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

Spencer D. Watts, Katherine L. Suchland, Susan G. Amara, Susan Ingram

Research output: Contribution to journalArticle

7 Citations (Scopus)

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 (k 50 = 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 GABA A agonist 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.

Original languageEnglish (US)
Article numbere35373
JournalPLoS One
Volume7
Issue number4
DOIs
StatePublished - Apr 10 2012

Fingerprint

biosensors
Biosensing Techniques
Biosensors
Neurons
Chlorides
Mesencephalon
chlorides
neurons
Membranes
Monitoring
monitoring
Fluorescence
brain
dopamine
Dopamine Plasma Membrane Transport Proteins
fluorescence
Cell membranes
transporters
Cell Membrane
Lipoylation

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

A sensitive membrane-targeted biosensor for monitoring changes in intracellular chloride in neuronal processes. / Watts, Spencer D.; Suchland, Katherine L.; Amara, Susan G.; Ingram, Susan.

In: PLoS One, Vol. 7, No. 4, e35373, 10.04.2012.

Research output: Contribution to journalArticle

Watts, Spencer D. ; Suchland, Katherine L. ; Amara, Susan G. ; Ingram, Susan. / A sensitive membrane-targeted biosensor for monitoring changes in intracellular chloride in neuronal processes. In: PLoS One. 2012 ; Vol. 7, No. 4.
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