Thesis (M.Sc.)--University of Alberta, 1997. / Submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Master of Science, Division of Neuroscience. Also available online.

<p></p><p>The<b> </b>L-type VGCC subtypes, including subtypes Ca_v1.1-1.4, have been shown to play critical roles in various cellular activities, including muscle contraction, hormone secretion, and neurotransmitter release. Recent research indicates the potential involvement of Ca_v1.3 in various neurological and psychiatric disorders, such as the early onset of Parkinson’s disease and substance abuse disorders. Non-selective L-VGCC subtype blockers such as dihydropyridines (DHPs) are used to treat hypertension and angina because they potently inhibit Ca_v1.2, but no selective Ca_v1.3 inhibitors have been developed yet. We resolved the molecular determinants to differentiate Ca_v1.2 and Ca_v1.3 in response to DHP nifedipine. Nifedipine IC₅₀ for Ca_v1.2 and Ca_v1.3 are 22nM and 289nM determined by whole-cell patch-clamp. We identified two significant amino acids, Ca_v1.3/M1030 to Ca_v1.2/V1036 in the transmembrane IIIS5 and Ca_v1.3/S1100 Ca_v1.2/A1106 in the extracellular IIIS-3P loop, to differentiate the subtype affinity to nifedipine. </p> <p>We found that the Ca_v1.3/II-III loop fused to eGFP decreased glucose-activated action potential (GSAP) frequency by ~80% in the pancreatic β-cell. We introduced several synthetic peptides, and peptide P3-1 from C-terminal induced a -16mV shift in V_1/2 inactivation with an EC₅₀ of 231nM. P3-1 contains a protein kinase G (PKG) phosphorylation site (RRISE) required for PKG inhibition of Ca_v1.3 current but not conserved in Ca_v1.2. We found that the shift in V_1/2 inactivation induced by co-expression of Ca_v1.3 with the Ca_v1.3/II-III loop/GFP requires the presence of a Ca_vβ subunit, and Ca_vβ₃ also exhibits selectivity over other β subunits. Significantly, P3-1 shifts the Ca_v1.2 inactivation to a more positive voltage when co-expressed with either Ca_vβ_2aor Ca_vβ₃, demonstrating the ability of P3-1 to differentiate Ca_v1.2 and Ca_v1.3 in a Ca_vβ-dependent manner.</p> <p><b> </b>Failure of pancreatic β-cells to secrete enough insulin to maintain glucose homeostasis is a hallmark of Type 2 diabetes. However, the consequences of the dysregulation of the endoplasmic reticulum (ER) Ca²⁺ channel ryanodine receptor-2 (RyR2) in pancreatic β-cells are not fully understood. Therefore, we characterized the electrical activity in INS-1 in which RyR2 has been deleted via CRISPR/Cas9 gene editing. We observed a decreased level of IP₃ receptor binding protein (IRBIT) in RyR2^KO INS-1 cells and generated IRBIT^KO INS-1 cells. VGCC current density in RyR2^KO doubled compared to controls and was also elevated in IRBIT^KOcompared to control cells. All HVA Ca²⁺ channels were upregulated, determined by fractional current blocked by nifedipine. We also found that GSAP frequency is doubled by RyR2 deletion due to failure to activate apamin sensitive SK (small conductance calcium-activated potassium) channels. </p><br><p></p>

No description available.

Mutations of the α1A subunit of CaV 2.1 voltage gated calcium (VGCC) channels are responsible for several inherited disorders affecting humans, including familial hemiplegic migraine, episodic ataxia type and spinocerebellar ataxia type. The leaner mouse also carries an autosomal recessive mutation in the α1A subunit of CaV 2.1 VGCCs, which, in the homozygous condition, results in a severe cerebellar atrophy and ataxia. The leaner mutation results in reduced calcium influx through CaV 2.1 VGCCs. To better understand cerebellar neurodegeneration and cerebellar dysfunction we focused our research on elucidating the relationship between mitochondrial function/dysfunction and calcium channel mutations. The aims of this dissertation were: 1) to estimate the extent of neuronal cell death, basal intracellular calcium and mitochondrial (dys)function in cerebellar granule cells (CGC) of adult leaner mice; 2) to analyze the role of the leaner calcium channel mutation on postnatal development of CGCs; and 3) to test whether inducing increased calcium influx by exposing cultured granule cells to potassium chloride can eliminate or reduce the CGC death. By using mechanism independent Fluoro-Jade staining and apoptosis specific TUNEL staining, we demonstrated that leaner CGC death continues into adulthood and the spatial pattern of granule cell death observed during postnatal development also continues into adulthood. The present investigation showed a reduced resting intracellular calcium in CGC from leaner mice as compared to age matched wild type mice, and tottering mice. The tottering mouse is another mutant mouse that carries a mutation in the α1A subunit of CaV 2.1 VGCCs like leaner mouse. However, these mice do not show any neurodegeneration and therefore they were used as a second control. Our results also showed that even though CGC of leaner mice have dysfunctional CaV2.1 channels, there is no change in depolarization induced Ca2+ influx, which suggests a functional compensation for CaV2.1 calcium channels by other VGCCs. Our results showed reduced mitochondrial membrane potential at the time of peak CGC death in leaner mice as compared to wild type CGCs and tottering CGCs. The results of this investigation suggest mitochondrial mediated but reactive oxygen species independent cell death in CGCs of leaner mice.

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, August 1999. / Includes bibliographical references. Also available on the Internet.

Ca2+ entry through voltage-gated calcium channels (CaVs) triggers a range of physiological events, including synaptic neurotransmission and muscular excito-contraction coupling. CaVs are often localised to discrete membrane microdomains and are required to be targeted to such fine structures in order to perform their cellular functions. CaVs are multi-subunit protein complexes that consist of a core, pore-forming α1 subunit and auxiliary β and α2/δ subunits. The α2/δ subunit is required for the optimal cell surface expression and function of CaVs and is itself localised to cholesterol-rich membrane microdomains called lipid rafts. What is unclear is whether the α2/δ subunit is required for whole CaV complexes to be localised to lipid rafts and what effects lipid raft association has on the cell surface distribution and function of CaVs. By a combination of cellular imaging, biochemistry and electrophysiology, this project shows that the auxiliary α2/δ-1 subunit is both necessary and sufficient to target CaV2.2 to lipid rafts in the COS-7 cell heterologous expression system (Robinson et al, 2010). In addition, α2/δ is localised at the cell surface in discrete puncta and co-localises with two endogenous lipid raft resident proteins, caveolin and flotillin-1. While the punctate cell surface distribution of α2/δ is co-incident with that of caveolin and flotillin-1, its distribution is not dependent on cellular cholesterol, but rather the integrity of the actin cytoskeleton. Additional structure-function analysis by employment of the pIN-α2/δ series of deletion and substitution mutants has shown that the association of α2/δ with lipid rafts is bestowed by an extracellular region of the delta peptide, contrary to other evidence supporting the notion that α2/δ may be a GPI-anchored protein. The exact physiological and functional significance of α2/δ and CaV association with lipid rafts remains poorly understood, but the fact that CaVs are enriched within these fine structures provides a potential mechanism for targeting and access to lipid raft associated signalling pathways.

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