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Neuronal Calcium Sensor-1 Regulation of Calcium Channels, Secretion, and Neuronal Outgrowth

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Abstract

Calcium (Ca2+) is an important intracellular messenger underlying cell physiology. Ca2+ channels are the main entry route for Ca2+ into excitable cells, and regulate processes such as neurotransmitter release and neuronal outgrowth. Neuronal Calcium Sensor-1 (NCS-1) is a member of the Calmodulin superfamily of EF-hand Ca2+ sensing proteins residing in the subfamily of NCS proteins. NCS-1 was originally discovered in Drosophila as an overexpression mutant (Frequenin), having an increased frequency of Ca2+-evoked neurotransmission. NCS-1 is N-terminally myristoylated, can bind intracellular membranes, and has a Ca2+ affinity of 0.3 μM. Over 10 years ago it was discovered that NCS-1 overexpression enhances Ca2+-evoked secretion in bovine adrenal chromaffin cells. The mechanism was unclear, but there was no apparent direct effect on the exocytotic machinery. It was revealed, again in chromaffin cells, that NCS-1 regulates voltage-gated Ca2+ channels (Cavs) in G-Protein Coupled Receptor (GPCR) signaling pathways. This work in chromaffin cells highlighted NCS-1 as an important modulator of neurotransmission. NCS-1 has since been shown to regulate and/or directly interact with many proteins including Cavs (P/Q, N, and L), TRPC1/5 channels, GPCRs, IP3R, and PI4 kinase type IIIβ. NCS-1 also affects neuronal outgrowth having roles in learning and memory affecting both short- and long-term synaptic plasticity. It is not known if NCS-1 affects neurotransmission and synaptic plasticity via its effect on PIP2 levels, and/or via a direct interaction with Ca2+ channels or their signaling complexes. This review gives a historical account of NCS-1 function, examining contributions from chromaffin cells, PC12 cells and other models, to describe how NCS-1’s regulation of Ca2+ channels allows it to exert its physiological effects.

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Authors and Affiliations

  1. Department of Biology, William Paterson University, 300 Pompton Road, Wayne, NJ, 07470, USA

    Jamie L. Weiss

  2. Department of Pain Medicine, Nanshan Hospital, 89 Taoyuan Road, Nanshan, Shenzhen, 518052, China

    Hui Hui

  3. The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK

    Robert D. Burgoyne

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Correspondence to Jamie L. Weiss.

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A commentary to this article can be found at doi:10.1007/s10571-010-9611-z.

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10571_2010_9588_MOESM1_ESM.tiff

(a) Endogenous NCS-1 Staining in K+ Differentiated Chromaffin Cells. Left panel transmitted light image of living differentiated bovine adrenal chromaffin cells by K+ (55 mM) depolarization for 48 h in the presence of FBS. Bovine chromaffin cells were electroporated with DN TRPC5 plasmid and incubated for 48 h. 55 mM K+ was added into the growth media to differentiate them for 48 h prior to co-immunostaining. Cells were co-stained with anti-DBH (Dopamine β-Hydroxlase) to label chromaffin cell exocytotic vesicles (TRITC, A), anti-NCS-1 (FITC, B) and phalloidin (Alexa Flour® 350, C). D is the merged image of A, B and C, in which both NCS-1 and DBH are enriched in the cell body and the growth cone areas. E, F and G are enlarged image of growth cones from the merged areas in boxes from picture D. Arrows point to the neurite filopodia tips, which are stained with phalloidin labeling the polymerized actin. Note NCS-1 staining is not present in the filopodia tips. (b) DN NCS-1 in NGF Differentiated Double Mutant Expressing Chromaffin Cells. Left panel living differentiated bovine adrenal chromaffin cells differentiated by NGF (100 ng/ml) for 10 days in the absence of FBS. Bovine adrenal chromaffin cells were electroporated with DN NCS-1 and DN TRPC5 plasmids and incubated for 48 h. 100 ng/ml NGF was used in a FBS free growth media to differentiate them for 10 days prior to co-immunostaining. Cells were co-stained with anti-DBH (TRITC, A), anti-NCS-1 (FITC, B) and phalloidin (C). D is the merged image of A, B and C, in which both NCS-1 and DBH are enriched in the cell body and the growth cone areas. E, F and G are enlarged images of growth cones from the merged areas in boxes from picture D. Arrows point to filopodia tips to show lack of phalloidin staining (compare to (a)) and enrichment in NCS-1 and DBH staining overlay. H. Hui and J.L. Weiss, data in preparation. (TIFF 2380 kb)

10571_2010_9588_MOESM2_ESM.tiff

(a) Quantification of NCS-1 Enrichment in Tips of Differentiated PC12 Cell Filopodia in DN/DN TRPC5/NCS-1 combination. PC12 cells transfected with cDNA constructs as indicated and fluorescence intensity of NCS-1 and tubulin staining was measured from digital photos using ImageJ software. Mean ± SEM **p < 0.001 shows significant differences via a students t-test of increased ratio value compared to the other cell groups (n = 25 cells/condition). H. Hui and J.L. Weiss, data in preparation. (b) Differential effects of NCS-1 over-expression on reporter growth hormone (GH) release from PC12. Cells were transfected with control or NCS-1 plasmid along with a GH encoding plasmid. After 3 days the cells were washed and challenged with (A) 300 mM ATP or (B) 55 mM KCl. Other cells (C) were permeabilized for 6 min by incubation in 20 mM digitonin and then challenged without (basal) or with 10 mM free Ca2+. After incubation for 15 min cellular GH and GH present in the medium were assayed and released GH expressed as a percentage of total GH. The extent of release was then normalized to the mean value of release for control stimulated cells. The data are shown as mean + SEM (n = 6). M.E. Graham and R.D. Burgoyne, data in preparation. (TIFF 2380 kb)

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Weiss, J.L., Hui, H. & Burgoyne, R.D. Neuronal Calcium Sensor-1 Regulation of Calcium Channels, Secretion, and Neuronal Outgrowth. Cell Mol Neurobiol 30, 1283–1292 (2010). https://doi.org/10.1007/s10571-010-9588-7

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