UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Identification of a novel protein interaction which contributes to N-type calcium channel G-protein and PKC-dependent modulation Guthrie, Heather

Abstract

The N-type calcium channel plays a critical role in neurotransmission and is known to associate with proteins involved in vesicle release such as syntaxin, synaptotagmin and SNAP-25. In addition, block of N-type channels has been shown to abolish neurotransmission in both the peripheral and central nervous systems. Modulation of calcium channels can alter calcium entry into a cell and therefore understanding these processes is crucial towards determining the factors governing N-type channel regulation of neurotransmission. In order to identify factors which may influence the activity of the N-type channels in neuronal physiology, I screened a rat brain yeast two-hybrid library with the cytoplasmic regions of the rat N-type am subunit. Using this approach clones encoding the protein Nell2 were identified that bound to the C-terminus of the N-type CXIB subunit. Nell2 is a PKC binding protein and a member of a family of proteins known as Nels (neuronally expressed proteins containing EGFlike repeats). RT-PCR of various rat tissues determined that Nell2 and N-type calcium channel RNA were expressed in many of the same tissues, including brain stem, cerebellum, cortex and pituitary with trace amounts in eye and heart. A full-length Nell2 clone was generated from rat brain RNA by RT-PCR with and without an N-terminal Flag epitope tag. Co-expression of Nell2 and N-type calcium channels was analyzed in ITEK cells and neurons by immunohistochemical techniques. Both proteins were found to be membrane associated and localized to the same regions within each cell type. In order to understand the functional significance of the Nell2/N-type channel interaction, electrophysiological analysis was carried out on HEK cells co-expressing Nell2 and N-type channels. The presence of Nell2 enhanced both the PKC and G-protein-dependent modulation of N-type channels. In the presence of Nell2, N-type channels became more sensitive to phorbol esters and co-expression with Nell2 antisense RNA abolished the characteristic PKC-dependent upregulation of N-type channel peak currents. The G-protein-dependent inhibition of N-type channel currents was further enhanced in the presence of Nell2. Deletion analysis of the Nell2 gene product showed that the first 20 amino acids largely contributed to the enhanced G-proteindependent inhibition. A model is proposed to describe how Nell2 may participate in neurotransmission by anchoring PKC at presynaptic sites with the N-type calcium channels.

Item Media

Item Citations and Data

Rights

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

Usage Statistics