Sodium Ions Affect GPR17 Conformational States and Functionality

Sodium is an allosteric modulator of several class-A GPCR regulating active versus inactive conformation state and may be a critical issue for the optimization of crystallization procedures. Herein, the role of Na+ in the regulation of GPR17 conformational states was studied through MD simulations of GPR17 wild-type and mutated on two different residues relevant for sodium interaction (D77A and D293N), in inactive and active conformations. We observed that Na+ stabilizes the inactive wild-type conformation more than the active conformation since in the latter the interaction between Na+ and D77/D293, fundamental in receptor stabilization, breaks. In addition to modeling studies, the role of sodium in GPR17 binding properties and functional responses was investigated in vitro using receptor variants carrying mutations at the sodium-binding residues D77 and D293. Radioligand binding studies, using GPR17 selective ligands, did not show any significant alteration of GPR17 binding properties in mutated variants with respect to wild-type receptor. In contrast, a significant increase in receptor coupling to G proteins was noticed for Asinex1 and LTD4 in the receptor variants D77, D293, and D77/D293 with respect to the wild-type form. Finally, all the receptor ligands showed a significant increase in their potency to inhibit cAMP accumulation in D77, D293, and D77-D293 GPR17 with respect to WT receptor form. Overall, these data, in agreement with modeling studies, suggest that ionic sodium stabilizes an inactive conformation of the receptor. When residues in the sodium-binding site are mutated, the ion can no longer bind, thereby favoring an active conformation of GPR17. These mutagenized variants, stabilizing an active receptor state, may be the most suitable for homogeneous receptor purification and crystallization.