ABSTRACT
K+-H+ exchange activity in hamster brown adipose tissue mitochondria is activated following depletion of matrix Mg2+ with the divalent cation ionophore A23187. Quinine inhibits K+-H+ exchange reversibly with an I50 of 22 microM, whereas mild treatment with N,N'-dicyclohexylcarbodiimide (DCCD) inhibits this activity irreversibly. In an attempt to label and identify the K+-H+ antiporter protein, brown adipose tissue mitochondria were incubated with [14C]DCCD and subjected to denaturing polyacrylamide gel electrophoresis and fluorography. We observed a labeled band of relative mol wt, 78,000, which satisfies criteria established in rat liver mitochondria for the identification of this carrier (W. H. Martin et al., J. Biol. Chem. 259: 2062-2065, 1984). Thus Mg2+ and quinine each protect the K+-H+ exchanger against both inhibition and binding by DCCD. Volume homeostasis in brown adipose tissue mitochondria, as in other mitochondria, requires a balance between K+ influx and efflux. We propose that regulation of the K+-H+ antiporter, the primary K+ efflux mechanism, plays a major role in this process.
Subject(s)
Adipose Tissue, Brown/ultrastructure , Homeostasis , Hydrogen/metabolism , Mitochondria/metabolism , Potassium/metabolism , Adipose Tissue, Brown/metabolism , Animals , Carrier Proteins/metabolism , Cricetinae , Dicyclohexylcarbodiimide/metabolism , Dicyclohexylcarbodiimide/pharmacology , Ion Exchange , Magnesium/pharmacology , Mesocricetus , Potassium-Hydrogen Antiporters , Quinine/pharmacologyABSTRACT
The murine S49 lymphoma cell transports Mg2+ by a system distinct from systems responsible for Ca2+ influx (J. Physiol. London 337: 351-371, 1983). We have now determined the ability of various cations, anions, and drugs to modulate Mg2+ influx. Neither sulfate, nitrate, phosphate, nor bicarbonate altered Mg2+ influx. Among cations only T1+, Ba2+, Zn2+, Mn2+, Sc3+, and La3+ potently inhibited Mg2+ influx without causing obvious cell toxicity. Seventeen other cations were ineffective at maximal nontoxic concentrations. T1+ inhibition (Ki = 300 micron) is noncompetitive and apparently derives from its ability to dissipate membrane potential. The noncompetitive nature of and the rather poor inhibition constants for Ca2+ (Ki approximately equal to 5 mM) and Mn2+ (Ki = 200 micron) indicate that neither cation is an effective physiological antagonist of Mg2+ influx. Only Ba2+ exhibited competitive inhibition of Mg2+ influx (Ki = 1 mM). Cisplatin and Ca2+ channel antagonists also did not inhibit Mg2+ influx. These data further differentiate Mg2+ transport systems from those for Ca2+. In addition, the selectivity series for group IIa cation inhibition of influx (Mg2+ greater than Ba2+ much greater than Ca2+ greater than or equal to Sr2+) has not been observed previously in biological systems and is indicative of a very high anionic field strength at the Mg2+ recognition site.
