Quarterly Journal of Experimental Physiology (1986) 71, 467-473
Printed in Great Britain

FREE INTRACELLULAR MAGNESIUM
CONCENTRATION IN FERRET VENTRICULAR
MUSCLE MEASURED WITH ION SELECTIVE
MICRO-ELECTRODES
L. A. BLATTER AND JOHN A. S. McGUIGAN
Department of Physiology, University of Berne, Buehlplatz 5, 3012 Berne, Switzerland
(RECEIVED FOR PUBLICATION 2 AUGUST 1985)

SUMMARY

The free Mg concentration ([Mg]i) was measured in ferret ventricular muscle using singlebarrelled Mg-sensitive micro-electrodes. The electrodes were calibrated in solutions containing the [K]i and [Na]i measured in this tissue. In thirteen measurements from seven experiments conforming to strict criteria for calibration and penetration the estimated mean [Mg]i was
04 mmol/l. This is to be regarded as an upper limit since it was estimated by linear extrapolation of the calibration curve.
INTRODUCTION

Ionized Mg plays an essential role in the regulation of a large number of cellular functions.
It is a co-factor in many enzymatic reactions, influences tension development in muscle and modulates amongst others the ionic transport systems in mitochondria (see reviews by
Gunther, 1977; Shine, 1979; and Ackerman & Nicholls, 1983). Despite its importance in cellular mechanisms, estimations of the free Mg ([Mg]i) by various methods range from
0- 1 mmol/l to 6 mmol/l (reviews: Flatman, 1984; Gupta, Gupta & Moore, 1984) mainly because of the lack of a simple direct method for its measurement.
Direct measurement of [Mg]i is now possible using micro-electrodes filled with the Mg resin described by Lanter, Erne, Ammann & Simon (1980) and measurements have been carried out in both cardiac and skeletal muscle (Hess, Metzger & Weingart, 1982; Lopez,
Alamo, Caputo, Vergara & DiPolo, 1984), and in nerve cells of Helix aspersa (AlvarezLeefmans, Gamifno & Rink, 1984). This resin has however, the disadvantage that it reacts to other cations. Ca and H present no problem since intracellularly their concentrations are less than 1,umol/l. The main intracellular interference comes from Na and K ions.
This paper presents measurements of [Mg]i using Mg-sensitive micro-electrodes in ferret ventricle a tissue in which the intracellular concentrations of both Na and K have been previously measured (Na, Chapman, Coray & McGuigan, 1983; K, Reverdin, Illanes &
McGuigan, 1986). The mean value of [Mg]i found in these experiments was 0 4 mmol/l.
METHODS

General
The preparation, mounting, tension measurement and recording from the ferret ventricular trabeculae were identical to that described in the preceding paper (Reverdin et al. 1986). The experiments were carried out at room temperature (24 °C).

L. A. BLATTER AND J. A. S. McGUIGAN

468

B
0

A
Before
0 ;, -10 -

E

-20L'

Tyrode

Tyrode

2

1

05

4

[MgJmmoll

_

[Nal = 14-6 mmol/I
[K] = 140-5 mmol/I

-5
-10 _

10 mm

-15
After
-10
> -20
-30 L

~~~~~~~~~~~~-20

0 -

Tyrode
05

1

2

...........
10 mm

4

[Mg]mmol/l

-25

L__,,_____.........
0-1

05 1

2 4

10

[Mg] (mmol/l)

Fig. 1. A, calibration of Mg micro-electrode before and after an intracellular impalement. UMg measured in Tyrode is defined as zero mV. B, calibration curve of the electrode. The mean value of UMg is plotted against log [Mg].

Mg-sensitive micro-electrodes
Manufacture. The electrodes were prepared as described in Chapman et al. (1983). It was found that washing and hydration did not influence the performance of the electrodes so this procedure was discontinued. The micro-electrodes were filled with the liquid Mg sensor, based on the neutral ligand
ETH 1117 (Lanter et al. 1980), backfilled with 100 mmol MgCl2/l and then stored by immersing their tips in 100 mmol MgCl2/l for a few days. This improved the performance of the electrodes. They had a tip diameter of less than 1 ,um (visual control) and a resistance of 20-30 GfQ.

Calibration. Calibration was carried out differentially between a 3 mol KCl/l electrode and the Mg micro-electrode in the chamber described in the preceding paper. The composition of the calibrating solutions was in mmol/l: KCI, 138-0; KOH, 2-5; NaCl, 14 6; Hepes, 5; and MgCl2, 0 5, 1, 2, or 4.
The solutions were titrated to pH 7-4 with 1 mol HCl/l.
The value of 140 5 mmol/l for [K]i was taken from the average of earlier K measurements.
In all experiments calibration was carried out before and after impalement and sufficient time was allowed for the calibration curve to reach a steady state. Typical calibration curves before and after an impalement are shown in Fig. 1 A. Fig. 1 B shows a plot of the magnesium potential against the log [Mg].
As mentioned in the introduction the resin ETH 11 17 also reacts to Ca ions and to test if the Ca contamination in the Mg solutions affected the calibration, we compared the behaviour of five electrodes in calibrating solutions, nominally Ca-free and in calibrating solutions containing
0-1 mmol EGTA/1. No difference was found and EGTA was not added routinely to the solutions.
These findings are similar to those of Hess et al. (1982), Alvarez-Leefmans et al. (1984), and Lopez et al. (1984).
Solutions. These are described in the preceding paper. MgCl2 was added to the calibrating solutions from a 1 mol/l stock obtained from BDH Chemical Ltd, Poole, England. In the 14 mmol/l K-HEPES
Tyrode Na was reduced from 155 mmol/l to 146 mmol/l. Mg cocktail was initially obtained as a gift from Dr Ammann, ETH Zurich. It was later bought from Fluka AG, 9470 Buchs, Switzerland.
Terminology. The membrane potential is designated as Em, the signal of the Mg micro-electrode
VMg and the Mg signal corresponding to the [Mg]i, UMg. Since the Mg micro-electrode measures both
Em and UMg, VMg = Em+ UMg.
The Mg micro-electrodes measure Mg activity but since there is no general agreement as to the calculation ofthe activity coefficient of a divalent ion, the results have been expressed as corresponding to the Mg concentration in the calibrating solutions.

[Mg]i

IN FERRET VENTRICLE

469

L

0

-25

2 -100

