SPECIAL COMMUNICATION
Using gadolinium to identify stretch-activated channels: technical considerations

Ray A. Caldwell1, Henry F. Clemo1,2, and Clive M. Baumgarten1

Departments of 1 Physiology and 2 Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298

    ABSTRACT
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Abstract
Introduction
Results & Discussion
References

Gadolinium (Gd3+) blocks cation-selective stretch-activated ion channels (SACs) and thereby inhibits a variety of physiological and pathophysiological processes. Gd3+ sensitivity has become a simple and widely used method for detecting the involvement of SACs, and, conversely, Gd3+ insensitivity has been used to infer that processes are not dependent on SACs. The limitations of this approach are not adequately appreciated, however. Avid binding of Gd3+ to anions commonly present in physiological salt solutions and culture media, including phosphate- and bicarbonate-buffered solutions and EGTA in intracellular solutions, often is not taken into account. Failure to detect an effect of Gd3+ in such solutions may reflect the vanishingly low concentrations of free Gd3+ rather than the lack of a role for SACs. Moreover, certain SACs are insensitive to Gd3+, and Gd3+ also blocks other ion channels. Gd3+ remains a useful tool for studying SACs, but appropriate care must be taken in experimental design and interpretation to avoid both false negative and false positive conclusions.

mechanosensitive channels; mechanoelectrical feedback; lanthanides; chelation

    INTRODUCTION
Top
Abstract
Introduction
Results & Discussion
References

THE IDENTIFICATION OF stretch-activated channels (SACs) in bacteria, plant, and animal cells has led to intense efforts to elucidate their physiological and pathophysiological roles (9, 27, 32). SACs are implicated in a wide range of responses to mechanical perturbations, including cell volume regulation, increased intracellular Ca2+, cell proliferation, gene expression, DNA synthesis, baroreceptor discharge, altered cardiac electrical activity, and release of atrial natriuretic factor (for review, see Ref. 9).

Gadolinium (Gd3+), a trivalent lanthanide, has emerged as the most commonly used tool to identify phenomena dependent on SACs (9). Millet and Pickard (25) originally postulated that Gd3+ blocks mechanosensitive ion channels on the basis of its ability to inhibit orientation of the roots of pea plants (Zea mays) in response to surface contact, thigmotropism, and gravity, geotropism. Direct evidence was provided by Yang and Sachs (41) who showed that Gd3+ blocks stretch-activated cation channels in Xenopus laevis oocytes. Often, 10 µM Gd3+ is sufficient to largely block cation SACs and thereby inhibit mechanosensitive processes (9, 27, 32). By extension, the inability of Gd3+ to modulate certain stretch-dependent events has been taken to imply that SACs are not involved.

The purpose of this communication is to highlight several methodological concerns. A review of recent literature indicates that Gd3+ sometimes is applied in the presence of anions that avidly bind free Gd3+ and effectively remove it from the experimental solution (4, 11, 13-16, 20-23, 29, 31, 33, 40). Notable among these anions are phosphate, carbonate, EGTA, sulfate, carboxylic acids, and albumin, which often are contained in physiological salt solutions and culture media. The use of Gd3+ with anions that avidly bind it can lead to false negative conclusions regarding the role of SACs in physiological processes. False negatives also can arise because several SACs are not blocked by Gd3+ (34, 41). On the other hand, false positives can arise because Gd3+ is not very specific (9).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Results & Discussion
References

Binding of Gd3+. Martell and Smith (24) and Evans (7) have provided critically reviewed stability constants for the interaction of Gd3+ with a number of inorganic anions, carboxylic acids, and amine polycarboxylic acid chelators (e.g., EGTA). Selected constants are listed in Table 1. For example, the equilibrium dissociation constants for PO3-4 and CO2-3 are given as 10-22.3 and 10-32.2, respectively. These values indicate that physiological anions are high-affinity ligands for Gd3+.

                              
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Table 1.   Selected dissociation constants for Gd3+ salts with commonly used anions

Calculation of the free Gd3+ concentration in phosphate- or bicarbonate-buffered physiological solutions is straightforward because Gd3+ binds with high affinity, there is a large excess of anionic ligand, pH is set at 7.4, and total phosphates or carbonates are known. At pH 7.4, the vast majority of inorganic phosphates and carbonates are not present in the forms that bind Gd3+ avidly, PO3-4 and CO2-3, but rather are present as protonated species.

To calculate the free Gd3+ concentration, the distribution of anionic species first was calculated from the apparent acidic dissociation constant (pKa) values and solubility of CO2 (Q'CO2) at an ionic strength of 0.16 M and 25°C, assuming the experimental solutions contained either 1 mM total inorganic phosphate or were equilibrated with 5% CO2. For carbonates, the adopted pKa values were 6.22 and 9.94 (30), and Q'CO2 was taken as 0.0331 mM/mmHg (5). Gassing with 5% CO2 gives a PCO2 of 36.8 mmHg in the face of a water vapor pressure of 23.8 mmHg (39). For phosphates, the pKa values were 2.02, 6.81, and 11.68 (30). The interaction of HPO2-4 and Na+ was accounted for by using a dissociation constant of 10-0.6 (24) and assuming Na+ in the experimental solutions was 130 mM. The dissociation constant for K+, 10-0.49 (24), is similar to that of Na+, and for present purposes, K+ was not considered separately.

