Calcium influx in Trypanosoma brucei can be induced by amphiphilic peptides and amines

The following study was undertaken to determine whether an inducible calcium influx pathway is present in intact bloodstream forms of Trypanosoma brucei. Fura-2 fluorescence was used to demonstrate that amphiphilic peptides and amines, including melittin, mastoparan and compound 48/80, each produced a dose dependent calcium influx across the plasma membrane. Calcium influx did not result from general disruption of membrane integrity, since a corresponding influx of ethidium bromide or other divalent cations was not observed. Instead, the calcium influx was selectively blocked by the calcium channel antagonists, La3+, Cd2+ or Ni2+, and was not affected by the Na+ channel antagonists, tetrodotoxin or amiloride. Activation of the trypanosome calcium influx pathway was dependent upon an intact membrane potential, and the rise in intracellular free calcium concentration ([Ca2+]i) was reversed upon membrane depolarization with gramicidin D. Changes in Ins(1,4,5)P3 did not accompany the calcium influx. Overall, these data provide the first evidence of an inducible calcium influx pathway in T. brucei, and describe methods to selectively manipulate this pathway.