Student Theses and Dissertations

Date of Award

2010

Document Type

Thesis

RU Laboratory

Bargmann Laboratory

Keywords

calcium channel biogenesis, CALF-1, CAV2 channel, pqn-53, UNC-2, c. elegans, presynaptic voltage-gated calcium channels

Abstract

Neurotransmitter release at nerve terminals is a fundamental aspect of communication in the nervous system. Voltage changes in the presynaptic membrane are sensed by presynaptic voltage-gated calcium channels that mediate calcium influx at the nerve terminals to execute exocytosis of various kinds of neurotransmitters. Defects in presynaptic calcium channels lead to many neurological disorders, emphasizing the importance of these channels in the regulation of neuronal activities in the brain. Previous physiological studies have focused on opening kinetics of the channels and their modulation by auxiliary subunits. Cell biological questions such as trafficking and clustering of the channels at the presynaptic site, however, have remained largely unanswered, partly due to a lack of an in vivo assay system to monitor calcium channel biogenesis in intact animals. In my thesis, I established an in vivo system to visualize the UNC-2 alpha1 subunit of the C. elegans CaV2 channel. I showed that GFP-tagged UNC-2 is localized to presynaptic active zones of sensory and motor neurons. Using this system, I conducted a direct visual genetic screen for mutants that are defective in UNC-2 localization, identifying three genes, calf-1, unc-36 and pqn-53/calf-2. CALF-1 is a neuronal-specific one-pass transmembrane protein that resides in the endoplasmic reticulum, and is required for endoplasmic reticulum exit of UNC-2. Structure-function analysis revealed that the transmembrane domain and the cytosolic arginine-based basic motifs are important for the function of CALF-1. Acute induction of calf-1 mobilizes preexisting UNC-2 for delivery to synapses, consistent with a direct trafficking role. The calcium channel alpha2-delta auxiliary subunit UNC-36 is also required for endoplasmic reticulum exit of UNC-2, but has additional functions. The polyglutamine protein pqn-53 is localized to the nucleus; PQN-53 inhibits a non-canonical unfolded protein response pathway, and activation of this pathway in pqn-53 mutants leads to the reduced expression of UNC-2 and other transmembrane proteins. Furthermore, pqn-53 mutants are resistant to killing by pathogenic bacteria, potentially due to constitutive activation of the non-canonical unfolded protein response. pqn-53 couples multipass transmembrane protein biogenesis with endoplasmic reticulum stress, and provides insights into the regulation of pathogen resistance by the endoplasmic reticulum stress-sensing pathway.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/359

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