~~~__j ___

[K ]o= 14 mmol/I
Fig. 2. From above downwards are shown the membrane potential Em, the Mg signal UMg and the signal of the
Mg-sensitive micro-electrode VMg. The arrow (middle trace) marks the end of a 22 min recording gap. During this time the Mg micro-electrode drifted down by 3 mV. Em, - 76 4 mV, UMg - 19-4 mV, and VMg - 95 8 mV.
Preparation diameter 350,um.

Fmin

RESULTS

As shown in Fig. 1 B the difference between a concentration of 0-5 mmol/l and 4 mmol/l is around 6 mV which means that a few mV in the UMg signal can make a large difference in the [Mg] estimated from the calibration curve. Thus, in order to ensure adequate calibration and penetration of the electrodes the following criteria were adapted for each impalement: (1) The calibration curves before and after impalement had to differ by 1 mV or less. (2) The drift of the micro-electrodes during the experiment had to be less than
0-25 mV/min. (3) Sufficient time was allowed for the Mg-sensitive micro-electrode to reach a plateau value. This could take up to 30 min. (4) As a test for adequate impalement of both electrodes the trabecula was depolarized by increasing the external K concentration
([K]0) from 5 mmol/l to 14 mmol/l. A deviation on the difference signal (UMg) of less than
1 mV was deemed acceptable. (5) The membrane potential (3 mol KCI/l electrode) had to be more negative than -74 mV. This is within one half of a S.D. of the mean value of
-78 mV (Chapman et al. 1983).
From twenty experiments, thirteen measurements in seven experiments met all these criteria. A typical example of such a measurement is shown in Fig. 2. The gap in the three curves corresponds to a time lapse of 22 min. During this time the Mg micro-electrode slowly sealed into the cell, the potential increasing by around 3 mV. Depolarization by increasing
[K]0 from 5 mmol/l to 14 mmol/l caused no deviation on the UMg trace. The [Mg]i in this experiment was 0 87 mmol/l.
It must be emphasized that a depolarizing test was carried out for every impalement.
Despite the appearance of an adequate impalement of both electrodes in some experiments, the depolarization test showed that at least one of the electrodes was not adequately in the cell. Such experiments were discarded.
A histogram ofthe thirteen measurements is shown in Fig. 3 A. Seven of the measurements were below 0-5 mmol/l and the largest value measured was 1.9 mmol/l. The mean value + S.D. ofEm, UMg, and VMgwererespectively, -77 7 mV + 2-1 mV, -21 7 mV + 2 3 mV and -99 4 mV+3 4 mV.

