Ca2+ is a critical link between many physiological stimuli and their intracellular effectors. In neurons, stimulus-evoked changes in the cytosolic free Ca2+ concentration ([Ca2+]i) influence enzyme activity, membrane excitability, exocytosis, and gene expression, and cytotoxic [Ca2+]i levels contribute to cell death in neurologic disease. Functional and molecular studies have described Ca2+ channels, pumps and exchangers that regulate [Ca2+]i, and enzymes whose activities depend on [Ca2+]i. However, little is known about how Ca2+ transporters define the time course of [Ca2+]i during stimulation, or the way time-varying [Ca2+] signals influence enzyme activity. The proposed research will examine how various Ca2+ transport systems shape the time course of [Ca2+]i during stimulation, and how a Ca2+-sensitive enzyme is influenced by [Ca2+]i dynamics. Bullfrog sympathetic neurons (BFSN's) will provide the model cell, Ca2+/calmodulin-dependent protein kinase II (CaMKII) will serve as the model enzyme, and Ca2+ indicator dyes, electrophysiological techniques, and protein kinase activity assays will be employed to examine the following topics: * Plasma membrane Ca2+ transport during stimulation. During membrane depolarization, [Ca2+]i rises under the influence of voltage-dependent Ca2+ entry, but the time-course of [Ca2+]i reflects other Ca2+ transporters as well. The net Ca2+ flux across the plasma membrane will be separated into contributions from Ca2+ entry through channels and Ca2+ extrusion via pumps and exchangers. Transport rates and their dependence on Ca2+ concentration, membrane potential and time will be examined to establish macroscopic transport rate laws. * Contributions from intracellular pools. I have proposed that a caffeine- and ryanodine-sensitive store (CSS) modulates [Ca2+]i responses to depolarization in BFSN's. The net Ca2+ flux between the CSS and cytosol will be separated into contributions from Ca2+ uptake and release, including Ca2+-induced Ca2+ release. Measurements will also be made of changes in the free Ca2+ level within the CSS during stimulation and the underlying Ca2+ fluxes. Along with information about plasma membrane transport. these data will elucidate how the time course of [Ca2+] during stimulation is defined, and its sensitivity to transport regulation. * Temporal control of CaMKII by [Ca2+]i. CaMKII is a ubiquitous [Ca2+]i- sensitive enzyme, and depolarization changes its activity by elevating (Ca2+]i. The kinetics of enzyme activation and deactivation will be examined in cell populations following imposed changes in [Ca2+]i. Predicted effects on CaMKII activity of temporal patterns of stimulation and Ca2+ transport modulation will also be tested.
| Ovsepian, Saak V; Friel, David D (2012) Enhanced synaptic inhibition disrupts the efferent code of cerebellar Purkinje neurons in leaner Cav2.1 Ca 2+ channel mutant mice. Cerebellum 11:666-80 |
