Intracellular Mg2+ (Mgi) is a cofactor for hundreds of enzymes and modulates membrane receptors, ionic channels, and transporters. Hormones induce massive efflux of Mgi from cells but the underlying mechanism of plasmalemmal transport of Mg2+ is unknown. Squid exons, because of their large size, are ideally suited to study Mg2+ transport: internal perfusion and voltage-clamping permits measurement and control of all the relevant driving forces for Mg2+ transport. In these cells, the concentration of intracellular free Mg2+ ([Mg2+]i) is about 600 times lower than expected if Mg2+ ions were at electrochemical equilibrium across the plasmalemma. Active extrusion of Mg2+ maintains [Mg2+]i under steady-state conditions. An ATP-dependent Na/Mg exchanger has been proposed as the sole mechanism responsible for Mg2+ extrusion. This hypothesis explains numerous experimental observations but leaves others unexplained: electrophysiological evidence indicates that Na/Mg exchange is voltage insensitive (consistent with a 2 Na+:1 Mg2+ exchange). However, thermodynamic considerations suggest that neither a stoichiometry of 1, 2, or 3 Na+:1 Mg2+ can predict the measured [Mg2+]; in excitable cells. Numerous observations in various cell types suggest that in addition to Na+, K+ and C1- may also be involved in the regulation of [Mg2+]i. Likewise, we have found preliminary evidence that, in squid axons, the electrochemical gradients of K+ and C1- may be coupled to Mg2+ transport: i) intracellular K+ and C1- are required for Mg2+-dependent Na+ fluxes; and ii) extracellular Mg2+ promotes simultaneous equimolar efflux of Na+-K+ and of Na+-C1-. However, an stoichiometry of 1Na+ + 1K+ = 1C1-:1 Mg2+ does not predict the measured steady-state [Mg2+]i and is inconsistent with an electroneutral exchanger. On the other hand, an stoichiometry of 2Na+ + 2K+ + 2C1-:1 Mg2+ explains the body of available information about Mg2+ transport and accurately predicts the steady-state [Mg2+]i in squid exons.
Our aim i s to assess if a 2Na+ + 2K+ + 2C1-:1 Mg2+ exchanger is the mechanism responsible for transporting Mg2+ across the plasmalemma and for maintaining [Mg2+]i under steady-state conditions in squid axons. Two main studies will be performed: i) determine the ionic-dependence and stoichiometry of the expected ionic fluxes (using radiolabelled ions); and ii) assess the effect of changes in the electrochemical gradients of Na+,K+ and C1- on the steady-state free and total [Mg]i (measured with flame photometry and fluorescent dyes). Intact, injected and internally dialyzed and voltage-clamped squid axons will be used.
| Nwoga, J C; Sniffen, J C; Pena-Rasgado, C et al. (1996) Effect of pentachlorophenol on calcium accumulation in barnacle muscle cells. J Physiol 491 ( Pt 1):13-20 |
| Gonzalez-Serratos, H; Hilgemann, D W; Rozycka, M et al. (1996) Na-Ca exchange studies in sarcolemmal skeletal muscle. Ann N Y Acad Sci 779:556-60 |
| Rasgado-Flores, H; Espinosa-Tanguma, R; Tie, J et al. (1996) Voltage dependence of Na-Ca exchange in barnacle muscle cells. I. Na-Na exchange activated by alpha-chymotrypsin. Ann N Y Acad Sci 779:236-48 |
