Physiological analysis of structural/functional features of neuronal calcium sensor-1

Martin, Victoria
Physiological analysis of structural/functional features of neuronal calcium sensor-1. PhD thesis, University of Liverpool.

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Calcium (Ca2+) signalling regulates many neuronal functions including neurotransmission, axonal growth and development. Neuronal calcium sensor-1 (NCS-1) has been shown to be involved in many of these processes. On Ca2+ binding, NCS-1 changes conformation and exposes a hydrophobic binding pocket. In yeast, NCS-1 binds to a PI4-kinase orthologue required for survival. In mammalian cells, NCS-1 is localised to the Golgi and plasma membranes and has been linked to multiple target proteins that have roles in neuronal signalling. NCS-1 has been shown to regulate the P/Q Ca2+ channel subunit Cav2.1; although no direct binding interaction has been identified between the proteins. The Cav2.1 C-terminal tail contains two Ca2+-sensor binding regions, the IQ domain and the calmodulin (CaM) binding domain (CBD). The first part of this study investigated NCS-1 or CaM and Cav2.1 interactions using biochemical and biophysical interactions. Pull-down analysis found that NCS-1 binds to a Cav2.1 C-terminal peptide in a Ca2+-dependent manner. Use of nuclear magnetic resonance spectroscopy also showed that the IQ domain of Cav2.1 bound to NCS-1 in the presence of Ca2+, though the NCS-1 region involved in this interaction could not be identified. The second part of this study investigated NCS-1 in the model organism C. elegans. In the worm, NCS-1 is expressed predominantly in sensory neurons. An ncs-1 null mutant worm strain (XA406) was previously shown to be defective in isothermal tracking and this was linked to a requirement for NCS-1 in memory and learning. To ensure this behaviour was not caused by a locomotion or neurotransmission phenotype, these behaviours were quantified and compared to the wild-type strain. No effect of the ncs-1 null mutation was found in a quantitative body-bend assay or in an assay of aldicarb resistance. The temperature-linked behaviour was further characterised using an acute assay for temperature-dependent locomotion (TDL). In this assay, the rate of locomotion of wild-type worms decreased when the temperature was elevated from 20oC to 28oC. In contrast, the rate of locomotion of the ncs-1 null worm was significantly increased at the higher temperature. This distinct phenotype was exploited to quantify the rescue of the null strain by expression of wild-type NCS-1 and to identify potential mechanisms involved in NCS-1 function. It was established that NCS-1 regulated TDL when expressed in AIY neurons. Using information from previous studies, key structural elements of NCS-1 were investigated by expressing NCS-1 with specific point mutations or deletions. N-terminal myristoylation of NCS-1 was not functionally required. In contrast, the N- and C-terminal clefts of the hydrophobic pocket of NCS-1 were shown to be physiologically important while the C-terminal tail was not essential for function in the TDL assay. These findings allowed discrimination between two potential modes of interaction of NCS-1 with its target proteins in a physiological context.

Item Type: Thesis (PhD)
Additional Information: Date: 2013-09 (completed)
Subjects: ?? QP ??
Divisions: Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 31 Jul 2014 09:42
Last Modified: 16 Dec 2022 04:41
DOI: 10.17638/00016345