Mg2+-Malate Co-Transport, a Mechanism for Na+-Independent Mg2+Transport in Neurons of the LeechHirudo medicinalis

Abstract

Mg2+-extrusion from Mg2+-loaded neurons of the leech, Hirudo medicinalis, is mediated mainly by Na+/Mg2+antiport. However, in a number of leech neurons, Mg2+is extruded in the nominal absence of extracellular Na+, indicating the existence of an additional, Na+-independent Mg2+transport mechanism. This mechanism was investigated using electrophysiological and microfluorimetrical techniques. The rate of Na+-independent Mg2+extrusion from Mg2+-loaded leech neurons was found to be independent of extracellular Ca2+, K+, NO3−, HCO3−, SO42−, HPO42−, and of intra- and extracellular pH. Na+-independent Mg2+extrusion was not inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), furosemide, ouabain, vanadate, iodoacetate, 4-amino-hippurate, or α-cyano-4-hydroxycinnamate and was not influenced by changes in the membrane potential in voltage-clamp experiments. Na+-independent Mg2+extrusion was, however, inhibited by the application of 2 mM probenecid, a blocker of organic anion transporters, suggesting that Mg2+might be co-transported with organic anions. Extracellularly, of all organic anions tested (malate, citrate, lactate, α-ketoglutarate, and 4-amino-hippurate) only high, but physiological, concentrations of malate (30 mM) had a significant inhibitory effect on Na+-independent Mg2+extrusion. Intracellularly, iontophoretically injected malate, citrate, or fura-2, but not Cl−, α-ketoglutarate, glutamate, succinate, or urate, were stimulating Na+-independent Mg2+extrusion from those neurons that initially did not extrude Mg2+in Na+-free solutions. Our data indicate that Mg2+is co-transported with organic anions, preferably with malate, the predominant extracellular anion in the leech. The proposed model implies that, under experimental conditions, malate drives Mg2+extrusion, whereas under physiological conditions, malate is actively taken up, driven by Mg2+, so that malate can be metabolized.