470

L. A. BLATTER AND J. A. S. McGUIGAN
B

A

0
7

6~~~~~

4
4O

0

-20

3

~~~~UMg -21-7

z

1
0-

-30

"-

,

>

v~~~

e

0.1

0-5

1

2

4

10

[Mg]i mmol/l
[Mg]i mmol/l
Fig. 3. A, histogram of the thirteen measurements from seven experiments. B, average calibration curve of the thirteen measurements (mean+ S.D.). The average Umg signal of -21-7 mV corresponds to a mean [Mg], of
0-4 mmol/l.

More than half of the measurements were less than 0 5 mmol/l and in order to arrive at a mean value for [Mg]i the thirteen calibration curves were averaged and the mean + S.D. for the mean curve is given in Fig. 3B. The mean value of UMg (-21-7 mV) corresponds to a mean [Mg]i of just over 0-4 mmol/l.
DISCUSSION

Micro-electrode measurements of [Mg]i
Previous measurements of [Mg]i in heart and skeletal muscle using micro-electrodes (Hess et al. 1982; Lopez et al. 1984) have given values of between 3 and 4 mmol/l. There are at least two reasons why these authors measured higher values than those reported in this paper. Firstly, the Mg resin reacts to both Na and K in the cell and the difference this can make to the estimation of the [Mg]i is demonstrated in Fig. 4. This gives a comparison of the calibration curves in two background solutions, namely the solutions, based on measured values as used in this paper, and the values assumed by Hess et al. (1982). A value of
0-4 mmol/I for [Mg]i with our calibrating solutions would correspond to a value of
2 1 mmol/l with the solutions of Hess et al. (1982).
The second reason is that it could take up to 30 min for the Mg micro-electrode to seal into the cell. This is shown in Fig. 2 and while initially a plateau appeared to have been reached, with time the Mg micro-electrode potential drifted down increasing the UMg signal by around 3 mV. In the published figures from both Hess et al. (1982) and Lopez et al.
(1984) only short time impalements are shown. We also found it essential to control each impalement. Even experiments that gave seemingly stable impalements had to be discarded when they failed the depolarization test (cf. Fig. 2).
In these experiments calibration was only carried out down to 0 5 mmol Mg/l, because at the concentrations of Na and K in the background solution the Mg micro-electrodes are near the limit of detection levels (see Lanter et al. 1980). Thus the electrodes are not very suitable for an exact measurement of Mg values of less than 0 5 mmol/l. Despite this

[Mg]i IN FERRET VENTRICLE

471

- [Na] = 14-6 mmol/I [K] = 140-5 mmol/l
* [Na] = 7 mmoVI
[K] = 120 mmoI/l
-10

-20-

Um
UMg -2[ 7

_- _ _
_

-30r
I

0-1

I

III
111, ,
0-5
1

I

2

1,111
4
10

[Mg]1i mmol/l
Fig. 4. Calibration response of Mg micro-electrode in two different background solutions. The upper curve
(*) is the mean+ S.D. from twenty-seven measurements (twelve micro-electrodes) in solutions with [K],
140-5 mmol/l and [Na], 14-6 mmol/l, i.e. the solution used in these experiments. The lower curve (0
0)
shows mean -S.D. for twelve measurements (eleven micro-electrodes) in the background solution of Hess et al.
(1982), [K], 120 mmol/I and [Na], 7 mmol/l. A [Mg]i of 0 4 mmol/l on the upper curve corresponds to
2-1 mmol/l on the lower curve.

an estimation of the mean [Mg]i could be obtained by linear extrapolation of the calibration curve. Since the curve becomes flat in this region the estimated mean value must be regarded as an upper limit.

Comparison with other [Mg]i measurements
[Mg]i measurements have been recently reviewed by Gupta et al. (1984) and Flatman
(1984) and of the various methods used we wish only to comment on the recent nuclear magnetic resonance (n.m.r.) measurements. Gupta and co-workers obtained values of
0'6 mmol/l for skeletal muscle and 0-8 mmol/l for heart muscle (Gupta & Moore, 1980;
Gupta et al. 1984). Wu, Pieper, Salhany & Eliot (1981) made similar measurements to Gupta
& Moore (1980) but interpreted the results to give a [Mg]i of 2-5 mmol/l. The calculations of Wu et al. (1981) have recently been shown to be erroneous and recalculation gave a value of 1-6 mmol/l (Garfinkel & Garfinkel, 1984). Cohen & Burt (1977) found a high value of
4-4 mmol/l for [Mg]i in frog skeletal muscle but this value has been strongly criticized on methodological grounds by Gupta et al. (1984). In conclusion, it now appears that the n.m.r. measurements are all around 1 mmol/l, and are in reasonable agreement with the measurements reported in this paper.
The lower values of [Mg]i for muscle tissue are also supported by other estimations in the literature. Maughan (1983) found a value of 0-2 mmol/l for frog skeletal muscle. Baylor,
Chandler & Marshall (1982) using the Mg-sensitive metallochronic indicator dyes Arsenazo
III and Dichlorophosphonazo III found values ranging between 0-2 and 1 -2 mmol/l for frog skeletal muscle. Palaty (1971) estimated an [Mg]i of less than 0.1 mmol/l in the smooth muscle of arteries by Mg efflux experiments.

[Mg]i and cellular function
An [Mg]i of 0-4 mmol/l corresponds to a reversal potential for Mg at 24 °C of + 2-9 mV and Mg is thus far from equilibrium. However, no attempt was made to investigate possible

472

L. A. BLATTER AND J. A. S. McGUIGAN

control mechanisms since alteration of the composition of the Tyrode would cause simultaneous alteration in the ionic composition of the cell and thus change the calibration of the electrode.
The low value for [Mg]i means that Mg concentration is near the optimal value for various enzymatic reactions (see Polimeni & Page, 1973; Gunther, 1977). It also supports a physiological role for Ca release of Ca, and Ca uptake into the mitochondria (Fabiato &
Fabiato, 1975; Fry, Powell, Twist & Ward, 1984a, b).
We wish to thank Marlis Jordi for technical help and Dr D. Ammann, ETH Zurich for the gift of the Mg cocktail. This work was supported by the Swiss National Science Foundation Grant number
3.320-0.82. Professor S. Weidmann and Dr M. Pressler made helpful comments on the manuscript.
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