Simultaneous bidirectional magnesium ion flux measurements in single barnacle muscle cells by mass spectrometry

Abstract

Stable isotopes of Mg were used to measure bidirectional magnesium ion fluxes in single barnacle giant muscle fibers immersed in Ca- and Na-free, isosmotic media. Measurements were made using a mass spectrometric technique, thermal ionization mass spectrometry (TIMS), in conjunction with atomic absorption spectroscopy. Kinetic relations based on a first-order model were developed that permit the determination of unidirectional rate coefficients for Mg influx, ki, and efflux, ke, in the same experiment from knowledge of initial conditions and the initial and final ratios of 26Mg/24Mg and 25Mg/24Mg in ambient solutions (i.e., by isotope dilution). Such determinations were made for three values of the external Mg ion concentration: 5, 25, and 60 mM. At the concentration [Mg+2]o = 5 mM, ki and ke were about equal at a value of 0.01 min-1. At the higher values of [Mg+2]o, the values of ke increased along a curve suggesting saturation, whereas the values of ki remained essentially constant. As could be expected on the basis of a constant ki, the initial influx rate varied in direct linear proportion to [Mg+2]o, and was 11.8 pmol/cm2s when [Mg+2]o was 5 mM. However, the initial efflux rate appeared to increase nonlinearly with [Mg+2]o, varying from 13.4 pmol/cm2s ([ Mg+2]o = 5 mM) to approximately 80 pmol/cm2s ([ Mg+2]o = 60 mM). The results are consistent with a model that assumes Mg influx to be mainly an electrodiffusive inward leak with PMg = 0.07 cm/s and Mg efflux to be almost entirely by active transport processes. Where comparisons can be made, the rate coefficients determined from stable isotope measurements agree with those previously obtained using radioactive Mg. The rate coefficients can be used to correctly predict time-dependent changes in total fiber Mg content. The results support the conclusion that nonradioactive tracers can be used to measure ion fluxes and ion flux ratios in excitable cells; it is expected that this method will greatly assist in the study of Mg regulation in general.

Intrinsic gating of inward rectifier in bovine pulmonary artery endothelial cells in the presence or absence of internal Mg2+.

“…In order to estimate the [MgZ+]i just under the membrane, we have applied a compartmental diffusion model analagous to that described by Hille (1977). We assumed a cell diameter of 10 #m, a pipette tip diameter of 1 t~m, a diffusion coefficient of 0.71 x 10 -5 cm2/s (Weast, 1987), and a membrane permeability of 0.07 x 10 -6 cm/s determined in barnacle muscle cells (Montes et al, 1989). With initial concentrations of 2 mM Mg 2+ in the bath, 10 mM Mg ~+ in the cellular compartments and 0 mM Mg 2+ in the pipette, the model predicts that [Mg2+]~ in the compartment just inside the membrane would drop with a time constant of 2.3 s to 1.9 #M even without considering the effects of Mg 2+ buffers used.…”

“…Consideration of the possibility that external Mg 2+ may cross the membrane and accumulate just under the membrane in a simple model described in Methods indicates that diffusion of Mg 2+ into the pipette will reduce submembrane [Mg ~+] to a few micromolar even without including the effects of Mg 2+ buffers. The critical assumption in this calculation is that the Mg 2+ permeability of barnacle muscle cells (Montes et al, 1989) is comparable to that in endothelial cells. If endothelial cells have a special Mg-transport mechanism then greater accumulation of Mg inside the membrane might occur.…”

Retrospective on the development of aequorin and aequorin‐based imaging to visualize changes in intracellular free [Ca2+]

“…Ridgway and Ashley () published the first report describing aequorin‐generated Ca 2+ transients in single cells when they microinjected 0.3–0.5 µl of holo‐aequorin (dissolved in 300 mM KCl) into giant muscle fibers (∼2–3 cm in length and ∼1.0–1.5 mm at its widest point) (Montes et al, ) of the acorn barnacle ( Balanus nubilus ). Following microinjection, they waited for some 30 min to allow the holo‐aequorin to diffuse throughout the muscle fiber prior to the start of the experiment.…”

“…The crystal structure of aequorin was determined via X-ray crystallography and four EF-hand domains were identified Head et al (2000) The crystal structures of four semi-synthetic aequorins were derived Toma et al (2006 Used theoretical methods to determine the origin and varying characteristics of aequorin chemiluminescence, bioluminescence, and fluorescence Chen et al (2013) RETROSPECTIVE ON AEQUORIN-BASED IMAGING HL60 leukemic cells, which range from 25 mm to just 12 mm in diameter (Cobbold et al, 1983(Cobbold et al, , 1987Cobbold and Lee, 1991). Ridgway and Ashley (1967) published the first report describing aequorin-generated Ca 2þ transients in single cells when they microinjected 0.3À0.5 ml of holo-aequorin (dissolved in 300 mM KCl) into giant muscle fibers ($2À3 cm in length and $1.0À1.5 mm at its widest point) (Montes et al, 1989) of the acorn barnacle (Balanus nubilus). Following microinjection, they waited for some 30 min to allow the holo-aequorin to diffuse throughout the muscle fiber prior to the start of the experiment.…”

Plasmalemmal transport of magnesium in excitable cells

“…Activation of FAS on B-cell lymphomas causes an increase in [Mg 2+ ] i that appears to be required for apoptosis (10 ] I handling may be a critical effector of non-insulin dependent diabetes mellitus (16,17). In addition, hypomagnesemia may produce nervous hyperexcitability (see, 18,19), tetanic syndrome (20), and meningo encephalic syndrome (21 (22,(35)(36)(37)(38)(39)(40). Among these preparations, barnacle muscle cells and squid giant axons offer the advantage that, owing to their large size, they can be internally perfused or dialyzed, and voltageclamped.…”