Amyotrophic Lateral Sclerosis (ALS) is a fatal, adult-onset progressive degenerative motor neuron disease that is characterized by muscle atrophy and weakness due to the loss of upper and lower motor neurons. Average survival time for individuals diagnosed with the disease is three to five years; currently there is no cure and only one drug approved by the Food and Administration (FDA). Scientists have proposed various theories in order to solve the mystery which surrounds ALS. One of these theories hypothesizes how hyperexcitability and excitotoxicity leads to the death of motor neurons. In this study, we will address ways of combatting the effects of hyperexcitability as well as excitotoxicity by targeting a specific channel type. The channels in question are small conductance calcium activated potassium channels (SK channels). We chose to target these channels because they directly affect the medium after-hyperpolarization (mAHP) of the cell which controls firing rate. We postulate that SK channels are being altered in such a way that cell firing rate has been increased, leading to phenotypes associated with the disease such as abnormal excitability, mitochondrial dysfunction, axonal loss motor impairment, muscle atrophy as well as excitotoxicity, thus leading to the spread of motor neuron death. Upon administration with a specific SK channel activator in the form of CyPPA; improvements in motor function and survival were found. These improvements suggest that SK channels are indeed viable drug targets and specific SK channel activators may be treatment options for individuals suffering from ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease that currently has no cure and extremely limited treatment options. The specific mechanisms that underlie motoneuron degeneration and death, which are classical features of this disease, are mostly unknown. This thesis tests the hypothesis that small-conductance calcium-activated potassium channels (SK) may be downregulated in ALS motoneurons, as suggested by computational modelling. SK channel expression was measured in spinal alpha-motoneuron cell bodies or somata of wildtype (WT) and mutant (mt) SOD1-G93A mice, a transgenic animal model of ALS. Quantitative immunohistochemical analysis of the developmental expression of SK channel isoforms SK2 and SK3 at various postnatal time points was performed to assess the effects of motoneuron degeneration on the level and/or pattern of protein expression on the somata of lower lumbar motor nuclei. Results indicate that the selective expression of SK3 may be gradually reduced over development in WT and mutant SOD1 mice but is affected by disease pathogenesis. In addition, SK channels appear to be clustered in both WT and mutant SOD1 motoneurons throughout development. However, SK clusters appear to be significantly smaller in mutant SOD1 motoneurons compared to their WT littermates. These changes indicate that the activity of SK channels, which regulate the firing rate of motoneurons, may be affected in ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease which targets motoneurons (MNs), yet underlying disease mechanisms are not well understood. Evaluating the intrinsic excitability of MNs in ALS could lead to a better understanding of mechanisms causing neurodegeneration. The SOD1-G93A (SOD) model is the most commonly studied animal model of ALS. However, past studies have shown highly conflicting results on SOD MN excitability. Interestingly, I show that depending on the level of membrane depolarization, SOD MNs show opposite results of both hyper- and hypo-excitability. This reveals that differences in the methodology of measuring excitability can heavily impact the study results. Finally, I have investigated the firing abnormalities leading to hypoexcitability in SOD MNs at high levels of membrane depolarization. Results indicate that these firing abnormalities are due to decreased Kv2.1 channel conductance. Furthermore, these firing abnormalities could be the basis for developing a biomarker which could be used to diagnose ALS earlier.

Neuroinflammation and demyelination in multiple sclerosis (MS) lead first to neuronal dysfunction, then to the potential for neurodegeneration. Voltage-gated potassium ion channels have key roles in maintaining the resting membrane potential of a neuron in readiness for neurotransmission. This includes repolarization of the membrane following an action potential, which involves a depolarization event mediated by voltage-gated sodium channels. Myelin, the many-layered lipid sheath surrounding axons, is known to participate in molecular interactions with the axonal membrane to target ion channels to important, specialized locations along the axon. The water channel aquaporin-4 (AQP4), expressed in astrocyte endfeet, also plays an important role in maintaining ionic and fluid homeostasis in the neuronal environment. Because progression of disease and permanent disability in MS appears related to the degree of neuronal loss, neuroprotective treatments are needed in MS. Although a nonspecific Kv channel blocker is currently approved for symptomatic treatment of MS, it is not known to provide any neuroprotective effect, it has significant side effects, and its mechanism of action is not clear. In my dissertation research, I used two animal models of MS, chronic or relapsing-remitting experimental autoimmune encephalomyelitis (chEAE or rrEAE) to mimic progressive or relapsing-remitting MS, respectively, and to characterize the effect of inflammatory, demyelinating lesions upon the expression and localization patterns of AQP4 and key Kv channels. I found that Kv 1.2, expressed in myelinated axons in spinal cord (SC) white matter (WM), was redistributed in lesioned areas from its normal location at the juxtaparanode (JXP). The JXP localization could be recovered in remitting rrEAE, but not late chEAE. Kv 2.1, normally clustered on the soma and proximal dendrites of alpha motor neurons located in SC ventral gray matter (GM), was declustered and reduced in expressi (open full item for complete abstract)

