Ca2+ transport in digitonin-permeabilized trypanosomatids

The use of digitonin to permeabilize Leishmania mexicana mexicana, Leishmania agamae, and Crithidia fasciculata plasma membranes enabled us to study Ca2+ transport in situ. The present results show that the mitochondria of these trypanosomatids are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Ca2+ uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Ca2+ uptake was partially inhibited by ruthenium red, almost totally inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and stimulated by inorganic phosphate. Large amounts of Ca2+ were retained by C. fasciculata mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butylhydroperoxide and diamide. In contrast, Ca2+ was not retained in the matrix of Leishmania sp. mitochondria for long periods of time. In addition to the mitochondrial Ca2+ uptake, a vanadate-sensitive Ca2(+)-transporting system was also detectable in these trypanosomatids.