Measurement of the limiting equivalent conductivities and mobilities of the most prevalent ionic species of EGTA (EGTA2− and EGTA3−) for use in electrophysiological experiments

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Abstract

In many experimental biological situations, chelating agents like EGTA (ethylene glycol-bis-(β-amino-ethyl ether) N,N,N′,N′-tetra-acetic acid) are commonly used to control or suppress the concentration of divalent ions like Ca2+. The evaluation of liquid junction potentials in electrophysiological measurements, and particularly in patch-clamp situations, requires information about the ions within the solution. Where there is a significant concentration of EGTA present, it is necessary to know the values of the relative mobility of at least the most predominant ionic species of EGTA in order to complete these calculations. EGTA, with four negative charges with different pKas, can therefore exist as four differently charged ions in solution (EGTA−, EGTA2−, EGTA3− and EGTA4−) or as uncharged, although between pH 5.5 and 8 it is almost exclusively EGTA2−. We have measured limiting equivalent conductivities of the most common ionic forms of EGTA (EGTA2−and EGTA3−) encountered at physiological pHs. These were 35.9±0.7 and 56±2.5 S cm2 equiv−1 respectively. Their mobilities relative to K+ were 0.24±0.01 for EGTA2− and 0.25±0.01 for EGTA3−. Thus for typical electrophysiological solutions, the contribution of EGTA to the liquid junction potential should be small (e.g. ∼0.4 mV).

Introduction

Liquid junction potentials arise whenever two solutions of different ionic composition or concentration are in contact and can significantly contribute errors to the measurements of membrane potential, unless appropriate corrections for them are applied. In patch-clamp and bilayer situations especially, liquid junction potential corrections are often up to 10 mV or even more (see discussion in Barry and Lynch, 1991, Neher, 1992 and Neher (1994) and Ng and Barry (1995)). These junction potential corrections can either be estimated by measurement (Neher, 1992, Neher, 1994), although even these measurements need additional corrections (Barry and Diamond, 1970), or they can simply be calculated directly from a knowledge of (or at least estimates of) the ionic mobilities of the ions present in the solutions at any significant concentration. These mobilities can be determined from limiting equivalent conductivity data (e.g. see data in Robinson and Stokes, 1965, Dean, 1992, Vanysek, 1995). Relative mobilities of the more common ions have been published (Zuidema et al., 1985, Barry and Lynch, 1991) and more recently the mobilities of some less common, but now frequently used, organic ions have also been measured (Ng and Barry, 1995). These values have now been incorporated into a program, JPCalc, that has been developed Barry (1994) together with a full Windows version, JPCalcW, produced in conjunction with Axon Instruments, Foster City, CA, USA) to calculate junction potentials for a full range of patch-clamp and other electrophysiological situations using the generalised Henderson equation (Morf, 1981, Barry and Lynch, 1991, Ng and Barry, 1995).

The limiting equivalent conductivities and mobilities of the ionic species contributed by EGTA (ethylene glycol-bis-(β-amino-ethyl ether) N,N,N′,N′-tetra-acetic acid; C14H24N2O10), however, have not been measured, in spite of the fact that EGTA is very widely used to control or suppress the concentrations of divalent ions like Ca2+, though generally used at a reasonably low concentration. EGTA has four possible negative charges with different pKas, and can therefore potentially exist as four different ions (EGTA1−, EGTA2−, EGTA3− and EGTA4−) or in uncharged form (see Table 1). Hence it is not simple to calculate its contribution to a junction potential and one must first know the relative proportions of the EGTA ions.

The aim of this paper was to measure the limiting equivalent conductivities of the EGTA species most prevalent at physiologically relevant pHs (EGTA2− and EGTA3−) and to calculate the mobility of each species relative to K+. The experimentally obtained values could then be used to predict the liquid junction potentials generated by simple EGTA containing solutions and be compared to experimentally measured values using these solutions.

Section snippets

Theoretical background

The molal conductivity, Λm, of a solution (the conductivity/molal concentration) is related to the molal concentration, m, of solute according to the Onsager relationship (MacInnes, 1961):Λm=Λom+smwhere Λom is the limiting molal conductivity as the salt concentration tends to zero and s is the slope of the line (s being independent of concentration), and:Λo=Λom/zwhere Λo is the limiting equivalent conductivity and z is the ionic valency.

Preparation of solutions

CaCl2 was obtained from UNIVAR-AJAX (Auburn, NSW, Australia) and NaCl and NaOH from BDH (Kilsyth, VIC, Australia). EGTA was obtained from Sigma (St. Louis, MO, USA). All reagents were at least of analytical grade and the solutions were prepared using double demineralised water as the solvent.

The 10 mM EGTA solution was prepared and pH adjusted with a stock solution of 1 mol l−1 NaOH. For EGTA solutions at a pH of 6, dilutions of 1, 3 and 5 mmol kg−1 H2O were prepared using the 10 mmol kg−1 H2O

Results and calculations

As already mentioned, the procedure was to initially measure limiting molal conductivities for the NaEGTA solutions at two pH values: at a pH of about 6 which was almost exclusively only contributed to by EGTA2− and precisely at pH 8.80 at which there was a known fraction of EGTA2− and EGTA3− and virtually no other EGTA ionic species. From these measurements, the limiting equivalent conductivity of Na+ would first be subtracted, the limiting molal conductivity of EGTA determined and finally the

Discussion

This paper has reported measurements of the limiting equivalent conductivities and relative mobilities of the most prevalent ionic species of EGTA, EGTA2− and EGTA3−, in the typical physiological pH range. It has also clearly demonstrated that such measurements for pH-dependent polyvalent ions like EGTA are by no means trivial exercises and require considerable care and theoretical underpinning. It should be noted that for a chelating agent like EDTA, the experimental measurement of its

Acknowledgements

We would particularly like to express our appreciation to Professor Donald Bers of Loyola University, Chicago, for discussions and reading through the original draft of the theoretical section on the calculations of the prevalencies of the different ionic species of EGTA and for similar discussions with Professor George Stephenson of Latrobe University, Australia. The help of Elaine Bonnet was also much appreciated, as was the support of the Australian Research Council and the National Health

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