Voltage-gated potassium channels have enchanted electrophysiologists for over 60 years since the pioneering work of Hodgkin and Huxley. Potassium channel inactivation is interesting biophysically since it comprises multiple distinct molecular mechanisms that can be characterized. However, inactivation is also interesting physiologically since it can impact the excitability of tissues as diverse as central neurons, pancreatic beta cells, and photoreceptors. Elucidating the molecular mechanisms underlying slow inactivation in Shaker IR and Kv2.1 channels has been the focus of this thesis. A single point mutation in Shaker IR (T449) has been shown to affect the kinetics of slow inactivation up to 100-fold. Originally, this effect was ascribed to C-type inactivation but Shaker is now known to possess two forms of slow inactivation: C-type (preferential open-state inactivation) and U-type (preferential closed-state inactivation). C-type inactivation occurs via outer-pore constriction while the mechanism of U-type inactivation remains unknown. This thesis uses established techniques of electrophysiology, pharmacology, and mutagenesis to demonstrate that mutants of Kv2.1 outer pore residue Y380 (homologous to Shaker T449) undergo U-type inactivation alone, and that mutants of Shaker T449 affect C-type inactivation alone. The study also hypothesizes that Kv2.1 channels lack C-type inactivation as it exists in Shaker IR and that C- and U-type inactivation have different molecular mechanisms. Furthermore, this study advances pore mutation as yet another tool (in addition to pharmacological and electrophysiological approaches) to help separate C- from U-type inactivation in channels with complex slow inactivation. In both Shaker and Kv2.1 channels, slow inactivation in wild-type and mutant channels was characterized with a 12-state Markov model concluding that C-type inactivation is not exclusive open-state inactivation and U-type inactivation is not exclusive closed-sta (open full item for complete abstract)

T cell receptor (TCR) engagement by an antigen presenting cell (APC) results in reorganization of intracellular and membrane molecules at the T/APC interface, forming a “signalosome”, the immunological synapse (IS). An early event associated with T/APC interaction is Ca2+ influx. K+ channels, Kv1.3 and KCa3.1, modulate Ca2+ signaling in human T cells. Resting and activated human T cells express both channels, albeit to different degrees. Kv1.3 channels modulate Ca2+ in resting, while KCa3.1 channels do so in activated, T cells. Although these channels play such an important role in Ca2+ homeostasis, very little is known about their localization in the IS. Furthermore, aberrant T cell responses in IS formation and Ca2+ influx have been documented in T cells from patients with systemic lupus erythematosus (SLE). The potential involvement of K+ channels in the etiology and progression of SLE remains unknown. Herein we determined K+ channel membrane distribution in resting and activated human T cells following TCR engagement and performed comparative studies with SLE T cells to decipher the role of K+ channels in the Ca2+ response anomaly. Our data show that in SLE T cells, Kv1.3 channels constitute the dominant channel and are functionally identical to their normal counterparts. We also found that resting SLE T cells show faster Kv1.3 kinetics out of the IS as compared to healthy T cells and comparable to healthy pre-activated T cells. However, normal pre-activated T cells recruit and maintain KCa3.1 channels in the IS after Kv1.3 channels leave, while SLE T cells do not express the appropriate KCa3.1 channel number to support this activated phenotype. This Kv1.3 mobility defect appears to be specific to SLE and not other autoimmune diseases, as it was not observed in rheumatoid arthritis (RA) patients. Further, transcription factor activation and gene expression relies on the shape of the Ca2+ response. Although SLE T cells demonstrate abnormal transcription fact (open full item for complete abstract)