Elevator-like movements of prestin mediate outer hair cell electromotility.

The outstanding acuity of the mammalian ear relies on cochlear amplification, an active mechanism based on the electromotility (eM) of outer hair cells. eM is a piezoelectric mechanism generated by little-understood, voltage-induced conformational changes of the anion transporter homolog prestin (SLC26A5). We used a combination of molecular dynamics (MD) simulations and biophysical approaches to identify the structural dynamics of prestin that mediate eM. MD simulations showed that prestin samples a vast conformational landscape with expanded (ES) and compact (CS) states beyond previously reported prestin structures. Transition from CS to ES is dominated by the translational-rotational movement of prestin's transport domain, akin to elevator-type substrate translocation by related solute carriers. Reversible transition between CS and ES states was supported experimentally by cysteine accessibility scanning, cysteine cross-linking between transport and scaffold domains, and voltage-clamp fluorometry (VCF). Our data demonstrate that prestin's piezoelectric dynamics recapitulate essential steps of a structurally conserved ion transport cycle.

Residency time of agonists does not affect the stability of GPCR-arrestin complexes.

BACKGROUND AND PURPOSE: The interaction of arrestins with G-protein coupled receptors (GPCRs) desensitizes agonist-dependent receptor responses and often leads to receptor internalization. GPCRs that internalize without arrestin have been classified as "class A" GPCRs whereas "class B" GPCRs co-internalize with arrestin into endosomes. The interaction of arrestins with GPCRs requires both agonist activation and receptor phosphorylation. Here, we ask the question whether agonists with very slow off-rates can cause the formation of particularly stable receptor-arrestin complexes. EXPERIMENTAL APPROACH: The stability of GPCR-arrestin-3 complexes at two class A GPCRs, the ß2 -adrenoceptor and the µ opioid receptor, was assessed using two different techniques, fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) employing several ligands with very different off-rates. Arrestin trafficking was determined by confocal microscopy. KEY RESULTS: Upon agonist washout, GPCR-arrestin-3 complexes showed markedly different dissociation rates in single-cell FRET experiments. In FRAP experiments, however, all full agonists led to the formation of receptor-arrestin complexes of identical stability whereas the complex between the µ receptor and arrestin-3 induced by the partial agonist morphine was less stable. Agonists with very slow off-rates could not mediate the co-internalization of arrestin-3 with class A GPCRs into endosomes. CONCLUSIONS AND IMPLICATIONS: Agonist off-rates do not affect the stability of GPCR-arrestin complexes but phosphorylation patterns do. Our results imply that orthosteric agonists are not able to pharmacologically convert class A into class B GPCRs.