ across the physiological range. CONCLUSION: The conductance of the CaV2.2 channel was determined across the range of 1-100 mM [Ca2+]EXT . With 2 mM [Ca2+]EXT, the conductance was determined to be 2.76 ± 0.24 pS.

Glycine receptors (GlyRs) are ligand-gated ion channels (LGICs) that, along with other members of the cys-loop superfamily of receptors, mediate a considerable portion of fast neurotransmission in the central nervous system (CNS). GlyRs are pentameric channels, organized quasi-symmetrically around an ion-conducting pore. Opening of the integral ion pore depends on ligand binding and transduction of this binding signal to the channel gate. Research presented in this dissertation describes a number of critical electrostatic interactions that play a role in conserving the closed-state stability of the receptor in the absence of ligand, ensuring that receptor activation occurs only upon neurotransmitter binding. These amino acids, aspartic acid at position 97 (D97), lysine 116 (K116), arginine 119 (R119) and arginine R131 (R131) are charged residues that interact with one another through electrostatic attraction. When D97 is replaced with any other amino acid this destabilizes the closed state of the channel and causes spontaneous GlyR channel opening. I show that restoration of this electrostatic interaction in GlyR bearing double mutations in which the charges are swapped (D97R/R119E and D97R/R131D) markedly decreases this spontaneous current. In addition, I investigate how these residues that interact at the interfaces between receptor subunits affect the efficacies of GlyR partial agonists. My work shows that the partial agonist taurine is converted into a full agonist at both D97R and R131D receptors. Furthermore, I analyzed the structure of the more extracellular part of the transmembrane (TM) 2 segment that lines the ion channel pore, showing that it is unlikely that this fragment (stretching from T13’ to S18’) is constrained in a true alpha helical conformation. From this work, using disulfide trapping and whole cell electrophysiology, I conclude that a significant level of flexibility characterizes this part of the TM2 domain. This segment includes residue S267, previously shown to be significant for alcohol and anesthetic actions, as well as residue Q266 that, when mutated, produces a hyperekplexia-like phenotype. The range of movement of residues in this region may therefore play an important role not only in channel gating but also in how modulators of GlyR function exert their actions. / text

Ultrathin fiberscopes typically have an imaging channel and an illumination channeland are available in diameters ranging from 0.5 mm to 2.5 mm. The minimum diam-eter can be reduced by combining the illumination and imaging paths into a singlefiberoptic channel. Constructing a single channel fiberscope requires a technique ofilluminating the tissue, while minimizing the Fresnel reflections and scatter withinthe common illumination and detection channel.A single channel fiberscope should image diffusely reflected light from tissue illu-minated with light filtered for the visible wavelength range (450 - 650 nm). Simplycombining the illumination and collection paths via a beamsplitter results in a lowobject to background signal ratio. The low contrast image is due to a low collectionefficiency of light from the ob ject as well as a high background signal from the Fresnelreflection at the proximal surface of the fiber bundle, where the illumination enters thefiber bundle. The focus of the dissertation is the investigation of methods to reducethe background signal from the proximal surface of the fiber bundle. Three systemswere tested. The first system uses a coherent fiber bundle with an AR-coating on theproximal face. The second system incorporates crossed polarizers into the light path.In addition, a technique was developed, whereby a portion of the image numericalaperture is devoted to illumination and a portion to image signal detection. Thistechnique is called numerical aperture sharing (NA sharing).This dissertation presents the design, construction, testing, and comparison ofthe three single channel fiberscopes. In addition, preliminary results of a study aimedat the usefulness of broadband diffuse reflectance imaging for the identification andtracking of disease progression in mouse esophagus are presented.