Modulation of Ca v 1.3 L-Type Calcium Channels by Arachidonic Acid and Muscarinic M 1 Receptors: A Dissertation

Abstract

Membrane excitability, gene expression, and neurotransmitter release are all controlled by voltage-gated L-type Ca 2+ (L- )channels. In turn, Ca 2+ channels are highly regulated by signal transduction cascades initiated by G protein-coupled receptor (GPCR) activation. In medium spiny neurons of the striatum, both the muscarinic M 1 receptors (M 1 R) and dopaminergic D 2 receptors (D 2 R) specifically inhibit the Ca v 1.3 L-channel. In Chapters III and IV, the pathways downstream of M 1 Rs and D 2 Rs are examined to determine whether an overlap or intersection in inhibition of Ca v 1.3 occurs by these two receptors. Transient transfection of Ca v 1.3 channels in HEK 293 cells, stably transfected with the M 1 R, and in ST14A cells were used as model systems. While a further characterization of ST14A cells determined that they exhibit a striatal profile, D 2 Rs or M 1 Rs did not inhibit Ca v 1.3. Lack of current inhibition may be due to the finding of no detectable expression of phospholipase Cβ-1 protein in ST14A cells. Ca 2+ channels are multiprotein complexes comprised of α 1 , β, and α 2 δ subunits. While the actions of arachidonic acid (AA) have been shown to mimic M 1 R inhibition of L-current in superior cervical ganglion neurons, the precise identity of the L-channel in these neurons -either Ca v 1.2 or Ca v 1.3 or both- is not known. The transfected model systems allowed for the analysis of whole-cells currents with different β subunit combinations as well as the study of only Ca v 1.3 channels. In Chapter III, I show that activation of M 1 Rs with the agonist Oxo-M inhibited Ca v 1.3 channels coexpressed with either β 1b , β 2a , β 3 , or β 4 subunits. Surprisingly, the magnitude of Ca v 1.3, β 2a currents was inhibited less than Ca v 1.3 currents with other β subunits. In Chapter V, AA is shown to mimic the profile of M 1 R stimulation on Ca v 1.3 currents. The magnitude of Ca v 1.3, β 2a currents was inhibited less than Ca v 1.3 currents with other β subunits by AA. This discovery points to a novel role for accessory β subunits in altering the magnitude of AA inhibition and kinetic changes of Ca v 1.3. Arachidonic acid (AA) inhibits Ca 2+ channels by an unknown mechanism at an unknown site. In Chapter V, I found that Ca v l.3 inhibition by AA was state-dependent and most likely stabilizes a closed channel conformation. The finding that the Ca 2+ channel accessory β subunit alters the magnitude of AA inhibition and kinetic changes of Ca v 1.3 suggests that AA could alter processes which rely on L-channels such as Ca 2+ -dependent gene expression, secretion and membrane excitability.