In:
The Journal of Physiology, Wiley, Vol. 592, No. 22 ( 2014-11-15), p. 4863-4875
Abstract:
The efficiency of neurotransmitter release is influenced by the presence of Ca 2+ binding molecules. In this study we show that the use of gluconate as the dominant anion in pipette filling solutions, together with the presence of ATP and GTP, influences the efficiency of Ca 2+ ‐mediated release during whole cell patch clamp measurements in the Calyx of Held. We introduce a novel fluorimetric titration procedure for low‐affinity calcium dyes, such as Fura2FF, that allows us to determine the calcium binding ratios of several commonly used pipette solutions. These results explain the influence of gluconate and nucleotides on neurotransmitter release, provide guidelines for calibrating pipette solutions and calcium indicators, and may provide a basis for studying the coupling between calcium sensors and sources. 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 3 m m 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, K D , 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 K D values for Fura2FF ranging from 11.5 ± 1.7 to 15.6 ± 7.47 μ m depending on the dominant anion used. For Fura6F and KCl‐based solutions we find K D = 17.8 ± 1.3 μ m . 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 Ca 2+ binding ratios and find a value of 3.55 for a solution containing 100 m m potassium gluconate and 4 m m 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 3 m m 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.
Type of Medium:
Online Resource
ISSN:
0022-3751
,
1469-7793
DOI:
10.1113/jphysiol.2014.592.issue-22
DOI:
10.1113/jphysiol.2014.281097
Language:
English
Publisher:
Wiley
Publication Date:
2014
detail.hit.zdb_id:
1475290-6
SSG:
12
