Link to the University of Pittsburgh Homepage
Link to the University Library System Homepage Link to the Contact Us Form

A sensitive membrane-targeted biosensor for monitoring changes in intracellular chloride in neuronal processes

UNSPECIFIED (2012) A sensitive membrane-targeted biosensor for monitoring changes in intracellular chloride in neuronal processes. PLoS ONE, 7 (4).

[img]
Preview
PDF
Published Version
Available under License : See the attached license file.

Download (3MB) | Preview
[img] Plain Text (licence)
Available under License : See the attached license file.

Download (1kB)

Abstract

Background: Regulation of chloride gradients is a major mechanism by which excitability is regulated in neurons. Disruption of these gradients is implicated in various diseases, including cystic fibrosis, neuropathic pain and epilepsy. Relatively few studies have addressed chloride regulation in neuronal processes because probes capable of detecting changes in small compartments over a physiological range are limited. Methodology/Principal Findings: In this study, a palmitoylation sequence was added to a variant of the yellow fluorescent protein previously described as a sensitive chloride indicator (YFPQS) to target the protein to the plasma membrane (mbYFPQS) of cultured midbrain neurons. The reporter partitions to the cytoplasmic face of the cellular membranes, including the plasma membrane throughout the neurons and fluorescence is stable over 30-40 min of repeated excitation showing less than 10% decrease in mbYFPQS fluorescence compared to baseline. The mbYFPQS has similar chloride sensitivity (k50= 41 mM) but has a shifted pKa compared to the unpalmitoylated YFPQS variant (cytYFPQS) that remains in the cytoplasm when expressed in midbrain neurons. Changes in mbYFPQS fluorescence were induced by the GABAAagonist muscimol and were similar in the soma and processes of the midbrain neurons. Amphetamine also increased mbYFPQS fluorescence in a subpopulation of cultured midbrain neurons that was reversed by the selective dopamine transporter (DAT) inhibitor, GBR12909, indicating that mbYFPQS is sensitive enough to detect endogenous DAT activity in midbrain dopamine (DA) neurons. Conclusions/Significance: The mbYFPQS biosensor is a sensitive tool to study modulation of intracellular chloride levels in neuronal processes and is particularly advantageous for simultaneous whole-cell patch clamp and live-cell imaging experiments. © 2012 Watts et al.


Share

Citation/Export:
Social Networking:
Share |

Details

Item Type: Article
Status: Published
Contributors:
ContributionContributors NameEmailPitt UsernameORCID
EditorNitabach, Michael N.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Date: 10 April 2012
Date Type: Publication
Journal or Publication Title: PLoS ONE
Volume: 7
Number: 4
DOI or Unique Handle: 10.1371/journal.pone.0035373
Schools and Programs: School of Medicine > Neurobiology
Refereed: Yes
Other ID: NLM PMC3323644
PubMed Central ID: PMC3323644
PubMed ID: 22506078
Date Deposited: 24 Sep 2012 20:03
Last Modified: 07 Jan 2019 21:55
URI: http://d-scholarship.pitt.edu/id/eprint/14168

Metrics

Monthly Views for the past 3 years

Plum Analytics

Altmetric.com


Actions (login required)

View Item View Item