Dispatched uses Na⁺ flux to power release of lipid-modified Hedgehog

The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters^1,2 and to H⁺-driven transporters of the RND family^3,4, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly -localized sites of embryonic expression^5-10. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na⁺ ions coordinated within a channel that traverses its transmembrane domain. We find that the rate of Hedgehog export is dependent on the Na⁺ gradient across the plasma membrane. The transmembrane channel and Na⁺ binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartate residues that each coordinate and neutralize the charge of one of the three Na⁺ ions. DISP1-NNN and variants that disrupt single Na⁺ sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. Interaction of the amino-terminal signalling domain of the Sonic hedgehog protein (ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. The bound and unbound DISP1 conformations display distinct Na⁺-site occupancies, which suggests a mechanism by which transmembrane Na⁺ flux may power extraction of the lipid-linked Hedgehog signal from the membrane. Na⁺-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na⁺ flux powers their conformationally driven activities.