Cooperativity of the responses activated by photolysis of caged cyclic nucleotides

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Contribution of Ca-activated Cl channels to the
current activated by photolysis of cyclic
nucleotides in mouse olfactory sensory neurons.
Anna Boccaccio
Anna Boccaccio Anna Menini
ISAS-SISSA, Trieste
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Odor response

• Odor response cationic and anionic curent

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High chloride !!!
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From Kurahashi Menini 1997
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Aim of the work evaluate the relative
contribution to the transduction current of the
CNG and chloride current
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Experimental approach

• Dissociated mouse olfactory
• sensory neurons
• Whole-cell patch clamp configuration
• Flash photolysis of cAMP, 8-Br-cAMP, caged Ca

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8-Br-cAMP
Vholding-50 mV
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Low Ca
Vholding-50 mV
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Low Ca
Ringer
t509 ms
Vholding-50 mV
t501.9 ms
Low Ca
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Cationic current
pipette gluconateVrev,chloride -50 mV
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Direct activation of the anionic current
caged Ca DM-nitrophen
Vholding-50 mV
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Pharmacology niflumic acid
Caged 8-Br-cAMP
Caged Ca DM-nitrophen
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Summary
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• Separate anionic from cation component
• Cl contributes to most of the transduction
  current
• shapes the current
• Cl current can be directly activated by Ca (caged
  Ca)
• Cl current is not completely blocked by niflumic
  acid

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Thanks to

• Trieste Anna Menini Simone Pifferi
• Lara Masten
• Michele Dibattista Juraj Rievaj
• Laura Lagostena
• Berlin Volker Hagen

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cationic current
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8-Br-cAMP
nh5.4
Vholding-50 mV
I / Imax Fn / (Fn Fn1/2)
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Low Ca
Vholding-50 mV
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Voltage dependence
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Concentration of cyclic nucleotide
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Solutions
Bath Ringer (mM) 140 NaCl, 5 KCl, 1 CaCl2, 1
MgCl2, 10 HEPES, 15 glucose, 1 Na-pyruvate, pH
7.4. Bath Low Ca2 (mM) 140 NaCl, 5 KCl, 10
EGTA, 4 MgCl2, 10 HEPES, 15 glucose,
1 Na-pyruvate, pH 7.4. Intracellular solution
high chloride (mM) 145 KCl, 4 MgCl2, 0.5 EGTA,
10 HEPES, 1 ATP, 0.1 GTP, pH 7.4. Intracellular
solution low chloride (mM) 133 K-gluconate,
12KCl, 4 MgCl2, 0.5 EGTA, 10 HEPES, 1 ATP, 0.1
GTP, pH 7.4. Intracellular solution caged Ca
(mM) 140 KCl, 3 DM-nitrophen, 1.5 CaCl2, 10
HEPES, pH 7.4.
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Adaptation
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Low Ca
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Fast Adaptation
Low Ca
Ringer
cAMP
Vholding -50 mV
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Voltage dependence
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Aim

• Separate anionic from cation component
• Reduction in driving force by shifting the
  reversal potential (ionic substitutions) or
  changing the holding potential
• Direct activation with caged compounds, cAMP,
• 8-Br-cAMP, caged Ca
• pharmacology

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