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

Content Provider	PubMed Central
Author	Woehler, Andrew Lin, Kun-han Neher, Erwin
Copyright Year	2014
Abstract	Significantly more Ca2+ influx is required for eliciting release of neurotransmitter during whole cell patch clamp recording in the Calyx of Held, when gluconate with 3 mm free ATP is used as pipette filling solution, as compared to a methanesulfonate-based solution with excess Mg2+. This reduction in efficiency of Ca2+ in eliciting release is due to low-affinity Ca2+ binding of both gluconate and ATP2− anions. To study these effects we developed a simple fluorimeteric titration procedure, which reports the dissociation constant, K D, of a given Ca2+ indicator dye, multiplied by 1 plus the sum of Ca2+ binding ratios of any anions, which act as low-affinity Ca2+ ligands. For solutions without Ca2+ binding anions we find K D values for Fura2FF ranging from 11.5 ± 1.7 to 15.6 ± 7.47 μm depending on the dominant anion used. For Fura6F and KCl-based solutions we find K D = 17.8 ± 1.3 μm. 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 K D of the indicator dye is equal to that of KCl-based solutions we calculate the summed Ca2+ binding ratios and find a value of 3.55 for a solution containing 100 mm potassium gluconate and 4 mm ATP. Gluconate contributes a value of 1.75 to this number, while the contribution of ATP depends strongly on the presence of Mg2+ and varies from 0.8 (with excess Mg2+) to 13.8 (in the presence of 3 mm free ATP). Methanesulfonate has negligible Ca2+ binding capacity. These results explain the reduced efficiency of Ca2+ influx in the presence of gluconate or nucleotides, as these anions are expected to intercept Ca2+ ions at short distance.
Related Links	http://dx.doi.org/10.1113/jphysiol.2014.281097
Ending Page	4875
Page Count	13
Starting Page	4863
File Format	PDF
ISSN	14697793
e-ISSN	14697793
Journal	The Journal of Physiology
Issue Number	Pt 22
Volume Number	592
Language	English
Publisher	BlackWell Publishing Ltd
Publisher Date	2014-11-15
Access Restriction	Open
Rights Holder	BlackWell Publishing Ltd
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Physiology Sports Science

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