Jamie L. Weiss

Neuronal Ca2+ Sensor 1 CHARACTERIZATION OF THE MYRISTOYLATED PROTEIN, ITS CELLULAR EFFECTS IN PERMEABILIZED ADRENAL CHROMAFFIN CELLS, Ca2+-INDEPENDENT MEMBRANE ASSOCIATION, AND INTERACTION WITH BINDING PROTEINS, SUGGESTING A ROLE IN RAPID Ca2+ SIGNAL TRANSDUCTION

Overexpression of frequenin and its orthologue neuronal Ca2+ sensor 1 (NCS-1) has been shown to increase evoked exocytosis in neurons and neuroendocrine cells. The site of action of NCS-1 and its biochemical targets that affect exocytosis are unknown. To allow further investigation of NCS-1 function, we have demonstrated that NCS-1 is a substrate forN-myristoyltransferase and generated recombinant myristoylated NCS-1. The bacterially expressed NCS-1 shows Ca2+-induced conformational changes. The possibility that NCS-1 directly interacts with the exocytotic machinery to enhance exocytosis was tested using digitonin-permeabilized chromaffin cells. Exogenous NCS-1 was retained in permeabilized cells but had no effect on Ca2+-dependent release of catecholamine. In addition, exogenous NCS-1 did not regulate cyclic nucleotide levels in this system. These data suggest that the effects of NCS-1 seen in intact cells are likely to be due to an action on the early steps of stimulus-secretion coupling or on Ca2+ homeostasis. Myristoylated NCS-1 bound to membranes in the absence of Ca2+ and endogenous NCS-1 was tightly membrane-associated. Using biotinylated NCS-1, a series of specific binding proteins were detected in cytosol, chromaffin granule membrane, and microsome fractions of adrenal medulla. These included proteins distinct from those detected by biotinylated calmodulin, demonstrating the presence of multiple specific Ca2+-independent and Ca2+-dependent binding proteins as putative targets for NCS-1 action. A model for NCS-1 function, from these data, indicates a constitutive membrane association independent of Ca2+. This differs from the Ca2+ myristoyl switch model for the closely related recoverin and suggests a possible action in rapid Ca2+ signal transduction in response to local Ca2+ signals.

Identification of Ca2+-dependent binding partners for the neuronal calcium sensor protein neurocalcin δ: interaction with actin, clathrin and tubulin

The neuronal calcium sensors are a family of EF-hand-containing Ca(2+)-binding proteins expressed predominantly in retinal photoreceptors and neurons. One of the family members is neurocalcin delta, the function of which is unknown. As an approach to elucidating the protein interactions made by neurocalcin delta, we have identified brain cytosolic proteins that bind to neurocalcin delta in a Ca(2+)-dependent manner. We used immobilized recombinant myristoylated neurocalcin delta combined with protein identification using MS. We demonstrate a specific interaction with clathrin heavy chain, alpha- and beta-tubulin, and actin. These interactions were dependent upon myristoylation of neurocalcin delta indicating that the N-terminal myristoyl group may be important for protein-protein interactions in addition to membrane association. Direct binding of neurocalcin delta to clathrin, tubulin and actin was confirmed using an overlay assay. These interactions were also demonstrated for endogenous neurocalcin delta by co-immunoprecipitation from rat brain cytosol. When expressed in HeLa cells, neurocalcin delta was cytosolic at resting Ca(2+) levels but translocated to membranes, including a perinuclear compartment (trans-Golgi network) where it co-localized with clathrin, following Ca(2+) elevation. These data suggest the possibility that neurocalcin delta functions in the control of clathrin-coated vesicle traffic.

Sense and sensibility in the regulation of voltage-gated Ca2+ channels

Voltage-gated Ca(2+) channels are crucial for neurotransmitter release and other neuronal functions, and their activity-dependent regulation could underlie various aspects of synaptic plasticity. Recent studies have identified Ca(2+)-sensing proteins involved in Ca(2+)-channel modulation. These have complex effects on channel gating, and data suggest that the actions of multiple Ca(2+) sensors are important for the fine-tuning of Ca(2+) channel activity.

CHAPTER 136 – EF-Hand Proteins and Calcium Sensing: The Neuronal Calcium Sensors

EF-hand calcium (Ca 2+ )-binding proteins play many important roles in Ca 2+ -homeostasis and Ca 2+ -signaling mechanisms. The EF-hand containing protein calmodulin has been most extensively studied but the EF-hand motif is the most prevalent and widely distributed protein domain in Ca 2+ signaling. The neuronal Ca 2+ -sensor (NCS) proteins are a closely-related family of Ca 2+ -binding proteins whose physiological functions have begun to emerge in recent years. The NCS proteins are already known to have multiple binding protein partners. More studies are needed to characterize these interactions so that we can place NCS proteins in known pathways. Further in vivo knockout and over-expression studies will reveal more about the roles of the NCS proteins and clarify their range of physiological functions in the regulation of neuronal activity.