Thesis (Ph.D.)--Indiana University, 2010. / Title from screen (viewed on April 1, 2010). Department of Pharmacology and Toxicology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Theodore R. Cummins, Grant D. Nicol, Gerry S. Oxford, Andy Hudmon, John H. Schild. Includes vitae. Includes bibliographical references (leaves 232-266).

Rett Syndrome (RTT) is a developmental disorder that affects numerous neuronal systems that underlie problems with breathing, movement, cognition and sleep. RTT is caused by mutations in the methyl-CpG-binding protein 2 (Mecp2) gene. MeCP2 is a ubiquitous protein that is found in all mature neurons and binds to methylated DNA to repress transcription; thus regulating protein expression levels in neurons. The mutations in Mecp2 affect a large number of proteins that are crucial for regulating neuronal activity. Despite the abnormal expression of many of these proteins, mice with a total loss of MeCP2 can live to adulthood and some people with RTT can live to a very late age as well. It is possible that mutations in the Mecp2 gene not only cause widespread defects, but also elicit neuroadaptive processes that may limit the impact of the MeCP2 dysfunction. To test this hypothesis we performed these studies in which we focused on how synaptic and membrane currents were altered to maintain normal neuronal activity in Mecp2-null mice. We show two examples from different neurons where neuroadaptations of ion channel expression allowed the neuron to remain viable. First, the properties of the nicotinic acetylcholine receptor (nAChR) current were altered in LC neurons in Mecp2-null mice. This was caused by changes in the nicotinic receptor subunit expression. Despite the changes in the nAChR current, the cholinergic modulation of LC neuron activity in WT and Mecp2-null mice were similar. Secondly, we show that the fast Na+ voltage-gated and the hyperpolarization-activated currents were altered in mesencephalic trigeminal V (Me5) propriosensory neurons. The changes in the hyperpolarization-activated current caused a smaller sag and post-inhibitory rebound. Opposite to what we expected, these cells were hyperexcitable. The hyperexcitability was due to changes in the fast Na+ voltage-gated current causing a decreased action potential threshold. Alterations in the ionic currents in Me5 neurons seem to be due to changes in subunit expression patterns. These results indicate that despite the complications caused by defects in the Mecp2 gene, neurons respond by rearranging receptor / ion channel expression. This reorganization allows neurons to remain viable despite the MeCP2 deficiency.

KCNE β-subunits modulate KCNQ1 (Q1) voltage-gate K+channels providing the current diversity required for Q1 channels to function in a wide variety of cell types and tissues. In the present thesis, the stoichiometry of KCNE1 (E1) β-subunits in functioning Q1 channels is investigated, along with the formation of heteromeric channel complexes, complexes containing 2 different KCNE β-subunits. The chemical approaches used to answer these questions were then expanded to generate a novel labeling reagent. To determine the stoichiometry of the Q1/E1 complex, I devised an iterative subunit counting approach that relies on a chemically releasable K+channel blocking reagent. The extracellularly applied reagent irreversibly blocks charybdotoxin (CTX) sensitive Q1 channels by chemically modifying E1 peptides that contain an N-terminal cysteine residue. Chemical release of the inhibitor and subsequent iterative applications of the reagent reported that Q1 channels partner with two KCNE β-subunits. To determine whether heteromeric Q1-KCNE complexes form, I synthesized a similar, but non-cleavable, K+channel blocking reagent that detects specific KCNE peptides in functioning complexes by irreversible channel inhibition. Using this “KCNE sensor”, heteromeric Q1/E1/E3, Q1/E1/E4 and Q1/E3/E4 complexes were shown to form, traffic to the cell surface and function. Using mathematical subtraction to visualize the irreversibly blocked current, the currents and gating kinetics of the different heteromeric complexes were revealed and a hierarchy of KCNE subunit modulation of Q1 channels was determined: E3>E1>>E4. Building on this technology, a chemically releasable K+ channel blocking reagent was created to specifically label KCNE β-subunits with biotin. The reagent delivers biotin to CTX sensitive Q1 channels and labeling occurs through free thiols provided by either cysteine residues or thiol modified sugars. This preliminary data demonstrates a novel strategy for labeling endogenous K+ channels in native cells.

The expansion of the voltage-gated calcium channel alpha 1 subunit families (CACNA1) of L and N types was investigated by combining phylogenetic analyses (neighbour-joining and maximum likelihood) with chromosomal data. Neighbouring gene families were analysed to see if the chromosomal regions duplicated through whole genome doublings in vertebrates. Results show that both types of CACNA1 expanded in two ancient whole genome duplications as parts of larger genomic regions. Many gene families in these regions obtained copies in an additional teleost-specific genome duplication. This diversification of CACNA1 genes probably contributed to evolutionary innovations in nervous system function.

