Extended Data Fig. 8: Electrostatic calculations and charge transfer of prestin across the membrane.

a, Mutation of the key residues in the anion binding pocket either completely abolishes the NLC (R399Q) or right shifts the V_1/2 by more than 80 mV (F101Y) to around +25 ± 5 mV (mean ± s.e.m.; n, is the number of independent cells. One-sided Student t-test, P=0.001); a similar effect has been observed in other prestin homologues using patch-clamp electrophysiology (51). b, Snapshots from the MD trajectories of the systems, and calculation of the electrostatic potential across the membrane at two states, the Down I state (with SO₄²⁻ in the left cavity, and without SO₄²⁻ in the right cavity) versus Up (with Cl⁻ in the left cavity and without any Cl⁻ in the right cavity). The x-z plane is crossing the two central anion-binding sites. In both models, the positive field is mainly focused around the transmembrane mid-plane and around the anion-binding site, creating an attractive (blue) field for the binding of the anion. However, in the Up state the field is more positive around the mid-plane compared to the corresponding region in the Intermediate state. In both cases, the presence of the anion only partially neutralizes (~35%) the positive field around the bilayer mid-plane. Note that the actual size of the simulation box is larger than what is illustrated here (see Methods). c, Averaged 1-D fraction of membrane potential in the z direction along the two central binding sites (shown as dashed blue lines in panel A with the central binding sites highlighted using the red cross symbols). The 1-D and 2-D maps were directly extracted from the ensemble averaged 3-D fraction of membrane potential map. The location of the phosphate atoms of the outer and inner lipid leaflets along the z axis was highlighted with dashed gray lines). d, Displacement of charge for prestin in the Up and Down I conformations at different transmembrane potentials. The gating charge between the two states is 0.38 +0.25 e calculated as the offset constant between the linear fits. (n = 3; data are mean ± SD; One-sided Student’s t-test; P=0.05). e, R399 in both monomers have been mutated to Q, S and E in different systems to see the contribution of R399 residue to the positive charge at the bilayer mid-plane using electrostatic calculations. R399 mutation to polar residues shows that R399 has almost ~40% contribution the positive charge of the field at the bilayer mid-plane. The remainder likely comes from the TM3-TM10 helical dipole and other positive charges in this area.