Mechanism of Ammonium Translocation in Rat Liver Mitochondria. Finger-printing of the Translocator

We have studied whether ammonia crosses the cristae membrane of rat liver mitochondria as a charged (NH4+) or an uncharged (NH3) species. Passive swelling of mitochondria suspended in ammonium and/or sodium acetates showed that: swelling depends upon ammonium concentration in the same way it depends on sodium (at constant acetate concentration). The curves reach a plateau at 115 mM ammonium and 100 mM sodium. A two units change of pH induces a two orders of magnitude change in the concentration of NH3, while NH4+ remains almost constant. However, the extent and initial rates of swelling are not significantly modified. The results are discussed as ammonia being translocated as a charged species via a transport system. The transport system was characterized studying the swelling reaction of inhibited rat liver mitochondria suspended in acetate salts of alkali, ammonium and nitrogenous cations: The selectivity pattern of the translocator is: NH4+ greater than Na+ greater than Li+ greater than K+ greater than Rb+ greater than Cs+ (Eisenman's sequence X). For homologous nitrogenous cations permeability decreases as molecular weight increases or free solution mobility decreases. Ions with Ladd Radius shorter than 3.7 to 3.8 A and cross sections below 15 A2 are permeable, those with longer radius or larger areas are not. As the chain of monosubstituted nitrogenous cations is lengthened, permeability decreases, passes through a minimum around NC = 4 and then increases. Nitrogenous cations which do not make hydrogen bonds do not enter mitochondria, those with one, two or three donor protons have similar permeabilities, and those with one, two or three oxygen acceptors increase their permeability almost linearly. Formamidine, acetamidine, TEA, guanidine derivatives, N,N-dicyclohexylcarbodiimide and N-ethyl maleimide failed to inhibit sodium-induced mitochondrial passive swelling.