Voltage-gated K+ (Kv) channels are known to cause serious disease upon their malfunction. Kv channels desensitised to voltage show inability to fully repolarise the membrane in excitable cells, which can make the membrane hyperexcited and in turn cause seizures such as in epilepsy, periodic ataxia or heart arrhythmia. Therefore, enhancers of Kv channels could serve as potential drugs. Some of these enhancers are polyunsaturated fatty acids and resin-acids which bind at the proteinlipid surface and affect the movement of the voltage sensor in the channel by a mechanism called the lipoelectric effect. To explore the lipoelectric modulation mechanism, we have performed an extensive computational study including docking and molecular dynamics simulations on resin-acid derivatives added to a model potassium channel called Shaker. Four derivatives, Wu32 and Wu50 that excite the channel and thus induce repolarisation of the membrane, as well as Wu18 and Wu27, who were found to be non-potent in previous experimental studies, have helped to point out a novel binding site in Shaker. The site is located between the pore and voltage-sensing domain of the channel and is in direct contact with the first gating charge arginine, R1, and the residue W454. We hypothesize that it is possible for resinacid derivatives to directly bind to the voltage-sensor when it is in an activated state, prolonging the time Shaker stays open. Further experimental studies on Shaker and human homologs are now needed to test our hypothesis. Therefore, we suggest recording the sensitivity of Shaker towards potent derivatives in combination with mutations of W454. If our findings of the novel binding site are correct, the suitability of Shaker as a model system for human Kv channel modulation by lipoelectric modulators can be questioned as W454 is replaced by small hydrophobic side chains in mammalian Shaker homologs.

No description available.

Voltage-gated Ca2+ (Cav) channels are activated upon depolarization. They specifically allow Ca2+ ions to come into the cell. These Ca2+ ions are bi-functional because they not only control cell excitability but also couple electrical activity to complex downstream signaling events, such as excitation-contraction coupling in muscles and neurotransmitter release in neurons. In the brain, Cav channels are expressed in the pre- or post-synaptic membrane of most excitable cells, neurons. In the past few years, their expression and function have also been characterized in many nonexcitable cells such as astrocytes. This dissertation focuses on the regulation of one subtype of postsynaptic Cav channels, Cav1.2, in neurons. In the first part of chapter I, I provide a literature overview of Cav channels in terms of their subtypes, localizations, physiological functions, and biophysical properties. For years, Cav channels were studied as single entities. But now, based on multiple proteomic studies, we know that these channels actually do not live alone. They interact with numerous proteins depending on the physiological conditions. Such interactions can anchor the channels to optimal sites of action, and tether Cav channels to their modulatory molecules. Therefore, it is crucial to understand how Cav channels are regulated by their macromolecular assembly. Among these protein partners, our lab studied the regulation of Cav channels by a subset of PDZ-domain containing proteins. Because these proteins play an important role in scaffolding and they colocalize with both pre- and post-synaptic Cav channels. Indeed, previous studies from our lab and other groups have revealed that PDZ proteins participate in a multitude of Cav regulation. The second part of chapter I introduces the diverse modulation of neuronal Cav channels by numerous PDZ proteins. In neurons, Cav1.2 channels regulate neuronal excitability and synaptic plasticity. Their functions have been implicated in learning, memory, and mood regulation. A study published in the journal Lancet showed that the gene encoding Cav1.2 is a common risk factor for five major psychiatric disorders. A PDZ protein, densin-180 (densin) is an excitatory synapse protein that promotes Ca2+-dependent facilitation of voltage-gated Cav1.3 Ca2+ channels in transfected cells. Mice lacking densin exhibit similar behavioral phenotypes that closely match those in mice lacking Cav1.2. In chapter II and III, we investigated the functional impact of densin on Cav1.2 channels and their auxiliary subunit β2a. Besides the regulation of Cav channels by their interactome, we have also known for a long time that Ca2+ currents undergo a negative feedback regulation. This regulation is called Ca2+-dependent inactivation (CDI) and it is mediated by Ca2+ that directly traverses the pore. CDI has been described for Cav channels in multiple cell types. In the heart, CDI prevents excessively long cardiac action potentials, which in turn can prevent activity-dependent arrhythmia. In neurons, CDI may be neuroprotective by preventing excitotoxic Ca2+ overloads. In the last 18 years, two essential components have been revealed in the mechanism of CDI. One is the protein calmodulin (CaM). CaM interacts directly with sites on the C-terminus of Cav channels. It binds to the incoming Ca2+ ions and produces a mysterious conformational change that determines the conductance of the channel. The other molecular player is Cavβ protein family. Cavβ comprises four subfamilies β1 through β4, which generally enhance the channel inactivation, except β2a. In chapter IV, Xiaohan Wang from Roger Colbran’s lab in Vanderbilt University, and I identified a new molecular determinant for Cav1.2 CDI. The α2δ subunit is an extracellular component of the Cav channel complex. Similar to Cavβ subunits, α2δ subunits are essential for the biophysical properties, surface level, and trafficking of Cavα1 subunits. There are four isoforms of α2δ subunits (α2δ1 to α2δ4). They display distinct tissue distributions. Although the roles of α2δ subunits in Cav channel regulation were studied extensively, studies have proposed that the function of α2δ subunits may be in part or entirely independent of Cav channel complex, such as synaptogenesis. Considering the important role of α2δ in physiology and pathology, it is imperative to identify the factors that regulate the properties of α2δ. In chapter V, I explored the trafficking dynamics of α2δ1 and revealed a potential regulator of α2δ1 for its protein stability and localization. One beauty of doing research is that it always motivates us to think and ask more questions on our journey of demystifying nature. While looking at the evidence that I find, I realize how much more we could do in the future. In chapter VI, I conclude the findings of each chapter and share my perspectives on the future direction for these research projects.

