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.