Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

Local activation and long-range inhibition are mechanisms conserved in self-organizing systems leading to biological patterns. A number of them involve the production by the developing cell of an inhibitory morphogen, but how this cell becomes immune to self-inhibition is rather unknown. Under combined nitrogen starvation, the multicellular cyanobacterium Nostoc PCC 7120 develops nitrogen-fixing heterocysts with a pattern of one heterocyst every 10-12 vegetative cells. Cell differentiation is regulated by HetR which activates the synthesis of its own inhibitory morphogens, diffusion of which establishes the differentiation pattern. Here we show that HetR interacts with HetL at the same interface as PatS, and that this interaction is necessary to suppress inhibition and to differentiate heterocysts. hetL expression is induced under nitrogen-starvation and is activated by HetR, suggesting that HetL provides immunity to the heterocyst. This protective mechanism might be conserved in other differentiating cyanobacteria as HetL homologues are spread across the phylum.