The brain network is a multiscale hierarchical organization from neurons and local circuits to macroscopic brain areas. The precise synaptic transmission at each synapse is therefore crucial for neural communication and the generation of orchestrated behaviors. Activation of presynaptic voltage-gated calcium channels (CaV2) initiates synaptic vesicle release and plays a key role in neurotransmission. In this dissertation, I have aimed to uncover how CaV2 activity affects synaptic transmission, circuit function and behavioral outcomes using Caenorhabditis elegans as a model. The C. elegans genome encodes an ensemble of highly conserved neurotransmission machinery, providing an opportunity to study the molecular mechanisms of synaptic function in a powerful genetic system. I identified a novel gain of function CaV2α1 mutation that causes CaV2 channels to activate at a lower membrane potential and slow the inactivation. Cell-specific expression of these gain-of-function CaV2 channels is sufficient to hyper-activate neurons of interest, offering a way to study their roles in a given circuit. CaV2(gf) mutants display behavioral hyperactivity and an excitation-dominant synaptic transmission. Imbalanced excitation and inhibition of the nervous system have been associated with several neurological disorders, including Familial Hemiplegic Migraine type 1 (FHM1) which is caused by gain- of-function mutations in the human CaV2.1α1 gene. I showed that animals carrying C. elegans CaV2α1 transgenes with corresponding human FHM1 mutations recapitulate the hyperactive behavioral phenotype exhibited by CaV2(gf) mutants, strongly suggesting the molecular function of CaV2 channels is highly conserved from C. elegans to human. Through performing a genome-wide forward genetic screen looking for CaV2α(gf) suppressors, we isolated new alleles of genes that required for CaV2 trafficking, localization and function. These regulators include subunits of CaV2 channel complex, components of synaptic and dense core vesicle release machinery as well as predicted extracellular proteins. Taken together, this work advances the understanding of CaV2 malfunction at both cellular and circuit levels, and provides a genetically amenable model for neurological disorders associated with excitation-inhibition imbalance. Additionally, through identifying regulators of CaV2, this research provides new avenues for understanding the CaV2 channel mediated neurotransmission and potential pharmacological targets for the treatments of calcium channelopathies.

