Ratiometric monitoring of intracellular

chlorideGlyR-YFP-4
using high sensitivity
CFP-YFP Cl-Sensor

Markova Olga

Bogomoletz institute of Physiology

Mediterranean institute of neurobiology (INMED)

2006
 Some dates of development of
fluorescent methods
• 1940 Coons uses antibiotics coupled to
fluorescent dyes to detect cellular antigens.
• 1994 Chalfie and collaborators introduce green
fluorescent protein (GFP) as a marker in
microscopy.
• 1995 Marshall transfects NMDA-GFP to
visualize spatial distribution of receptor in living
cells
• 2006 Fluorescent monitoring becomes one of
the most popular methods in physiology
 Family of GFP proteins
Mutants of GFP

• Yellow fluorescent
protein (YFP)

• Cyan fluorescent
protein (CFP)
The green fluorescent protein (GFP)
is a protein from the
jellyfish Aequorea victorea that
fluorescent green when exposed to blue light.
 F.R.E.T.
Fluorescence resonance energy transfer

FRET is a distance-dependent
interaction between the electronic
excited states of two dye
molecules in which excitation is
transferred from a donor molecule
to an acceptor molecule without
emission of a photon.
 Chloride functions
• Main vehicle of synaptic inhibition in mature neurons
• Developmental switch from excitation to inhibition
• A switch between inibition and excitation during
sustained activation of GABA receptors
• Regulation of transduction in olfactory neurons,
astrocytes and hypothalamic neurons
• Regulation of cell volume
• Regulation of intracellular pH
• Regulation of fluid secretion
• Stabilization of membrane resting potential
Human diseases due to dysfunction
of Cl homeostasis
• Cystic fibrosis
• Myotonia congenita
• Inherited hypercalciuric nephrolitiasis
• Batter syndrome
• Hyperekplexia/startle disease
 Fluorecent dyes for CI monitoring
Ratio measurements Ratiometric
are not feasible
• SPQ 6-Methoxy-N-(3- • Cl-sensor (Kd 70mM)
sulfopropyl)quinolinium excitation ratio
• MQAE N- • Clomeleon (Kd 160mM)
(Ethoxycarbonylmethyl)-6- emission ratio
methoxyquinolinium bromide
• MEQ 6-Methoxy-N-
ethylquinolinium iodide
• Lucigenin
• Cl-sensitive EYFP
 Fluorecent dyes for CI monitoring
Ratio measurements Ratiometric
are not feasible
• SPQ 6-Methoxy-N-(3- • Cl-sensor (Kd 70mM)
sulfopropyl)quinolinium excitation ratio
• MQAE N- • Clomeleon (Kd 160mM)
(Ethoxycarbonylmethyl)-6- emission ratio
methoxyquinolinium bromide
• MEQ 6-Methoxy-N-
ethylquinolinium iodide
• Lucigenin
• Cl-sensitive EYFP
Chemical structure of CFP/YFP-
based Cl-sensor
 Third mutation of EYFP

Luis J. V. FEBS Letters 2001

Construction of DNA vector
 Expression of Cl-sensor
HEK-293 Hippocampal neuron

10 µm 20 µm
Spectra of Cl-sensor
445nm 485nm
Diffusional exchange of Cl with
patch pipette
 Experimental procedure
• Co-transfection of CHO cells
Cl-sensor + GlyR

• Application of Glycine at different holding

potentials with simultaneous fluorescent
and electrophysiological recordings
Registration of intracellular Cl

300 mM
Whole
cell
130 mM

50 mM
50 mM 130 mM 300 mM
Estimation of IC50
 Cl in CHO cells

20mM
Cl distribution in neuron
Cl distribution in neuron
 Scheme of Bio-Sensor:
Cl-Sensor incorporated in glycine
receptor

A GlyR
B Bio-Sensor

SS
S
S

S
S
S
S

4 4
1 2 2 1

Cl­Sensor
 Expression in HEK cells
Cl - sensor Cl-sensor - GlyR

GlyR-YFP-4

10 µm 10 µm
Bio-sensor responses to Glycine
application
Vh Vh
-30mV -60mV
 Summary
• Estimation IC50 of new highly sensitive Cl-
sensor
• Measurement of Cl distribution in CHO
cells
• Simultaneous registration fluorescent
changes and glycinergic currents
 What we are planning to do in the
future:
Technical tasks
• Visualisation of Cl currents living cells
(synapses)
• Transfection of Cl-sensor into slices
Biological tasks
• Study of Cl changes in epileptical tissues
 Acknowledgments INMED

INMED, Marseille
• Piotr Bregestovsky
• Igor Medina
• Ilona Chudotvorova Piotr Igor

Institut Pasteur, Paris

• Yves Jacob
• Eleonore Real Yves Jacob
Ilona
(Institut Pasteur,
Paris)
Ville de Marseille