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  Calcium-buffering effects of gluconate and nucleotides, as determined by a novel fluorimetric titration method.

Woehler, A., Lin, K. H., & Neher, E. (2014). Calcium-buffering effects of gluconate and nucleotides, as determined by a novel fluorimetric titration method. Journal of Physiology, 592(22), 4863-4875. doi:10.1113/jphysiol.2014.281097.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0024-42B9-6 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-EE6D-6
Genre: Journal Article

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Woehler, A.1, Author              
Lin, K. H.2, Author              
Neher, E.1, Author              
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1Emeritus Group of Membrane Biophysics, MPI for Biophysical Chemistry, Max Planck Society, ou_1571137              
2Research Group of Activity-Dependent and Developmental Plasticity at the Calyx of Held, MPI for biophysical chemistry, Max Planck Society, ou_578581              

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 Abstract: Significantly more Ca(2+) influx is required for eliciting release of neurotransmitter during whole cell patch clamp recording in the Calyx of Held, when gluconate with 3mm free ATP is used as pipette filling solution, as compared to a methanesulfonate-based solution with excess Mg(2+). This reduction in efficiency of Ca(2+) in eliciting release is due to low-affinity Ca(2+) binding of both gluconate and ATP(2-) anions. To study these effects we developed a simple fluorimeteric titration procedure, which reports the dissociation constant, KD, of a given Ca(2+) indicator dye, multiplied by 1 plus the sum of Ca(2+) binding ratios of any anions, which act as low-affinity Ca(2+) ligands. For solutions without Ca(2+) binding anions we find KD values for Fura2FF ranging from 11.5±1.7 to 15.6±7.47mum depending on the dominant anion used. For Fura6F and KCl-based solutions we find KD=17.8±1.3mum. For solutions with gluconate as the main anion and for solutions that contain nucleotides, such as ATP and GTP, we find much higher values for the product. Assuming that the KD of the indicator dye is equal to that of KCl-based solutions we calculate the summed Ca(2+) binding ratios and find a value of 3.55 for a solution containing 100mm potassium gluconate and 4mm ATP. Gluconate contributes a value of 1.75 to this number, while the contribution of ATP depends strongly on the presence of Mg(2+) and varies from 0.8 (with excess Mg(2+)) to 13.8 (in the presence of 3mm free ATP). Methanesulfonate has negligible Ca(2+) binding capacity. These results explain the reduced efficiency of Ca(2+) influx in the presence of gluconate or nucleotides, as these anions are expected to intercept Ca(2+) ions at short distance.

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Language(s): eng - English
 Dates: 2014-10-022014-11-15
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1113/jphysiol.2014.281097
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Title: Journal of Physiology
Source Genre: Journal
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Pages: - Volume / Issue: 592 (22) Sequence Number: - Start / End Page: 4863 - 4875 Identifier: -