Philosophiae Doctor - PhD / Neurodegenerative disorders are characterised by progressive loss of the brain’s physiological functions as a result of gradual degeneration of neurons in the central nervous system. Even though they are classified as diseases of the elderly, occurrence earlier in life is possible, but that would suggest the influence of genetic and/or environmental factors. Due to the continuous rise in modernisation and industrialisation over the years, there has been an increase in incidence and prevalence of neurodegenerative disorders. With the advances in technology and life expectancy, the rates of the common forms (Alzheimer’s disease and Parkinson’s disease), are expected to increase exponentially by 2050. Unfortunately, there is still no clinically approved treatment or therapy to slow down or halt the degenerative process as most registered drugs only offer symptomatic relief. Confounding this issue is the lack of definite mechanism of neurodegeneration, which is still poorly defined and not completely understood. Nonetheless, the pathology of most neurodegenerative disorders is believed to be a combination of interrelated processes that eventually leads to neuronal cell death. Among the postulated processes, the impact of excitotoxicity mediated by NMDA receptor over-activation is prominent and it is implicated in virtually all neurodegenerative disorders. With this basic insight, it is believed that molecules capable of inhibiting NMDA receptors and associated calcium channels, without affecting the normal physiological functions of the brain, could potentially serve as good neuroprotective drugs. Competitive and uncompetitive blockers (MK-801 and ketamine) have been explored, but none were clinically accepted due to undesirable side effects such as hallucinations, sedation and depression. However, NGP1-01, a polycyclic cage molecule, has been shown to be neuroprotective through modulation of NMDA receptors and voltage gated calcium channels and attenuation of MPP+ -induced toxicity. A similar approach could be useful in the design and development of new neuroprotective drugs. The aim of this study was to synthesise a series of open and rearranged cage-like molecules and explore their neuroprotective potential in neuroblastoma SH-SY5Y cells. The proposed structures, with norbornane scaffolds that contained different moieties, were designed to structurally resemble NGP1-01 and MK-801. Once synthesised, the compounds were purified and characterised, and were evaluated for their biological activities. Compounds were first screened for cytotoxicity at different concentrations. Thereafter, they were evaluated for neuroprotective effects against MPP+ -induced excitotoxicity and for calcium flux modulatory effects on NMDA receptor and voltage gated calcium channels. The norbornane derivatives were synthesised and characterised, and all final products were afforded in sufficient yields. All compounds with the exception of two compounds displayed good cytotoxic profiles towards the SH-SY5Y neuroblastoma cells at 10 µM, 50 µM and 100 µM concentrations as they demonstrated percentage cell viabilities close to 100% (control treated cells). Only two compounds showed percentage cell viability of 51% and 59% at 100 µM. Utilising the same cell line, all compounds, tested at 10 µM, attenuated MPP+ -induced toxicity after 24 hours of exposure to a neurotoxin. This was evident in the 23% to 53% enhancement (significant with p < 0.05) in cell viability when compared to the MPP+ only treated cells. In comparison to known NMDA receptor and/or voltage gated calcium channel blockers (MK-801, NGP1-01 or nimodipine), the synthesised compounds demonstrated mono or dual inhibition of calcium channels as they effectively attenuated calcium influx by blocking NMDA receptors and/or voltage gated calcium channels expressed in neuroblastoma SHSY5Y cells. This group of compounds were found to be more potent NMDA receptor inhibitors, probably due to similarities with MK-801 and memantine, than voltage gated calcium channel inhibitors. All compounds demonstrated moderate to good calcium inhibitory effects at NMDA receptors in the range of 23% to 70% while a selected few displayed very little or no activity at the voltage gated calcium channels. In conclusion, 27 compounds with norbornane scaffolds were successfully synthesised and evaluated for cytotoxicity and neuroprotection. The abilities of the synthesised compounds to protect neurons from the neurotoxin MPP+ and reduce calcium flux into neuronal cells were successfully demonstrated. These characteristics are essential in neuroprotection as they may prove significant in halting or slowing down the disease progression. The compounds showing a good cytotoxicity profile, neuroprotective effects and ability to reduce calcium overload, could potentially act as neuroprotective agents with good safety profiles or contribute as lead structures to the development and design of structurally related molecules that could clinically benefit people with neurodegenerative disorders.

Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-gated sodium channels (VGSCs) are transmembrane proteins responsible for the initiation of action potentials in excitable tissues by selectively allowing Na+ to flow through the cell membrane. VGSC subtype Nav1.7 is highly expressed in nociceptive (pain-sensing) neurons. It has recently been shown that individuals lacking the Nav1.7 subtype do not experience pain but otherwise function normally. In addition, dysfunction of Nav1.7 caused by point mutations in the channel is involved in two inherited pain disorders, primary erythromelalgia (PE) and paroxysmal extreme pain disorder (PEPD). This indicates Nav1.7 is a very important component in nociception. The aims of this dissertation were to 1) investigate if the antipsychotic drug, trifluoperazine (TFP), could modulate Nav1.7 current; 2) examine changes in Nav1.7 properties produced by the PE mutation N395K including sensitivity to the local anesthetic (LA), lidocaine; and 3) determine how different inactivated conformations of Nav1.7 affect lidocaine inhibition on the channel using PEPD mutations (I1461T and T1464I) that alter transitions between the different inactivated configurations of Nav1.7. Standard whole-cell electrophysiology was used to determine electrophysiological and pharmacological changes in WT and mutant sodium currents. Results from this dissertation demonstrate 1) TFP inhibits Nav1.7 channels through the LA interaction site; 2) the N395K mutation alters electrophysiological properties of Nav1.7 and decreases channel sensitivity to the local anesthetic lidocaine; and 3) lidocaine stabilizes Nav1.7 in a configuration that decreases transition to the slow inactivated state of the channel. Overall, this dissertation answers important questions regarding the pharmacology of Nav1.7 and provides insight into the changes in Nav1.7 channel properties caused by point mutations that may contribute to abnormal pain sensations. The results of this dissertation on the function and pharmacology of the Nav1.7 channel are crucial to the understanding of pain pathophysiology and will provide insight for the advancement of pain management therapies.