Identifikation der Phosphatidylinositol-3,5-bisphosphat Bindetasche im humanen Zweiporenkanal 2

Zweiporenkanäle (TPCs) des endolysomalen Systems aus Säugetieren können durch Phosphatidylinositol 3,5 bisphosphat (PI(3,5)P2), ein Membranlipid welches in niedriger Konzentration in der Membran auftritt, aktiviert werden. Die beiden humanen Zweiporenkanäle 1 und 2 sind für das Trafficking und die Funktion interner Organellen von besonderer Bedeutung. Durch die Kombination aus in silico und in vivo Untersuchungen des PI(3,5)P2–insensitiven Zweiporenkanals 1 aus Arabidposis thaliana im Vergleich zum PI(3,5)P2-aktivierten humanen Zweiporenkanals 2 konnte eine cytosolischer Bereich des humanen Zweiporenkanals 2 identifiziert werden, der eine Bindetasche für (PI(3,5)P2) bildet. Diese Bindung wird zum einen durch positiv geladene Reste im Bereich des Linkers zwischen S4 und S5 vermittelt (K203, K204, K207) und zum anderen durch einen Rest im cytosolischen Loop zwischen S6 und S7 (S322). Die hier postulierte Bindetasche könnte somit bei Bindung des Liganden eine koordinierten Bewegung zwischen diesen Teilen des Proteins ermöglichen und damit die allosterische Umlagerung einleiten, die den Kanal in den geöffneten Zustand überführt.Mammalian two-pore channels (TPCs) are activated by the low-abundance membrane lipid phosphatidyl-(3,5)-bisphosphate (PI(3,5)P2) present in the endo-lysosomal system. Malfunction of human TPC1 or TPC2 (hTPC) results in severe organellar storage diseases and membrane trafficking defects. Here, we compared the lipid-binding characteristics of hTPC2 and of the PI(3,5)P2-insensitive TPC1 from the model plant Arabidopsis thaliana. Combination of simulations with functional analysis of channel mutants revealed the presence of an hTPC2-specific lipid-binding pocket mutually formed by two channel regions exposed to the cytosolic side of the membrane. We showed that PI(3,5)P2 is simultaneously stabilized by positively charged amino acids (K203, K204, and K207) in the linker between transmembrane helices S4 and S5 and by S322 in the cytosolic extension of S6. We suggest that PI(3,5)P2 cross links two parts of the channel, enabling their coordinated movement during channel gating

Phosphatidylinositol-3,5-bisphosphate lipid-binding-induced activation of the human two-pore channel 2

Mammalian two-pore channels (TPCs) are activated by the low-abundance membrane lipid phosphatidyl-(3,5)-bisphosphate (PI(3,5)P2) present in the endo-lysosomal system. Malfunction of human TPC1 or TPC2 (hTPC) results in severe organellar storage diseases and membrane trafficking defects. Here, we compared the lipid-binding characteristics of hTPC2 and of the PI(3,5)P2-insensitive TPC1 from the model plant Arabidopsis thaliana. Combination of simulations with functional analysis of channel mutants revealed the presence of an hTPC2-specific lipid-binding pocket mutually formed by two channel regions exposed to the cytosolic side of the membrane. We showed that PI(3,5)P2is simultaneously stabilized by positively charged amino acids (K203, K204, and K207) in the linker between transmembrane helices S4 and S5 and by S322 in the cytosolic extension of S6. We suggest that PI(3,5)P2cross links two parts of the channel, enabling their coordinated movement during channel gating