Functional Uncoupling of Pain-Linked Nav1.7/A1632E Dimers Partly Rescues Its Pain-Causing Phenotype

Abstract

The voltage-gated sodium channel Nav1.7 is essential for an adequate perception of pain in humans and its mutations cause various pain syndromes. The hNav1.7/A1632E mutation leads to a pain syndrome which combines symptoms of erythromelalgia and paroxysmal extreme pain disorder (PEPD). The mutation-induced gating changes include increased persistent current. Using 3D molecular simulations, we show that in hNav1.7/A1632E the inactivation particle (IFM motif) is hindered from binding by steric and hydrophobic mismatch within its binding pocket at the side of the pore module. This phenomenon is well in line with the recently proposed allosteric fast inactivation mechanism, in which the IFM motif no longer occludes the pore by a "hinged lid" mechanism but by binding-induced conformational changes. By using native polyacrylamide gel electrophoresis (PAGE), we show that hNav1.7 dimerizes similar to what has been reported for hNav1.5. Applying a patch-clamp approach, we reveal that the disease-linked persistent current depends on the channel's functional dimerization status. Difopein is an inhibitor of 14-3-3 protein and suggested to functionally uncouple dimerization of hNav1.5. Its co-transfection leads to a significant decrease in hNav1.7/A1632E persistent currents. Thus, channel dimerization seems to support the dramatic increase in the pain-causing persistent current induced by the hNav1.7/A1632E mutation's interference with the allosteric fast inactivation mechanism. We here reveal how functional uncoupling of hNav1.7/A1632E dimers partly rescues the pain-causing molecular phenotype. Our work hence supports the newly described allosteric fast inactivation mechanism and combines it for the first time with sodium channel dimerization, leading to new insights in human pain syndromes.