Although the calculated concentrations of PO3-4 and CO2-3 at pH 7.4 are very low under these conditions, 2.96 × 10-8 and 5.3 × 10-5 M, respectively, the large excess of phosphates and carbonates with respect to Gd3+ assures that mass action will maintain PO3-4 and CO2-3 virtually constant as the unprotonated species bind to Gd3+. With PO3-4 and CO2-3 known, the free Gd3+ concentration was directly calculated for 10 µM total Gd3+ from the equilibrium constant (Table 1). The calculations indicate that free Gd3+ is only ~2 × 10-20 M in the presence of phosphate and ~6 × 10-13 M in the presence of a bicarbonate buffer system. For simplicity, the Gd3+ dissociation constants in Martell and Smith (24) were not corrected for ionic strength. Moreover, ~10% of free Gd3+ was in the form GdOH2+ due to hydrolysis (30). Thus these calculations should be regarded as order of magnitude estimates only. Nevertheless, the calculated values are sufficiently accurate to show that the free Gd3+ concentration is vanishingly low in the presence of certain physiological anions. Therefore, failure to detect an effect of Gd3+ in phosphate- or bicarbonate-buffered solutions does not by itself provide credible evidence regarding the role of SACs.

Several studies on Ca2+ currents and muscle contraction previously led to the qualitative conclusion that phosphate and bicarbonate buffer systems interfered with the action of Gd3+ (3, 19, 38) and reduced the potency of Gd3+ as a blocker of SACs (9). However, the extent of the interaction between Gd3+ and these anions apparently was not fully appreciated, and calculations of the free Gd3+ concentration from equilibrium constants in the literature were not made.

Available evidence suggests that HEPES, PIPES, MOPS, and imidiazole are appropriate buffers for use with lanthanides because the affinity of lanthanides for organic sulfate groups and nitrogen donors is very weak (7). On the other hand, both TES and Tris interact with lanthanides to some extent, and phosphate and bicarbonate systems should be strenuously avoided.

Although the present discussion has focused on phosphate and bicarbonate, the equilibrium constant for EGTA (Table 1) indicates that it also should not be included in solutions designed to evaluate the effect of Gd3+. Failure to recognize this interaction may confound aspects of the interpretation of several recent studies (14-16, 23, 31). The consequence of inclusion of anions that bind Gd3+ with lower affinity is less clear, and case-by-case calculations of free Gd3+ concentration are necessary.

The concern that binding of Gd3+ to anions can lead to false negative conclusions is based on the premise that free Gd3+ is the species responsible for blocking SACs. This presumption has not been evaluated in detail, and the possibility that certain Gd3+-anion complexes can block SACs and other ion channels cannot be rigorously excluded. Such an interaction may in part explain several studies reporting effects of Gd3+ in the presence of phosphate and bicarbonate (10, 12, 35, 36).

Other sources of error. False negative conclusions also can arise because some SACs have been shown to be insensitive to Gd3+ in studies conducted in the absence of anions with high affinity for this lanthanide. Among the Gd3+-insensitive SACs are K+-selective SACs in rat astrocytes (41) and snail (Lymnaea) neurons (34).

A final concern in using Gd3+ as a marker for SACs is that false positive findings may emerge because Gd3+ is not a specific antagonist. Besides SACs, Gd3+ can block L-type (1, 17, 18, 33), T-type (1, 26), and N-type Ca2+ (2, 3), Na+(6), K+ (6, 12), and Ca2+-activated Cl- (37) channels, as well as purine P2X channels (28) and muscarinic receptor-mediated Ca2+ transients (8), at concentrations that may overlap those used to block SACs. Thus it is essential to confirm a positive response to Gd3+ with other experimental paradigms before concluding that SACs are responsible for the process under study.

As in all methods, the use of Gd3+ to identify processes that involve SACs has limitations. In this case, the limitations include the chemical interactions of Gd3+ with physiological anions, its imperfect selectivity, and the presence of Gd3+-insensitive SACs in some preparations. Recognition of these limitations allows the experimentalist to draw correct inferences.

    ACKNOWLEDGEMENTS

We thank Dr. Joseph J. Feher for comments on the manuscript.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-46764 and HL-02798.

Address for reprint requests: C. M. Baumgarten, Dept. of Physiology, Medical College of Virginia, Richmond, VA 23298-0551.

Received 19 November 1997; accepted in final form 29 April 1998.

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Abstract
Introduction
Results & Discussion
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