Hearing sensitivity in mammals is enhanced by more than 40 dB (that is, 100-fold) by mechanical amplification thought to be generated by one class of cochlear sensory cells, the outer hair cells. In addition to the mechano-electrical transduction required for auditory sensation, mammalian outer hair cells also perform electromechanical transduction, whereby transmembrane voltage drives cellular length changes at audio frequencies in vitro. This electromotility is thought to arise through voltage-gated conformational changes in a membrane protein, and prestin has been proposed as this molecular motor. Here we show that targeted deletion of prestin in mice results in loss of outer hair cell electromotility in vitro and a 40-60 dB loss of cochlear sensitivity in vivo, without disruption of mechano-electrical transduction in outer hair cells. In heterozygotes, electromotility is halved and there is a twofold (about 6 dB) increase in cochlear thresholds. These results suggest that prestin is indeed the motor protein, that there is a simple and direct coupling between electromotility and cochlear amplification, and that there is no need to invoke additional active processes to explain cochlear sensitivity in the mammalian ear.

Growing evidence suggests that close appositions between the endoplasmic reticulum (ER) and other membranes, including appositions with the plasma membrane (PM), mediate exchange of lipids between the two bilayers. The mechanisms of such exchange, which allows lipid transfer independently of vesicular transport, remain poorly understood. The presence of an SMP (synaptotagmin-like-mitochondrial-lipid binding protein) domain, a proposed lipid binding module, in several proteins localized at membrane contact sites raised the possibility that such domains may be implicated in lipid transport1,2. SMP-containing proteins include components of the ERMES complex, an ER-mitochondrial tether3, and the Extended-Synaptotagmins/tricalbins, which are ER-PM tethers4-6. Here we present at 2.44 Å resolution the crystal structure of a fragment of Extended-Synaptotagmin 2 (E-Syt2), including an SMP domain and two adjacent C2 domains. The SMP domain has a beta-barrel structure like protein modules in the TULIP superfamily. It dimerizes to form a ~90 Å long cylinder traversed by a channel lined entirely with hydrophobic residues, with the two C2A-C2B fragments forming arched structures flexibly linked to the SMP domain. Importantly, structural analysis complemented by mass spectrometry revealed the presence of glycerophospholipids in the E-Syt2 SMP channel, indicating a direct role for E-Syts in lipid transport. These findings provide strong evidence for a role of SMP domain containing proteins in the control of lipid transfer at membrane contact sites and have broad implication beyond the field of ER to PM appositions.

It is a central tenet of cochlear neurobiology that mammalian ears rely on a local, mechanical amplification process for their high sensitivity and sharp frequency selectivity. While it is generally agreed that outer hair cells provide the amplification, two mechanisms have been proposed: stereociliary motility and somatic motility. The latter is driven by the motor protein prestin. Electrophysiological phenotyping of a prestin knockout mouse intimated that somatic motility is the amplifier. However, outer hair cells of knockout mice have significantly altered mechanical properties, making this mouse model unsatisfactory. Here, we study a mouse model without alteration to outer hair cell and organ of Corti mechanics or to mechanoelectric transduction, but with diminished prestin function. These animals have knockout-like behavior, demonstrating that prestin-based electromotility is required for cochlear amplification.

Highlights d LDAF1 and seipin form an 600 kDa oligomeric complex in the ER bilayer d The LDAF1-seipin complex determines the sites of lipid droplet formation in the ER d LDAF1 dissociates from seipin and moves to the growing lipid droplet surface d LDAF1 facilitates lipid droplet formation at low ER triglyceride concentration